Scientific program DGP, 21st Annual Meeting March 17th – 20th, 2004 in Würzburg

Scientific Program DGP, 21st Annual Meeting March 17 th -20 th, 2004 in W~Jrzburg

DGP Congress in WLirzburg 2004

21. Jahrestagung der Deutschen Gesellschaft f0r Parasitologie (DGP) in Verbindung mit dem Treffen der DGP Arbeitsgruppe ,,Wirkstoffentwicklung"

March 17 th to 20 th, 2004 Wi.irzburg, Germany

Congress Venue: Philosophisches HSrsaalgeb~ude der Universit~t W0rzburg, Am Hubland

Welcome to the 21 st DGP Congress in W~irzburg

Dear colleagues,

On behalf of the German Society of Parasitology (DGP), the University of W0rzburg and the local Program Committee, we have the great pleasure and honour to welcome you to the 21 st DGP Congress in Wf3rzburg.

Worldwide, the numbers of people and animals suffering and dying from parasites are overwhelming. To unravel this problem, research on parasites and the diseases must include a variety of fields: cell biology, molecular biology, biochemistry, genetics and immunology. In each case, modern biology has provided not only basic insights, but also potential intervention strategies. In addition to basic science paradigms, the implementation of parasitic disease control strategies raises epidemiological issues. The scientific sessions of the 21 st DGP Congress in W0rzburg consider all these aspects. The programs provides you with three plenary sessions, 19 parallel workshops and the poster presentations.

We look forward to an interesting congress with stimulating discussions and wish you a pleasant stay in WQrzburg.

On behalf of the local Organizing Committee

Klaus Brehm, Matthias Frosch, Matthias Leippe, Heidrun Moll

Deutsche Gesellschafl fiir Parasitologie e.V.

A U F N A H M E A N T R A G

Hiermit beantrage ich meine Aufnahme in die ,,Deutsche Gesellschaft fiir Parasitologie e.V."

Name Vomame Datum > in Druckbuchstaben <

Unterschrift

Referenzen (DGP-Mitglieder, soweit m6glich)

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.. ......................................................................................................................................................

F R A G E B O G E N (far die Mitgliederdatei)

A

Titel m~innl. weibl. Fachrichtung (Naturwiss., Medizin usw.)

Institution (Universitgt, Finna, Beh6rde) Land (wenn nicht Deutschland)

Institut o.~i. Geburtsdatum (erscheint nicht im Mitgliederverz.)

Abt., AG, Fachgruppe o.g.

Stral3e und Hausnummer od. Geb~iudenummer

Postleitzahl (Code) und Stadt

Telefon

FAX

e-mail

Privatanschrift: Strage und Hausnummer

Privatanschrift: Postleitzahl und Stadt

Telefon privat

FAX privat

e-mail privat

Der Jahresbeitrag betrfigt e 25.-, Far Studierende C 9.-. Beitrgge und Spenden sind nach § 10b EStG, § 11 Ziff. 5 KStG abzugsf~ihig

Deutsche Gesellschaft fttr Parasitologie e.V., Schriftftthrerin: Prof. Dr. Brigitte Frank Universi~t Hohenheim, FG Parasitologie, D-70599 Stuttgart, Tel. 0711 / 459-2277, Fax 0711 / 459-2276

e-mail: [email protected]

Scientific Program DGP, 21st Annual Meeting March 17 th -20 th, 2004 in WQrzburg

Lageplan

Haltestel le Lini~ 14

5

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in W0rzburg

Sponsoren und Aussteller

Deutsche Forschungsgemeinschaft

SFB 479 ,,Erregervariabilit~t und Wirtsreaktion bei infektiSsen

Krankheitsprozessen".

SFB 630 ,,Erkennung, Gewinnung und funktionale Analyse von

Wirkstoffen gegen Infektionskrankheiten"

Universit~tsbund W0rzburg

Aventis Pharma Deutschland GmbH

BD Biosciences

GlaxoSmithKline GmbH & Co. KG

Intervet Deutschland GmbH

Janvier S.A.

Pfizer GmbH

6

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in W0rzburg

Veranstaltungsort: Philosophiezentrum am Hubland (Tel. 6rtl. TagungsbQro: 0171-960-4304)

Tagungsleitung: Prof. Dr. Matthias Frosch

Institut fGr Hygiene und Mikrobiologie U niversit~it WQrzburg Josef-Schneider-Strasse 2 97080 Werzburg Tel.: 0931-201-46160 Fax.: 0931-201-46445 Email: [email protected]

Tagungsb(iro: Frau Monika Ulrich

Institut for Hygiene und Mikrobiologie Josef-Schneider-Strasse 2 97080 WQrzburg Tel.: 0931-201-46161 Fax: 0931-201-46445 Email: [email protected]

Organisatoren: Zentrum for Infektionsforschung der Universit~t WGrzburg

(Dr. Klaus Brehm, Prof. Dr. Matthias Frosch, Prof. Dr. Heidrun Moll, Prof.

Dr. Matthias Leippe)

Prof. Dr. Peter K6hler (Universit~it ZGrich)

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in WiJrzburg

21 st DGP - C o n g r e s s 2004

Registration

Philosophy-Building 'Am Hubland'

Wednesday, March 17, 2004

Thursday, March 18, 2004

Friday, March 19, 2004

Saturday, March 20, 2004

17.00 - 20.00 h

08.00- 18.00 h

08.00- 18.00 h

08.00- 12.00 h

Opening

Lecture Hall 1

Wednesday, March 17, 2004 18.00 - 20.00 h

Welcome Address

Prof. Dr. A. Haase, President of the University of W0rzburg

Prof. Dr. M. Frosch, Organizing Committee

Prof. ~r. K. Lingelbach, President of the German Society of Parasitology

Awards Rudolphi Award to PD Dr. B. Sures

Presentation of the Leukart Medal to Prof. Dr. H. Mehlhorn

Leukart Medal Lecture

,Von Parasiten und Parasitologen - 0berlebensk0nstler unter sich'

Prof. Dr. H. Mehlhorn, DQsseldorf, Germany

8


Social Program

Wednesday, March 17, 2004

,Get Together' at the Philosophy-Building (Hubland)

20.00 h

Thursday, March 18, 2004

,Wine Tasting' at the Juliusspital W0rzburg

I

Friday, March 18, 2004

Dinner at the Fortress ,Marienberg' in WLirzburg

20.30 h

19.30 h

Please note,

pick up points and schedule for the shuttle bus transfer to the

Juliusspital and Fortress Marienberg will be announced at the

registration desk,

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in W~3rzburg

m m

~ 2

"0

C

~0 ~

n

o 0 G0

0 0 0

o o

O o

O O

! O o

o o

O O cD

o O o

O O

10

Scientific Program DGP, 21st Annual Meeting March 17 th - - 20 th, 2004 in Werzburg

o

" O

, ,c I-,-

11

Scientific Program DGP, 21st Annual Meeting March 17 t h - 20 th, 2004 in Werzburg

O

im i _

12

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in Werzburg

i "

~ " t "

' - --I

a .

n B

. m m

3 " - ~ =

C

3 " _~=

O

, , , , , , , .

"O

13


DGP-Congress 2004 in W0rzburg

Scientific Program

Thursday, 18/3/2004

08.30-10.00 Lecture Hall 3

Scientif ic Session: Defence M e c h a n i s m s & I m m u n o l o g y I

( w o r m s , m o s q u i t o e s , i n t e r m e d i a t e hos t s )

Chair: T. Jacobs

08.30 064VI

Antimicrobial peptides of the amoebapore superfamily identified in the soil nematode Caenorhabditis elegans. Roeder, T. ; Leippe, M.

08.45 065VI

Molecular characterization of an invertebrate defense protein with broad-spectrum activity. Bruhn, H. ; Winkelmann, J. ; Leippe, M.

09,00 066VI

The melanization response in Anopheles gambiae is controlled by SRPN2, Michel, K. ; Pelte, N. ; Mueller, H.M. ; Reichhart, J.M. ; Kafatos, F.C.

09.15 067VI

Regulation of superoxide dismutase and catalase in hemoctyes of schistosome intermediate host snails. Zelck, U. ; B~ssler, T. ; Janje, B.

09.30 068VI

Experimental serial passages of Sarcocystis singaporensis in intermediate hosts with different MHC class I background. Reck, C. ; J~kel, T. ; Luz, J. ; Kliemt, D. ; Mackenstedt, U.

09.45 068VIA

Molecular analysis of in vivo antigenic variation of Giardia lamblia clone GSIM-83-H7. M011er, N.; Bienz, M.; yon AIImen, N.

14

Scientific Program DGP, 21st Annual Meeting March 17 th -20 th, 2004 in Werzburg

Thursday, 18/3/2004

09.00 - 10.00 Lecture Hall 4

Scientific Session: Arthropods

Chair: H.M. M011er

09.00 014VE

Blood digestion in the human body louse Pediculus humanus and the reduviid bug Triatoma infestans. Kollien, A.; Waniek, P.; Hendgen-Cotta, U.; Stock, P.; Schaub, G.

09.15 017VE

Anticoagulatory activities of saliva and fluids from the different salivary glands and stomach of triatomines. Dimitrijevic, B.; Meiser, C.; Schaub, G.

09.30 016VE

Trypsins of the human body louse, Pediculus humanus. Waniek, P.J.; Kollien, A.H.; Pr~ls, F.; Habedank, B.; Schaub, G.A.

09.45 015VE

Characterisation of recombinant immunoreactive antigens from the scab mite Sarcoptes scabiei. von Witzendorff, C.; Matthes, H.F.; Lucius, R.; Reich, B.; Kalinna, B.

15

Scientific Program DGP, 21st Annual Meeting March 17 th -20 th, 2004 in W0rzburg

Thursday, 18/3/2004

10.30 - 12.00 Plenary Session I

Lecture Hall 1

Chair: R. Lucius

10.30 Comparing genes and genomes in freeliving and parasitic nematoda: the multiple origins of the parasitic phenotype.

Mark Blaxter, University of Edinburgh

11.15 The role for dendrit ic cells in Plasmodium falciparum malaria.

Britta Urban, University of Oxford

13.00 - 14.00 Foyer

Poster Presentation

13.00 - 14.00 Lecture Hall 3

Presentations of the ,,Piekarski Award" nominees

16

Scientific Program DGP, 21st Annual Meeting March 17 th - -20 th, 2004 in Werzburg

Thursday, 18/3/2004

14.00 - 16.00 Lecture Hall 3

Scientific Session: Genomics & Proteomics I (Leishmania, Helminths)

Chairs: I. Bruchhaus, C. Grevelding

14.00 116VG

Developmental regulation of mitochondrial targeted proteins in Leishmania donovani. Harder, S.; Bente, M.; Krause, E.; Bruchhaus, I.

14.15 117VG

Identification of stage-specific kinases in Leishmania donovani using a proteomic approach. Bente, M.; Harder, S.; Krause, E.; Bruchhaus, I.

14.30 119VG

Target identification and rational drug design based on ESTs from parasitic invertebrates. Krasky, A.; Rohwer, A.; Schroeder, J.; Seizer, P.M.

14.45 120VG

Structural and evolutionary analysis of the transcribed sequence of Boudicca, Schistosoma mansoni retrotransposon. Copeland, C.S.; Heyers, O.; Kalinna, B.H.; Bachmair, A.; Stadler, P.F.; Hofacker, I.L.; Brindley, P.J.

15.00 121VG

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts. Heyers, O.; Walduck, A.K.; Brindley, P.J.; Bleil3, W.; Lucius, R.; Dorbic, T.; Wittig, B.; Kalinna, B.H.

15.15 121VGA

Analyses of tissue-specific gene activity in transgenic schistosomes. Wippersteg, V.; Liedtke, S.; EI-Bahay, A.O.; MQnnich, M.; Philipp, C.; Ribeiro, F.; Kusel, J.R.; Rossi, A.; Klinkert, M.Q.; Grevelding, C.G.

15.30 122VG

Ten different mRNAs arising from trans- and alternative-splicing mechanisms code for the Onchocerca volvulus glutathione S- transferase 3 (Ov-GST3). H6ppner, J.; Walter, R.D.; Liebau, E.

15.45 123VG

Caenorhabditis elegans as a model for overexpression of filarial antigens. Pillai, S.; Hartmann, S.; Lucius, R.; Kalinna, B.H.

17


Thursday, 18/3/2004

14.00 - 16.00 Lecture Hall 4

Chairs:

14.00 038VE

14.15 039VE

14.30 040VE

14.45 041VE

15.00 042VE

15.15 043VE

15.30 044VE

15.45 045VE

Scientific Session: Epidemiology

U. Mackenstedt, P. Kern

Risk assessment and prevention of alveolar echinococcosis: Activities of the ongoing EU-FP5 project EchinoRisk(QLK2-CT- 2001-01995). Kern, P.; Romig, T.; The EchinoRisk Consortium

Echinococcus multilocularis and fox biology. Thoma, D.; Romig, T.; Heinel, S.; Janko, C.; Schreiber, T.; KSnig, A.; Dinkel, A.; Mackenstedt, U.

Is the neozoan raccoon dog epidemiologically relevant as a definitive host of Echinococcus multilocularis? Tackmann, K.; Goretzki, J.; Sutor, A.; Schwarz, S.; P5tzsch, C.; Conraths, F.J.

Strain characterization of cystic echinococcosis in l ivestock in Sudan. Omer, R.A.; Dinkel, A.; Romig, T.; Mackenstedt, U.; Aradaib, I.

Helminthological-serological surveys as a tool of preventive medicine - cost and benefit. Auer, H.; Asp~ck, H.

Bovine Neospora caninum infection: putative risk factors related to the farm location are not under the control of farmers. Schares, G.; B~rwald, A.; Staubach, C.; Ziller, M.; Kl~ss, D.; Schr~der, R.; Wurm, R.; Rauser, M.; Labohm, R.; Dr&ger, K.; Fasen, W.; HeE,, R.G.; Conraths, F.J.

Leishmania tropica in Jericho (A'riha) and its environs, a classical focus of Leishmania major. AI-Jawabreh, A.; Schoenian, G.

Babesia in domestic cats - an epidemiological study in South Africa. WOrth, S.; Zahler-Rinder, M.

18

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in WClrzburg

Thursday, 18/3/2004

14.00 - 16.00 Lecture Hall 5

Chairs:

14.00 133VB

14.15 134VB

14.30 135VB

14.45 136VB

15.00 137VB

15.15 138VB

15.30 139VB

15.45 140VB

Scientific Session: Cell Biology & Biochemistry I

( Plasmodium/Leishmania)

K. Becker-Brandeburg, M. Lanzer

Analysis of sequence elements responsible for trafficking of the P. falciparum stevor multi-gene family. Przyborski, J.; Miller, S.; Pfahler, J.; Brendan, C.; Lanzer, M.

Glyoxalase I of the malarial parasite Plasmodium falciparum: evidence for subunit fusion. Rahlfs, S.; Iozef, R.; Akoachere, M.; Schirmer, H.; Becker, K.

X-ray structure of glutathione S-transferase from the malarial parasite Plasmodium falciparum: a novel isoform of GST. Fritz, K.; Rahlfs, S.; Harwaldt, P.; Becker, A.; Schirmer, H.; Kabsch, W.; Becker, K.

Lipoic acid metabolism in the human malaria parasite Plasmodium falciparum. Wrenger, C.; M011er S.

Clonal variation in calcium homeostasis in Plasmodium falciparum, Rohrbach, P.; Friedrich, 0.; Lanzer, M.

The bifunctional PfAdoMetDC/ODC. the key enzymes of polyamine biosynthesis - is crucial for the survival of Plasmodium falciparum. MQIler, I.B.; Krnajski, Z.; Langer, C.; U3ersen, K.; Walter, R.D.

A multi-domain adhesion protein family expressed in Plasmodium falciparum gametocytes is essential for sporozoite midgut to salivary gland transition. Pradel, G.; Hayton, K.; Bonawitz, A.; Mejia, C.; Templeton, T.

A novel member of the GCN5-related N-acetyltransferase superfamily from Leishmania major catalyses the N6-acetylation of S-(aminoethyl)-L-cysteine. LQersen, K.; Clos, J.; Walter, R.

19

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in WQrzburg

Thursday, 18/3/2004

16.30 - 17.45 Lecture Hall 3

Scientific Session: Genomics & Proteomics II

(Helminths (contd.), Entamoeba, Plasmodium)

Chairs: E. Liebau

16.30 123VGA

Investigation of differentially transcribed genes in hypobiosis induced and not induced third-stage Dictyocaulus viviparus larvae by quantitative real-time PCR. Strube, C.; von Samson-Himmelstjerna, G.; Schnieder, T.

16.45 124VG

Novel saposin-like proteins of Entamoeba histolytica different from the amoebapores. Winkelmann, J.; Bruhn, H.; Leippe, M.

17.00 125VG

Proteomic approach towards the characterization of the parasitophorous vacuole in Plasmodium falciparum- infected erythrocytes. Nyalwidhe, J.; Baumeister, S.; Lingelbach, K.

17.15 126VG

Using cysteine protease inhibitors to target proteins involved in survival and egression of intraerythrocytic stages of Plasmodium falciparum. Gelhaus, C.; Vicik, R.; Schirmeister, T.; Leippe, M.

17.30 127VG

Generation of transgenic Plasmodium reporter parasites to study sporozoite and liver stage biology. Engelmann, S.; Matuschewski, K.

20


Thursday, 18/3/2004

16.30 - 18.00 Lecture Hall 4

Scientific Session: Vectors & Intermediate Hosts

Chairs: A. Kollien

16.30 018VE

A new lure for host-seeking anthropophilic mosquitoes and a novel type of a simple, non-CO2 mosquito trap. Geier, M.; Rose, A.; Eiras, A.E.

16.45 019VE

Discrimination of bovid species by PCR-RFLP analysis: a possible technique for the identification of blood meals from haematophagous arthropods. Steuber, S.; Ab EI-Rady, A.; Clausen, P.H.

17.00 020VE

Identification of symbiotic actinomycetes and other bacteria in the intestinal tract of Triatoma klugi, T. brasiliensis and Panstrongylus megistus. Hoffmann, T.; HQtt, S.; Rogl, A.; Schaub, G.

17.15 021VE

New tools for monitoring gravid females of the mosquitoes Aedes aegypti and Aedes albopictus (Diptera: Culicidae), vectors of Dengue and other arboviral diseases. Eiras, A.E.; Rose, A.; Geier, M.

17.30 022VE

Melanotic parasite encapsulation in the malaria mosquito Anopheles gambiae. Volz, J.; Steinb0chel, M.; Kafatos, F.C.; M011er, H.M.

17.45 023VE

Miracidial host-finding: a neglected factor in trematode transmission. Hertel, J.; Habed, B.; Kedves, K.; I_oy, C.; Haas, W.

21


Thursday, 18/3/2004

16.30 - 18.00 Lecture Hall 5

Scientific Session: Cell Biology & Biochemistry II

(Signal Transduction)

Chairs: K. Brehm

16.30 141VB

MAP kinases in defence cells of the schistosome intermediate host snail Lymnaea stagnalis. Edele, F.; Oberl~nder, M.; Zelck, U.

16.45 142VB

Gonad-preferential activity of protein tyrosine kinases in schistosomes. Grevelding, C.G.; Knobloch, J.; Kapp, K.; Philipp, C.; M0nnich, M.; Sch0131er, P.; Sroka, S.; Lammers, R.; Kunz, W.

17.00 143VB

Structural and functional characterization of TGF- signaling systems in Echinococcus multilocularis. Zavala-Gongora, R.; Kroner, A.; Wittek, B.; Knaus, P.; Brehm, K.

17.15 144VB

Characterization of a MAP kinase kinase homologue from L. mexicana. Kuhn, D.; Wiese, M.

17.30 145VB

A protein kinase involved in flagellar length control in Leishmania mexicana. Wiese, M.; Kuhn, D.; Scholz, A.; Gruenfelder, C.

17.45 146VB

Gametogenesis in Plasmodium berghei is controlled by calcium and a calcium-dependent protein kinase. Billker, O.

22


Friday, 19/3/2004

08.30 - 10.00 Lecture Hall 3

Scientific Session: Vaccines & Chemotherapy

Chairs: N. MOiler

08.30 091VC

Identification of Eimeria tenella secretory antigens for the development of a subunit vaccine against coccidiosis. Klotz, C.; Lucius, R.; Pogonka, T.

08.45 092VC

Vaccination of mice against experimental Neospora caninum infection using NcMICl-recombinant antigen and DNA-vaccination. Alaeddine, F.; Hemphill, A.

09.00 093VC

Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound. Stettler, M.; Rossignol, J.F.; Walker, M.; Gottstein, B.; Hemphill, A.

09.15 094VC

Differences in the genetics of benzimidazole resistance between sheep trichostrongyles and cyathostomins. yon Samson-Himmelstjerna, G.; Buschbaum, S.; Dr~gem011er, M.; Schnieder, T.

09.30 095VC

Identification of secreted molecules of Eimeria tenella by bioinformatic approaches. Klotz, C.; Lucius, R.; Marh0fer, R.; Seizer, P.; Pogonka, T.

09.45 096VC

Effect of antiretroviral protease inhibitors alone and in combination with paromomycin on the excystation, invasion, and in vitro-development of Cryptosporidium parvum. Hommer, V.; Eichholz, J.; Petry, F.

23

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in WOrzburg

Friday, 19/3/2004

08.30 - 10.00

Chairs:

08.30 O01VS

08.45 002VS

09.00 003VS

Lecture Hall 4

Scientific Session: Systematics & Morphology

I. Reiter-Owona; M. Zahler-Rinder

Strain variation in Leishmania majorfrom Central Asia and Middle East detected by multi locus sequence and microsatelite typing. Sch6nian, G. ; AI-Jawabreh, A. ; Schnur, L.F. ; Strelkova, M.V.

A molecular approach to assess heterogeneity of bovine Babesia in a world-wide collection of isolates. Vogl, S. ; Zahler-Rinder, M.

A molecular approach to the relationships among amoebozoan taxa. Walochnik, J. ; Michel, R. ; Asp6ck, H.

Scientific Session: Diagnostics

09.15 004VS

09.30 005VS

09.45 006VS

Species-specific real-time PCR for quantification of Toxoplasma gondii. Vorwerk, N. ; von Samson-Himmelstjerna, G. ; Tenter, A.M.

Specific detection of five species of Borrelia, the etiologic agens of Lyme disease, by means of quantitative real time PCR. Stoverock, M. ; yon Samson-Himmelstjerna, G. ; Epe, C. ; Schnieder, T.

Developing diagnostics for non-culturable parasites: the microsporidium Enterocytozoon bieneusi as a model. Wiedemann, M. ; Propping, S. ; Rinder, H.

24

Scientific Program DGP, 21st Annual Meeting March 17 th -- 2 0 th, 2004 in W0rzburg

Friday, 19/3/2004

08.30 - 10.00 Lecture Hall 5

Scientific Session: Cell Biology & Biochemistry III

(Amoeba/ Giardia)

Chairs: M. Leippe

08.30 147VB

The pathogenic amoeba Balamuthia mandrillaris possesses cell- associated phospholipase A, lysophospholipase A, and lipase activities. Shadrach, W.S.; Radam, E.; Flieger, A.; Kiderlen, A.F.

08.45 148VB

Several isoforms of pore-forming peptides of Naegleria fowled were proteolytically released from a precursor protein and display antimicrobial activity. Herbst, R.; Marciano-Cabral, F.; Leippe, M.

09.00 149VB

Identification of amoebastatin, a cysteine proteinase inhibitor in trophozoites Entamoeba histolytic. Scholze, H.; Riekenberg, S.; Witjes, B.; Key, G.; Bakker-Grunwald, T.

09.15 150VB

Identification and functional characterization of a -N- acetylhexosaminidase of Entamoeba histolytica. Riekenberg, S.; Flockenhaus, B.; Bakker-Grunwald, T.; Scholze, H.

09.30 151VB

Resistance against a cysteine proteinase inhibitor in Entamoeba histolytica correlates with secretion of unprocessed proteinases and reduced pathogenicity. Nowak, N.; Tannich, E.; Bruchhaus, I.

09.45 152VB

Characterization and subcellular localization of annexin E1 in trophozoites of Giardia lamblia. Vahrmann, A.; Szkodowska, A.; Mueller, M.C.; Bakker-Grunwald, T.; Scholze, H.

25


Friday, 19/3/2004

1 0 . 3 0 - 12.00

Chair:

10.30

Lecture Hall 1 Plenary Session II

T. Schnieder

Molecular studies on cyathostomins: bringing horse worms into the 21st century.

Jane Hodgkinson, University of Liverpool

11.15 G u i n e a w o r m eradication: the challenges of the final push.

Nevio Zagaria, World Health Organization

13.00- 14.00 Foyer

Poster Presentation

(authors should be present for questions and discussion)

2 6


Friday, 19/3/2004

14.00 - 16.00 Lecture Hall 3

Scientific Session: Chemotherapy -Antiinfectives (Wirkstoffseminar) I

Chairs: P. K6hler, P. Seizer

14.00 097vc

Effects of bisphosphonates on the growth of Entamoeba histolytica in vitro and in vivo. Bruchhaus, I.; Nozaki, T.; Meints, G.A.; Oldfield, E.

14.15 098VC

The trypanothione-dependent glyoxalase II as a drug target in african trypanosomes. Irsch, T.; Krauth-Siegel, R.L.

14.30 099VC

Irreversible inhibition of trypanothione reductase by unsaturated mannich bases. Lee, B.; Bauer, H.; Davioud-Charvet, E.; Krauth-Siegel, R.L

14.45 100VC

DNA topoisomerase inhibitors as anti-trypanosomal drugs. Steverding, D.; Deterding, A.; Dungey, F.A.; Thompson, K.A.

15.00 101VC

Targeting enzymes involved in spermidine metabolism of parasitic protozoa-a possible new strategy for antiparasitic treatment. Andrea, G.; Hamels, I.; H6rauf, A.; Kaiser, A.

15.15 102VC

Virtual screening for novel kinase inhibitors as potential drugs for the treatment of parasitic diseases. Beyer, C.; Cramer, J.; Seizer, P.M.

15.30 103VC

Interactions of methylene blue with the glutathione redox system of Plasmodium falciparum in vitro, Eubel, J.; Coulibaly, B.; Davioud-Charvet, E.; Becker, K.; Schirmer, R.H.

15.45 104VC

The threedimensional structure of a Mu-class related glutathione S-transferase from Plasmodium falciparum. Perbandt, M.; Burmeister, C.; Walter, R.D.; Betzel, C.; Liebau, E.

27


Friday, 19/3/2004

14.00 - 15.45 Lecture Hall 4

Scientific Session: Veterinary Parasitology I

Chairs: A. Tenter, G. yon Samson-Himmelstjerna

14.00 051 VV

Development of a viability assay for Cryptosporidium parvum oocysts. Najdrowski, M.; Wackwitz, C.; Joachim, A.

14.15 052W

Occurrence of Neospora caninum and Hammondia heydorni oocysts in faeces collected from naturally infected dogs in Germany. Schares, G.; B~rwald, A.; Conraths, F.J.; Barutzki, D.

14.30 053VV

Identification and characterization of Theileria lestoquardi proteins for use as diagnostic and immunization tools. Bakheit, M.A.; Seitzer, U.; Scholzen, T.; Ahmed, J.S.

14.45 054W

T cell proliferation and cytokine gene transcription in Eimeria bovis primary and reinfected calves. Taubert, A.; S0hwold, A.; Hermosilla, C.; Zahner, H.

15.00 055W

Toxoplasma gondii oocyst uptake by mussels (Mytilus galloprovincialis). Arkush, K.D.; Miller, M.A.; Leutenegger, C.M.; Conrad, P.A.; Tenter, A.M.

15.15 056W

The role of polar filament discharge in transmission of myxozoan actinospore stages. Kallert, D.; Estzerbauer, E.; EI-Matbouli, M.; Haas, W.

15.30 057W

Isolation and initial characterization of a protein from Culicoides nubeculosus relevant to summer eczema in horses. Langner, K.F.; Greiser-Wilke, I.; Schlote, S.; Heselhaus, J.; Leibold, W.

28

Scientific Program DGP, 21st Annual Meeting March 17 th -20 th, 2004 in WQrzburg

Friday, 19/3/2004

14.00 - 15.45 Lecture Hall 5

Scientific Session: Defence Mechanisms & Immunology II

(Cell Invasion, Innate Immunity, Immunomodulation)

Chairs: S. Hartmann, H. Zahner

14.00 069vi

Cell invasion and intracellular fate of the microsporidium Encephalitozoon cuniculi. Franzen, C. ; M011er, A. ; Hartmann, P. ; Salzberger, B.

14.15 070VI

Polymorphonuclear cell adhesion and adhesion molecule gene transcription in coccidia (Eimeria bovis, Neospora caninum, Toxoplasma gondil~ infected bovine endothelial cells. Hermosilla, C. ; Zahner, H. ; Taubert, A.

14.30 071vi

NK cells contribute to the control of Trypanosoma cruzi infection by killing free parasites by Perforin-independent mechanisms. Lieke, T. ; Graefe, S. ; Gaworski, I. ; Fleischer, B. ; Jacobs, T.

14.45 072vi

Involvement of natural killer T cells and CDld in Leishmania major-induced murine leishmaniasis. Mai, B. ;Moll, H.

15.00 073vi

Modulation of dendritic cells by Echinococcus multilocularis antigen. H~rter, G. ; Kern, P. ; Manfras, B.J.

15.15 074vi

Isolation and characterization of a secretory component of Echinococcus multilocularis metacestodes potentially involved in modulating the host-parasite interface. Walker, M. ; Dematteis, S. ; Baz, A. ; Gottstein, B. ; Hemphill, A.

15.30 075vi

Modulation of heterologous immune responses by a glycoprotein of Ascaris suum. Hartmann, S. ; Mueller, M. ;Adam, R. ; Lucius, R.

29


Friday, 19/3/2004

16.30 - 18.00 Lecture Hall 3

Scientific Session: Chemotherapy -Antiinfectives (Wirkstoffseminar) II

Chairs: P. K6hler, P. Seizer

16.30 105VC

The proteasome inhibitor MLN-273 inhibits cell cycle progression in Plasmodium falciparum parasites. Lindenthal, C.; Weich, N.; Klinkert, M.Q.

16.45 106VC

A chloroquine efflux pump is genetically linked with pfcrt and chloroquine resistance in Plasmodium falciparum. McLean, J.; Sanchez, C.; Stein, W.; Lanzer, M.

17.00 107VC

Functional analysis of the P. falciparum chloroquine resistance determinant pfcrt in Xenopus oocytes. Nessler, S.; Friedrich, O.; Planelles, G.; Sanchez, C.; Lanzer, M.

17.15 108VC

Carboxylic acid derivatives of menadione of Plasmodium falciparum glutathione reductase inhibitors and prodrugs. Bauer, H.; Biot, C.; Schirmer, R.H.; Davioud-Charvet, E.

17.30 109VC

Series of novel polyamine synthesis inhibitors show an antiproliferating effect on Plasmodium falciparum. Das Gupta, R.; Krause, T.; Khomutov, A.; M011er, S.; LQersen, K.; Walter, R.D.

17.45 109VCA

UIS2, a new Plasmodium candidate for multistage drug targeting. Sayed Ibrahim Aly, A.; Janse, C.; Waters, A.P.; Matuschewski, K.

30


Friday, 19/3/2004

16.30 - 17.00 Lecture Hall 4

Scient i f ic Session: Veter inary Paras i to logy II

Chairs: A. Tenter, G. yon Samson-Himmelstjerna

16.30 058W

Diagnostic potential and limitations of the PCR technique for the detection of trypanocidal failure in cattle, Gall, Y.; Clausen, P.H.

16.45 059W

Improving the management of trypanocide resistance in the cotton zone of West Africa: a coordinated regional study. Clausen, P.H.; Grace, D.; Diall, O.; Diallo, B.; Barry, M.; M0nstermann, S.; Bocoum, Z.; Diarra, B.; Sidibe, I.; Affognon, H.; Waibel, H.; Randolph, T.; McDermott, J.

31

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in W(Jrzburg

Friday, 19/3/2004

16.30 - 17.45 Lecture Hall 5

Scientific Session: Defence Mechanisms & Immunology III

(B and T Cell Immunity)

Chairs: H. Moll, K. Erb

16.30 076VI

Diverse mechanisms of Toxoplasma gondii to interfere with the IFN-A-induced MHC class II expression in murine macrophages. Lang, C. ; Algner, M. ; Gross, U. ; LQder, C.C.K.

16.45 077vi

Identification and characterisation of protective immune cells from Eimeria falciformis infected mice. Schelzke, K. ; Lucius, R. ; Kretschmer, U. ; Pogonka, T.

17.00 078VI

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Thl immune response in comparison to patients with localized cutaneous leishmaniasis. Trujillo-Vargas, C.M. ; Robledo, S.M. ; Velez, I.D. ; Patino, P.J. ; Erb, K.J.

17.15 079vi

17.30 080VI

The role of B- and T-cell-immunity in Toltrazuril-treated C57BL/6 WT, pMT and nude mice experimentally infected with Neospora caninum. Gottstein, B. ; Ammann, P. ; Mueller, N.

Protective and allergenic properties of Acanthocheilo~Tema viteae tropomyosin. Sereda, M.J. ; Lucius, R. ; Kranich, S. ; Stirati, R. ; Volkmer-Engert, R. ; Hartmann, S.

32


Saturday, 20/3/2004

08.30 - 10.30 Lecture Hall 3

Scientific Session: Cell Biology & Biochemistry IV

(Parasite-Host Cell- Interaction)

Chairs: U. Gross, M. Frosch

08.30 153VB

Identification and characterization of heme-interacting proteins in the malaria parasite, Plasmodium falciparum. Nickel, C.; Campanale, N.; Tilley, L.; Becker, K.

08.45 154VB

Two small secretory molecules affect development of Plasmodium sporozoites in hepatocytes. M011er, A.K.; Camargo, N.; Matuschewski, K.; Kappe, S.

09.00 155VB

Survival of host hepatocytes depends on the developmental stage of Plasmodium berghei parasites. Heussler, V.; Fr6hlke, U.

09.15 156VB

Identification and characterisation of Theileria annulata proteins as to their potential to interact with the proteins of transformed host cells. Schneider, I.; Hailer, D.; Seitzer, U.; Ahmed, J.

09,30 157VB

The sporozoan parasite Theileria annulata influences p53- mediated apoptotic events during infection of mononuclear cells. Hailer, D.; Schneider, I.; Ahmed, J.; Seitzer, U.

09.45 158VB

In vitro induction of Neospora caninum bradyzoites in Vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites. Vonlaufen, N.; Naguleswaran, A.; M011er, N.; Hemphill, A.

10.00 159VB

Characteristics of Leishmania major-harbouring vacuoles in murine dendritic cells. Fuss, V.; Steigerwald, J.; Moll, H.

10.15 160VB

Inhibition of caspase activity by Toxoplasma gondii in a cell-free system indicates novel mechanisms of interference with host cell apoptosis. Keller, P.; Goebel, S.; Fischer, S.F.; H~cker, G.; Gross, U.; LQder, C.C.K.

33


Saturday, 20/3/2004

08.30 - 10.30 Lecture Hall 4

Scientific Session: Ecology

Chairs: W. Haas, B. Sures

08.30 024VE

Following a parasite through its life cycle: how tough is it to cope with different immune systems? Hammerschmidt, K.; Kurtz, J.

08.45 025VE

Trematodes within host tissues: Diversity of orientation strategies. Grabe, K.; Wulff, C.; Meyer, V.; Haas, W.

09,00 026VE

Effect of the nematode Camallanus lacustris on male traits and female mate choice in three-spined sticklebacks. Skupch, K.C.; Kalbe, M.

09.15 027VE

The pathogenic amoeba Balamuthia mandrillaris is a host for intracellular multiplication of Legionella pneumophila bacteria. Shadrach, W.S.; Laube, U.; Holland, G.; Ozel, M.; Kiderlen, A.F.; Flieger, A.

09.30 028VE

The ameba Balamuthia mandrillaris infects immunodeficient mice also by cutaneous and oral routes. Kiderlen, A.F.; Laube, U.; Radam, E.; Matzk, P.; Tata, P.S.

09,45 029VE

Intestinal parasites as biological indicators for environmental pollution. Sures, B.

10.00 030VE

Parasites and automobile traffic: acanthocephalans accumulate platinum group metals (Pt, Pd, Rh) emitted from catalytic exhaust gas converters. Zimmermann, S.; Thielen, F.; Sures, B.

10.15 031VE

Eel parasite diversity: A tool to monitor aquatic ecosystems? Thielen, F.; MiJnderle, M.; Taraschewski, H.; Sures, B.

34

Scientific Program DGP, 21st Annual Meeting March 17 t h - 20 th, 2004 in W0rzburg

Saturday, 20/3/2004

1 1 . 0 0 - 12.30

Chair:

11.00

Plenary Session I I I

K. Lingelbach

Adaptation of African trypanosomes to man.

Etienne Pays, Free University of Brussels

Lecture Hall 1

11.45 Molecular and cellular analysis of ICAM-1 adhesion in Plasmodium falciparum malaria.

Alister Craig, Liverpool School of Tropical Medicine

1 2 . 3 0 - 13.30

,,Piekarski Award" Announcement

Concluding Remarks

13.30 Departure

Lecture Hall 1

3 5


Poster Sessions:

Systematics, Phylogeny, Morphology, Diagnostics

. 007PS Development and application of microsatellites for genetic analysis of Leishmania tropica. Schwenkenbecher, J.; Presber, W.; Sch~nian, G.

. 008PS Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile secretory organelle. Harris, J.R.; Adrian, M.; Perry, F.

. 009PS Phylogeny of the L. donovani complex based on analysis of microsatellite polymorphisms. Kuhls, K.; Sch~nian, G.

. 010PS Hooks, spines and crypts - specific morphological features of the acanthocephalan acanthor. Reitze, F.; Taraschewski, H.

. 011PS Detection and characterisation of anti-Cryptosporidiurn panmrn IgA- antibodies in human milk. Strathmann, K.P.; Petry, F.

. 012PS Quantitation of Microsporidia in Cultured Cells by Flow Cytometry. Franzen, C.; M011er, A.; Hartmann, P.; Salzberger, B.

. 013PS A PCR system for identification of Echinococcus species and genotypes, with reference to the epidemiological situation in eastern Africa. Dinkel, A.; Njoroge, E.M.; Zimmermann, A.; W~lz, M.; Zeyhle, E.; Elmahdi, I.E.; Mackenstedt, U.; Romig, T.

36


Arthropods, Vectors, Intermediate Hosts, Ecology

. 032PD A GIS-based study on the population dynamics of Ixodes ricinus in the Siebengebirge near Bonn. Schwarz, A.; Maier, W.A.; Kampen, H.

. 033PD Borrelia burgdorferi infection prevalences in questing Ixodes ricinus ticks in the city area and the outskirts of Bonn. Maetzel, D.; Maier, W.A.; Kampen, H.

10. 034PD Different parasite communities in parapatric host populations of three- spined sticklebacks ( Gasterosteus aculeatus). Kalbe, M.; Wegner, K.M.

11. 035PD Isolation and in vitro Cultivation of Nosema algerae from Drosophila melanogaster. Franzen, C.; Fischer, S.; Sch~lmerich, J.; Schneuwly, S.

12. 036PD Effects of trematode parasites on mortality in the common cockle Cerastoderma edule. Thieltges, D.W.

13. 037PD Parasites in native and introduced molluscs of the Wadden Sea. Krakau, M.; Thieltges, D.W.

Epidemiology

14. 046PE Prevalence of Neospora caninum infections in dairy herds in Rhineland- Palatinate. yon Blumr~der, D.; Schares, G.; Stambusch, R.; Labohm, R.; Klawonn, W.; Dr&ger, K.; Fasen, W.; Conraths, F.J.

15. 047PE A coproantigen survey of Echmococcus multilocularisin foxes in Baden- WLirttemberg. Weible, A.K.; Reule, M.; Pleydell, D.; Toumeux, F.P.; Renner, C.; Thoma, D.; Mackenstedt, U.; Deplazes, P.; Romig, T.

37

Scientific Program DGP, 21st Annual Meeting March 17 t h - - 20 th, 2004 in W0rzburg

16.

17.

18.

19.

048PE Intestinal parasites in Afghan residents employed in Camp Warehouse, Kabul. Scheid, P.L.; Thoma, B.R.

049PE Developmental aspects of Echinococcus multilocularis metacestodes in the common vole (Microtus arvalis). Merli, M; Romig, T.; Dinkel, A.; Mackenstedt, U.

050PE The function of wild nutria (Myocastor coypus) as intermediate hosts for Echinococcus multilocularis in comparison to wild muskrats (Ondatra zibethicus). Hartel, K.S.; Spittler, H.; Doering, H.; Winkelmann, J.; Hoerauf, A.; Reiter- Owona, I.

050PEA On the eradicability of filarial diseases. Duerr, H.P.; Dietz, K.; Eichner, M.

Veterinary Parasitology

20. 060PV Cyathostomin recombinant beta-tubulin expression. Blackball, W.; Pape, M.; von Samson-Himmelstjerna, G.; Schnieder, T

21. 061 PV Characterisation of susceptibility/resistance against Sarcocystis miescheriana in swine by phenotypic and genotypic variation of clinical and clinical-chemical traits in a F2-model. Hepp, S.; Hertrampf, B; Daugschies, A; Mackenstedt, U.; Zahner, H.; Reiner, G.

22. 062PV Co-infection by Onchocerca.filariae, Dermatophilus-bacteria and bovine papular stomatitis virus (BPSV) in zebu-cattle of North-Cameroon. Lay, K.; Geiger, S; Achu-Kwi, D; B0ttner, M.; Renz, A.

23. 063PV Cross-sectional survey on ectoparasite infestations in scavenging chickens in Bali, Indonesia. Damriyasa, I.M.; Ardana, I.B.; Prelezov, P.; Bauer, C.

38


Defence Mechanisms & Immunology

24. 081PI Chemokines, GM-SCF, COX-2 and iNOS gene transcription in coccidia (Eimeria bows, Neospora caninum, Toxoplasma gondil~ infected bovine endothelial cells. Taubert, A. ; Zahner, H. ; Hermosilla, C.

25. 082PI Immunomodulatory activity of antigens of the rodent filariae Acanthocheilonema viteae. Rausch, S. ; Lucius, R. ; Sonnenburg, B. ; M011er, M. ; Hartmann, S.

26. 083PI Nitric oxide production in spleen and liver of Calomys callosus infected with Trypanosoma cruzi. Dost, C.K. ; Saraiva, J. ; Zentgraf, U. ; Engels, W. ; Albuquerque, S.

27. 084PI In vitro-screening of anti- inf lammatory compounds released by parasitic worms. H0bner, M.P. ; Herrmann, K.U. ; Hoffmann, W.H. ; Soboslay, P.T. ; Schumacher, S. ; Laufer, S. ; Kalbacher, H. ; Schulz-Key, H.

28. 085PI Identif ication of immunomodulatory proteins of the gastrointestinal nematode Heligmosomoides polygyrus. Rzepecka, J. ; Lucius, R. ; Doligalska, M. ; Rausch, S. ; Hartmann, S.

29. 086PI Eimeria bovis infection reduces adhesion of polymorphous polynuclear neutrophils to infected endothelial cells by downregulat ing adhesion molecule gene transcription. Taubert, A. ; Zahnert, H. ; Hermosilla, C.

30. 087PI Chemokine and chemokine receptor dynamics in dendrit ic cells exposed to Leishmania major. Steigerwald, M. ;Moll, H.

31. 088PI Membrane-permeabilization by saposin-like proteins - variations on a common fold. Bruhn, H. ; Hecht, O. ; Gr~tzinger, J. ; Leippe, M.

39

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 t", 2004 in W0rzburg

32.

33.

089PI Molecular cloning of Acanthocheilonema viteae serpin: a potential immunomodulator in filarial infections. Hermann, A.J. ; Lucius, R. ; M011er, M. ; Sonnenburg, B. ; Hartmann, S.

090PI Immune response against primary and secondary alveolar echinococcosis in the mouse model. SchSnfelder, K. ; Merli, M. ; Traub, K. ; Mackenstedt, U.

Vaccines, Chemotherapy, Antiinfectives (Wirkstoffseminar)

34. 110PC In vitro effect mannosamine on glycosylphosphatidylinositol synthesis in Plasmodium falciparum. Santos de Macedo, C.; Schwarz, R.T.; Azzouz, N.

35. 111PC Ultrastructural studies on Wolbachia-endobacteria in Onchocerca ochengi after tetracycline treatment. Plappert, S.; Alfons, R.; Trees, S.; Mackenstedt, U.

36. 112PC In vitro efficacy of antimicrobials against Balamuthia mandrillaris, free- living ameba and opportunistic agent of encephalitis. Tata, P.S.; Radam, E.; Laube, U.; Kayser, O.; Kiderlen, A.F.

37. 113PC A screen for genes that can confer resistance to miltefosine in Leishmania. Choudhury, K.; Clos, J.

38. 114PC Trypanothione-dependent peroxidases in African trypanosomes. Schlecker, T.; Voncken, F.; Clayton, C.E.; Krauth-Siegel, R.L.

39. 115PC Identification of cysteine proteases of intraerythrocytic life stages of Plasmodium falciparum by a biotinylated small-molecule probe. Schirmeister, T.; Gelhaus, C.; Vicik, R.; Leippe, M.

40


Genomics & Proteomics

40. 128PG Is the differential gene expression in Trypanosoma cruzi strains group specific? Dost, C.K.; Saraiva, J.; Monesi, N.; Engels, W.; Albuquerque, S.

41. 129PG Cloning and characterization of a defensin-encoding cDNA of Triatoma brasiliensis. Araujo, C.A.; Waniek, P.J.; Jansen, A.M.; Kollien, A.K.; Schaub, G.A.

42. 131PG Differential gene expression in Cryptosporidium parvum infected intestinal epithelial cells. HQbner, M.; Petry, F.; Hommer, V.; Najdrowski, M.; Zelck, U.

43. 132PG Cysteine proteinases in the digestive tract of triatomines. Hendgen-Cotta, U.; Kollien, A.; Schaub, G.

Cell Biology & Biochemistry

44. 161PB The effect of Troglitazone in Trypanosoma brucei. Denninger, V.; Figarella, K.; Duszenko, M.

45. 162PB Aquaglyceroporins of Trypanosoma brucei. Uzcategui, N.; Palmada, M.; Szallies, A.; Lang, F.; Duszenko, M.

46. 163PB Establishment of knock-down approaches in adult Drosophila melanogaster. Gerber, S.; HQbner, K.; Gunkel, N.; Seizer, P.M.; Wolf, C.

47. 164PB Conformation and function of a lipid binding protein AgFABP from the parasitic nematode Ascaridia galli. Jordanova, R.; Radoslavov, G.; Fischer, P.; Liebau, E.; Walter, R.D.; Bankov, I.; Boteva, R.

48. 165PB Caenorhabditis elegans as a model system for the investigation of stress responsive proteins of parasitic nematodes. Burmeister, C.; Leiers, B.; Walter, R.D.; Liebau, E.

41

Scientific Program DGP, 21st Annual Meeting March 17 t h - 20 th, 2004 in W0rzburg

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

166PB Unraveling the role of the Leishmania 100 kD Heat Shock Protein. Klaholz, L.; Wiesgigl, M.; Clos, J.

167PB Changes in substrate preferences, glucose uptake and enzymatic activities in a glibenclamide-resistant Leishmania mexicana strain. Uzcategui, N.; Figarella, K.; Camacho, N.; Ponte-Sucre, A.

168PB Trypanosoma brucei transferrin receptor mRNA contains iron- responsive elements in the 5"-UTR. Ngazoa, E.S.; Kabiri, M.; Becker, K.; Steverding, D.

169PB Expression and transport of Toxoplasma gondii protein-complexes in Giardia duodenalis. Gaechter, V.; Deplazes, P.; Hehl, A.

170PB Biochemical characterization of LmxMPK1, a mitogen-activated protein (MAP) kinase homologue essential for survival of Leishmania mexicana amastigotes - attempts to identify protein kinase substrates. Melzer, I.M.; Schuldt, K.; Wiese, M.

171PB Glutathione synthesis in parasitic nematodes: Caenorhabditis elegans as a model system. Ajonina, C.; Sommer, A.; L0ersen, K.; Liebau, E.; Walter, R.D.

172PB Comparative examination of the expression of tissue cyst markers in Hammondia sp. isolated from dogs and foxes. Meyer, J.; Riebe, R.; Conraths, F.J.; Bohne, W.; Rohn, K.; Peters, M.; Schares, G.

173PB Structure-based mutational analyses of the glyoxalase I from Plasmodium falciparum. Haase, S.; KQhnl, J.; Walter, R.; Torda, A.; Liebau, E.

174PB Role of PUF-proteins in gene expression in Trypanosoma bruceL Luu, V.D.; Clayton, C.; Guilbride, L.

175PB Functional characterization of TLP-1, a novel member of the TRAP-family in Plasmodium. Hei6, K.; M011er, A.K.; Matuschewski, K.

42


59.

60.

61.

62.

63.

64.

176PB Characterisation of the gonad-preferentially expressed diaphanous homolog from Schistosoma mansoni. M0nnich, M.; Knobloch, J.; EI-Bahay, A.; Kapp, K.; Lammers, R.; Grevelding, C.G.

177PB Identification of binding partners of the protein tyrosine kinases TK3 and TK4 of Schistosoma mansonL Philipp, C.; Knobloch, J.; Sroka, S.; Grevelding, C.G.

178PB In vivo ratiometric pH measurements of Plasmodium falciparum. Kuhn, Y.; Rohrbach, P.; Lanzer, M.

179PB Development of an axenic in vitro cultivation system for Echinococcus multilocularis larvae. Spiliotis, M.; Tappe, D.; Sesterhenn, L.; Brehm, K.

180PB Identification and characterization of an insulin receptor homologue from Echinococcus multilocularis. Konrad, C.; Kroner, A.; Tappe, D.; Spiliotis, M.; Zavala-Gongora, R.; Brehm, K.

181PB Functional characterization of ERM (ezrin/radixin/moesin) signaling mechanisms in the Echinococcus multilocularis larval stage. Hubert, K.; Zavala-Gongora, R.; Bergmann, M.; Frosch, M.; Brehm, K.

43

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in WOrzburg

Abstracts

Systematics, Phylogeny, Morphology, Diagnostics

Strain var ia t ion in Leishmania major f rom central asia and middle east detected by mult i locus sequence and microsatel l i te typing. Sch~nian, G} t, AI-Jawabreh, A. ~, Schnur, L. F. 2, Strelkova, M. V. 3 iInstitut fear Mikrobiologie und Hygiene; Universit~tsmedizin Charit~ Berlin; Berlin; Germany 2Department of Parasitology, Hadassah Medical School; Hebrew University Jerusalem; Jerusalem; Israel; 3Martsinovsky Institute of Medical Parasitology and Tropical Medicine; Sechenov Medical Academy Moscow; Moscow; Russia

The species L.rnajor was shown to be epidemiologically quite uniform despite the widespread geographical distribution and the involvement of different rodent hosts and sand flies vectors. Gerbils (Rhombomys opimus) and fat sand rats (Psarnrnornys obesus) are the main reservoir hosts in Central Asia and Middle East, respectively. Host-parasite relationship appears to be very specific because Central Asian parasites failed to infect Middle East rodent hosts in the laboratory, and vice versa. Sequence polymorphisms suggesting genetic separation of strains of different geographical origin were observed by PCR-SSCP in following genomic targets: a) NADH-dehydrogenase gene, b) 6 phosphogluconate dehydrogenase gene, c) gp63 coding sequences, d) ribosomal internal transcribed spacer 1, and e) two different anonymous DNA sequences originally amplified with random primers. Microsatellite repeats were identified within sequences of L.major available from data bases. Primers annealing to the unique flanking regions have been designed and PCR products obtained were separated in polyacrylamide gels and screened for length polymorphism. All strains from Central Asia had identical genotypes whereas considerable heterogeneity and further geographical subdivision was observed in strains isolated in Middle East. Molecular markers and host-parasite relationships confirmed that strains of L.major from Central Asia and Middle East represent genetically and biologically distinct sub-populations.

A molecular approach to assess heterogenei ty of bovine Babesia in a wor ld -wide collection of isolates Vogl, S} t, Zahler-Rinder, M. ~ ~Institut for Vergleichende Tropenmedizin und Parasitologie; Ludwig-Maxirnilians-Universit~t MfJnchen; MEmchen; Germany

Babesia of cattle are known to be very variable in immunogeneity, pathogenicity and vector specifity. Within this diverse group of parasites there are limited morphological and biological characters that can be used to recognize intraspecific relationships or even to distinguish species. Therefore a new molecular approach demonstrating heterogeneity of bovine Babesia was introduced: In this study the ITS region was characterized and analysed for the first time for bovine Babesia. 50 blood samples containing Babesia of different geographic origin, deriving from naturally infected animals or vaccine strains were examined. All samples belong to one of the four species of veterinary importance, B. bovis, B. bigernina, B. divergens and B. ovata. The ITS region of the ribosomal DNA consisting of the first and the second internal transcribed spacers and the coding 5,8S gene was amplified using polymerase chain reaction (PCR), cloned and sequenced. Phylogenetic relationships between the pathogens were inferred by three independent methods of phylogenetic tree construction using the algorithms Distance Matrix, Maximum Parsimony and Maximum Likelihood. To detect possible intraisolate variation several clones for each sample - altogether 95 clones - were analysed. The phylogenetic trees revealed quite good agreement with the current taxonomy of bovine Babesia. B. divergens appeared very homogenous, but within B. bovis and B. bigemina intraindividual and intraspecific variations were observed. This heterogeneity within the two species was confirmed by low identity scores for B. bigemina with 92,7 % and B. bows with 82,7 %. Phylogenetic relations were correlated with the geographical origin of the isolates. The Southern African isolates had a closer relationship to Australia than to isolates of North Africa. In contrast, the homogeneity of the B. divergens clones was indicated by intraindividual and intraspecific identities of at least 99,1%. Possible reasons for high intraspecific variation in B. bovis and B. bigernina, but not in B. divergens are discussed.

A molecular approach to the re lat ionships among amoebozoan taxa Walochnik, j.1 ,, Michel, R. 2, Asp6ck, H. ~ 1Klinisches Institut fElr Hygiene und Medizinische Mikrobiologie, Abteilung for Medizinische Parasitologie; Universit~t Wien; Wien; Austria 2Zentrales Institut BW-Koblenz, Abteilung f[Jr Mikrobiologie; BW-Koblenz; Koblenz; Germany

Several representatives of the amoebozoa, including Entamoeba histolytica, Acantharnoeba spp., Balamuthia mandrillaris and Sappinia diploidea are involved into human disease, however, the intra-amoebozoan relationships are still not completely resolved. The amoebozoa consisting of organisms with scarce

44

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in Werzburg

morphological characters and basically no fossil record are difficult to classify. Moreover, the asexual mode of reproduction in most of the amoebozoan taxa questions the applicability of the binary nomenclature. During the past years electron optical and molecular biological data have helped to solve many problems in protozoan phylogeny and to unravel previously unidentified relationships, but up to now only of few taxa sufficient molecular data are available.

The aim of the current study was to contribute to the elucidation of intra-amoebozoan phylogeny. Therefore, the 18S rRNA gene of 16 new amoebozoan isolates belonging to various different taxa was sequenced and a cluster analysis was performed including other amoebozoans of which sequence data are available.

I t was shown, that while our analysis was in concordance with the gross amoebozoan groups, several amoebozoan genera, including Acanthamoeba, Vannella, Mastigamoeba, Rhizamoeba, Hyperamoeba and Physarum do not appear as monophyla in the present state. Altogether, this study indicates, that some amoebozoan genera might need to be reviewed and once again points up the difficulties in intra-amoebozoan phylogeny and classification.

Species-specific rea l - t ime PCR for quantif ication of Toxoplasma gondii Vorwerk, N. l, von Samson-Himmelstjerna, G. ~, Tenter, A. M} t ~Institut fDr Parasitologie; 7-ier~rztliche Hochschule Hannover; Hannover; Germany

Toxoplasma gondii is a versatile protozoon that may infect all warm-blooded animals, i.e. mammals and birds. Transmission from animals to humans may involve the ingestion of three infectious stages: sporulated oocysts from the environment, tissue cysts contained in meat or offal of chronically infected hosts, and tachyzoites contained in milk or offal of recently infected hosts. However, because most studies on infectious stages of T. gondii in meat-producing animals, in products of animal origin, or in environmental samples report only qualitative data, it is difficult to assess the true risk of infection for humans in individual cases. Hence, there is a need for a method that enables the quantitative detection of the parasite in epidemiological studies and risk analyses. Here we developed a real-time PCR based on the detection of the 7-. gondii B1 gene. Sensitivity assessment of this method with 3 virulent strains (BK, ENT, RH), 7 non-virulent strains (Beverley, C, DX, Fukaya, GTP, ME49, TPR), and the vaccine strain ($48) of T. gondii showed that quantification is possible between 101 and 104 haploid stages of T. gondii per test sample, with a detection limit of 1-10 haploid stages per test sample. No cross-reactions were observed with genomic DNA templates derived from parasites that are phylogenetically related to T. gondii and may also be present in food or environmental samples (Hammondia hammondi, Neospora caninum, Isospora spp., Sarcocystis spp., Eimeria spp., Cryptosporidium spp.). This method is currently being evaluated with respect to quantification of T. gondii in environmental samples.

Specific detection of five species of Borrelia, the etiologic agens of Lyme disease, by means of quant i tat ive real t ime PCR Stoverock, M. l, yon Samson-Himmelstjerna, G. 1 ~, Epe, C. l, Schnieder, T. 1 lInstitut for Parasitologie; Tier~rztliche Hochschule Hannover; Hannover; Germany

Lyme disease is the most prevalent tick-borne disease in Europe and North America. Its etiologic agents, spirochetes of the genus Borrelia, can be detected either by dark field microscopy or by serology. However, both methods lack satisfactory degrees of sensitivity and specificity. PCR provides a powerful tool for the directto detection of Borrelia spp. Furthermore, quantitative real time PCR allows a quantification of the parasite burden. In Europe there are five species of Borrelia: B. burgdorferi sensu stricto., B. afzelii, B. garinii, (together B, burgdorferi sensu latu) B, valaisiana and B. lusitaniae. The pathogenicity of B. lusitaniae is not yet determined and controversely discussed. Species-specific detection of Borreliae was carried out by amplification of fragments of the RNA polymerase [3. Specific bands were detected using pure borrelial DNA as well as DNA isolated from naturally infected ixodid ticks. The detection limit was as low as 1-2 single spirochetes per tick. In addition we designed primers to amplify the internal transcribed spacer (ITS) ribosomal DNA region of all five Borrelia species in a conventional PCR. This allows the fast, secure detection of all borreliae in one single PCR. Due to a positive correlation between borrelial burden and manifestation of clinical symptoms the quantification of the borreliae can be relevant for medical treatment. Therefore, in addition to the conventional PCR we established a quantitative real time PCR using fluorescent labelled minor groove binder probes specific to the ITS region. One probe hybridized with B. burgdorferi s.I. and B. valaisiana, the other solely with B. lusitanie. The aim was a separate detection of pathogenic and potencially apathogenic species. We could detect borrelial DNA to a minimum of ten copies. The application of the method in questing field ticks is planned for the future. For all both DNA isolated from cultured Borreliae as well as DNA from infected ticks were suitable. No cross- reaction with other spirochetes could be observed in any of our PCRs.

45


Developing diagnostics for non-culturable parasites: the microsporidium Enterocytozoon bieneusi as a model Wiedemann, M. 1 t Propping, S. 1, Rinder, H} 1Department of Tropical Medicine and Infectious Diseases; University of Munich; MCJnchen; Germany

The Enzyme Immunoassay (EIA) is widely recognized as a simple, rapid, sensitive, cheap and high capacity method to detect infections; either by directly demonstrating antigens or the antibodies produced by the host. In both cases, large quantities of pure antigen are needed, either for the detection of the immune response of the host or for immunization and the subsequent production of specific mono- or polyclonal antibodies to be used in a Sandwich ELISA. As a consequence, the development of these tools is greatly impeded if the infectious agent cannot be cultured and antigen cannot be produced in sufficient amounts and purity. An example is the most frequent microsporidium in human infections, Enterocytozoon bieneusi, a non-culturable protozoon. We describe here a generally applicable approach by which, first, an expression library of another, if necessary distantly related, but culturable organism, in this example Encephalitozoon cuniculi, is screened with polyclonal antibodies produced against that species. Immunoreactive clones are then characterized to facilitate the construction of degenerate PCR primers for the amplification of homologous genes from closely related organisms, in this case E. intestinalis and E. hellem. In the next step, alignment of these sequences identifies evolutionary conserved regions against which

Development and application of microsatellites for genetic analysis of Leishmania tropica Schwenkenbecher, J.1 t, Presber, W. ~, Sch6nian, G. 1 l Inst i tut for Mikrobiologie und Hygiene, Abt. Parasitologie; Humboldt-Universit~t/Charite; Berlin; Germany

Microsatellite markers were designed for Leishmania tropica for studies of epidemiology and population genetics. Markers originally designed for L. major amplified from DNA of L. tropica only at a small percentage. Consequently, we constructed and screened a genomic library, isolated microsatellite loci and tested them for their ability to differentiate between strains of L. tropica. 16 polymorphic markers were developed, 10 of which consisted of dinucleotide repeats. A BLAST search on sequences of closely related L. major revealed their location on different chromosomes, supporting their usefulness for genetic studies. The markers were applied to analyze 104 different strains, showing a high degree of genetic variation, which neither correlates with geographical distribution nor with disease pattern. Genetic distances among and between subpopulations were estimated using Arlequin, Genetix and Microsat programs. The alignment of cloned L. tropica sequences with genebank information for L. major revealed a similarity between 90 and 98%. Microsatellite flanking regions, which are described as very conserved in literature, show differences in Leishmania. To investigate this further, markers were tested for their ability to differentiate between strains of L. major, L. infantum and L. donovani. Only 30% of these markers were informative for Leishmania species others then L. tropica. Thus, specific microsatellite markers have to be designed and tested for each single species of Leishmania.

Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile secretory organelle Harris, J. R. 1, Adrian, M. 2, Perry, F. 3 ~ IInstitute of Zoology; Johannes Gutenberg-University; Mainz; Germany 2Department of Biology; University of Lausanne; Lausanne-Dorigny; Switzerland 3Institute of Medical Microbiology & Hygiene; Johannes Gutenberg-University; Mainz; Germany

Invasive stages of Apicomplexan parasites possess secretory organelles, namely rhoptries, dense granules and micronemes, which are involved in host cell invasion. We have performed a detailed structural analysis of micronemes from Cryptosporidium parvum sporozoites. From an EM study of thin sections, the rod-like micronemes have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro. This correlates with the shape change of intact sporozoites, from viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast microscopy and thin section EM. From negatively stained EM specimens of sporozoites the cellular shape change has been confirmed as has the rod to sphere micronemal shape change. Intact micronemes released directly from sporozoites exclude negative stain and appear as smooth-surfaced electron transparent particles. Biochemically purified rod-shaped C. parvum micronemes are shown to be fragile organelles that inevitably undergo variable damage during preparation for EM study. In the absence of glutaraldehyde fixation, damaged micronemes allow the negative stain to enter and loose their contents and during storage undergo the shape transformation from rod to spherical. Fixed micronemes maintain the rod shape; intact fixed micronemes still exclude negative stain but damaged micronemes reveal a helical arrangement of internal protein within the rod-like micronemes. Loss of this internal organised structure appears to be responsible for the micronemal shape change. Once released from the oocyst C. parvum sporozoites have a limited life-span and need to find a host cell quickly for successful invasion. Our analysis shows that the parasite undergoes structural changes in the overall shape of the cell which is paralleled by the change and disintegration of the microneme organelle indicating a relationship of structure and function of the invasive parasite stage.

46


Phylogeny of the L. donovani complex based on analysis of microsatellite polymorphisms Kuhls, K} t, Sch6nian, G. 1 ~Institut fElr Mikrobiologie und Hygiene; Charlte - Universit~tsmedizin Berlin; Berlin; Germany

Species of the Leishmania donovani complex (L. infantum/L, chagasi, L. donovani, L. archibaldi) are the causative agents of visceral (VL) leishmaniasis. Differentiation between species and strains is based mostly on zymodeme characterization. Phylogenetic relationships between the species are still not clear. We have analized 23 strains of L. infantum, 3 L. chagasi, 22 L. donovani and 6 L. archibaldi of different zymodemes and geographic origin in order to understand genetic heterogeneity at the species and strain level and to elucidade phylogenetic relationships between different geographic groups of strains. Independent markers containing polymorphic microsatellites (ribosomal internal transcribed spacer ITS1/ITS2, Lm2, Lm4, the a-tubulin intergenic region, specific microsatellite markers) were studied applying sequence analysis, SSCP and microsatellite PAGE analysis. Microsatellite data were combined and used for phylogenetic inference. Four distinct phylogenetic groups of strains could be differentiated based on the combined data set: (1) L. donovani (Kenya, India), (2) L. donovani (Sudan, Ethiopia) + L. infantum (Sudan) + L. archibaldi (Sudan, Ethiopia), (3) L. donovani (Sudan, Ethiopia, China) + L. archibaldi (Sudan), (4) L. infantum (Mediterranean basin, China) + L. chagasi (Brazil, Panama). These groups are not consistent with previous species definitions based on isoenzyme analyses. L. infantum is polyphyletic and L. archibaldi is not supported as a distinct species. Synonymy of L. infantum and L. chagasi has been confirmed. The Indian and Kenyan strains of L. donovani are distinct from strains from Sudan and Ethiopia and imply a possible Kenyan ancestor of the Indian strains. Interestingly, two groups of Indian strains could be differentiated, one of which is more closely related to the L. donovani strains from Kenya. Eight of the microsatellite based markers discriminate MON-1 strains and can thus be used for epidemiological studies.

Hooks, spines and crypts - specific morphological features of the acanthocephalan acanthor Reitze, F. ~ t, Taraschewski, H. 1 iZoologie I, Okologie - Parasitologie; Universit~t Karlsruhe; 76131 Karlsruhe; Germany

Thus far mainly acanthors still enclosed by four (or five) eggshells were studied by transmission electron microscopy. We investigated acanthors of the archiacanthocephalans Moniliformis moniliformis and Macracanthorhynchus hirudinaceus experimentally liberated from their enclosures and of the eoacanthocephalan Paratenuisentis ambiguus hatched inside the intestine of the intermediate host. The anterior part of the acanthocephalan larvae of all three species is armed with circular rows of hollow hooks which vary in dimension and shape. These huge anterior hooks seem to play the major role during the process of penetration through the intermediate host's intestinal wall. The larva assists this mechanical process by lengthening and shortening its complete posterior body. Further posterior the body of the larva is covered with numerous smaller body spines. In contrast to the hooks these body spines do not destroy the intestinal wall of the intermediate host but apparently prevent the larvae from slipping back into the intestinal lumen. In acanthors of all examined species the action of the hooks and spines is assisted by bundles of crypts of the outer membrane. The crypts are fused and form lacunas which are filled with small vesicles. From the anterior to the posterior end of the acanthor the number of crypts decreases clearly. Therefore it appears that these crypts do not function in the uptake of nutrients like in adult acanthocephalans but discharge enzymes contained in the vesicles. The hooks, spines and crypts on the surface of the acanthors are arranged in a species-specific pattern.

Detection and characterisation of anti-Cryptosporidium parvum IgA-antibodies in human milk Strathmann, K. p.1, Petry, F. 1 t 1Institute of Medical Microbiology & Hygiene; Johannes Gutenberg-University; Mainz; Germany

Cryptosporidium parvum is a coccidian parasite that has gained much attention in the last 20 years as a clinically important human pathogen. The organism infects the gastrointestinal epithelium to produce a diarrhoea that is self-limiting in immunocompetent persons but potentially life-threatening in immunocompromised individuals. Infection regularly leads to specific serum antibody response of the IgM, IgG, and IgA classes and serological data has been widely used in the epidemiology of cryptosporidiosis. The aim of this study was to detect and characterise C. parvum-specific IgA antibodies in human milk as a marker for exposure/infection. For the first time different test systems have been established for the detection of anti-C. parvum IgA including the use of the recombinant antigens Cp17 and P23. 386 human milk samples were tested in an enzyme-linked immunosorbent assay (ELISA) employing native C. pavum oocyst lysates as antigen. 32.6% of all samples investigated had a high reactivity (OD > 66% of the positive control). ELISA results were confirmed in an immunofluorescent test (IFT) showing reactivity against corpuscular structures of oocysts and sporozoites. To demonstrate specific reactivity against C. parvum antigens, western blots with oocyst antigen and sporozoite antigen have been performed. 43.6% of all samples with a high reactivity in the ELISA showed reactivity with immunologic relevant C. parvum antigens of < 19 kD and 64.1% with antigens of 20-29 kD. We

47

Scientific Program DGP, 21st Annual Meeting March 17 th - -20 th, 2004 in W0rzburg

developed a second ELISA system with recombinant Cp17 and P23 antigens and confirmed specific reactivity against Cp17 and P23 in a western blot. With these assays we showed specific reactivity of IgA antibodies in human milk against defined recombinant C. parvum antigens which might be superior to the use of total parasite lysate antigen.

Quantitation of Microsporidia in Cultured Cells by Flow Cytometry Franzen, C} t, M~ller, A. 2, Hartmann, P.~, Salzberger, B. ~ 1Klinik und Poliklinik fElr Innere Medizin I; Universit~t Regensburg; Regensburg; Germany 2Klinik for P~diatrie; Universit~t Bonn; Bonn; Germany

Background: Microsporidia are obligate intracellular protozoan parasites that emerged as major opportunistic pathogens in humans since the onset of the AIDS pandemic. In the present study, we investigated whether flow cytometry is a useful method for the quantitation of intracellular microsporidian spores in cultured cells. Methods: Microsporidia (Encephalitozoon cuniculi) were grown in cell cultures and various cell-lines were coincubated with microsporidian spores at different multiplicities of infection as well as for different periods of time. After permeabilization of the cells intracellular spores were stained with a polyclonal anti-E, cuniculi serum and a FITC-labeled secondary antibody. Stained cells were analyzed on a FACScan apparatus and results were compared with those of fluorescence microscopy. Results: Noninfected cells showed a lower fluorescence, while the relative fluorescence observed for infected cells was significantely higher. The cell population with the more intense fluorescence, representing cells with internalised microsporidian spores, increased with the multiplicity of infection as well as over time. Results of flow cytometry and fluorescence microscopy were in excellent agreement for all experiments. Conclusions: We have developed a flow cytometric assay to detect and quantify cells with intracellular microsporidian spores. This method is easy to use, highly reproducible, and should be useful for future research.

A PCR system for identification of Echinococcus species and genotypes, with reference to the epidemiological situation in eastern Africa Dinkel, A. ~ t, Njoroge, E. M. 2, Zimmermann, A. 1, W~Iz, M. 1, Zeyhle, E. 2, Elmahdi, L E?, Mackenstedt, U. 1, Romig, T. 1 1Department of Parasitology; University of Hohenheim; Stuttgart; Germany 2Clinical Department; African Medical and Research Foundation; Nairobi; Kenya 3Molecular Biology and Oncology; University of Gezira; Gezira; Sudan

We present a specific and sensitive PCR / semi nested PCR system for rapid diagnosis of E. granulosus genotype G1, E. granulosus genotype G6/7, and E. ortleppi (G5). This system includes a simple PCR specific for G1 as well as a PCR for diagnosis of G5/6/7. For subsequent discrimination between E. ortleppi and G6/7 we developed two semi nested PCRs. Target sequence for amplification is part of the mitochondrial 12S rRNA gene. Specificity of the PCRs was 100% when evaluated with isolates of 16 cestode species including E. multilocularis, E. equinus, E. ortleppi and 3 strains of E. granulosus (G1, G6 and G7). Sensitivity threshold was 0.25 pg DNA. This new approach was compared to published protocols of RFLP-PCR and sequencing of mitochondrial CO1 and ND1 genes using Echinococcus isolates of human, sheep, goat, camel, cattle and pig origin from Kenya and Sudan. Additionally, two internal DNA probes Were developed, one hybridizing only with G1, the other with G5, G6 and G7 amplification products. The system could also be used for strain specific diagnosis of Echinococcus spp. eggs contained in fecal samples of final hosts. The sensitivity of a G1 specific coprodiagnostic PCR was sufficient to give a positive result with a single egg. Preliminary epidemiological results obtained with this approach include the detection of a camel strain (G6) infection for the first time in a human patient from eastern Africa, and the first reports of E. ortleppi (G5) in livestock from Kenya and the Sudan.

48


Arthropods, Vectors, Intermediate Hosts, Ecology

Blood digestion in the human body louse Pediculus humanus and the reduviid bug Tr iatoma infestans Kollien, A. 1 t, Waniek, P.~, Hendgen-Cotta, U. ~, Stock, P.~, Schaub, G. ~ ~Department of Special Zoology; Ruhr-University; Bochum; Germany

The physiology of blood digestion in lice and triatomines is completely different. Lice spend their entire life cycle on the host, require frequent small blood meals and have a rapid digestion. Reduviid bugs, however, feed for only a short period, and ingest an enormous amount of blood which is digested very slowly. Like the majority of haematophagous insects, lice utilise an alkaline protease-based digestion and so far a leucine aminopeptidase and trypsin have been described. Bugs have an acidic midgut lumen and digest blood using cysteine and/or aspartic proteinases and then aminopeptidases and carboxypeptidases [summarized in 1]. From a cDNA library an aminopeptidase and a trypsin gene of Pediculus humanus have been cloned and sequenced. The glutamyl-

aminopeptidase gene has an open reading frame of 2,760 bp and is constitutively expressed after feeding. In situ hybridisations on whole guts show that the aminopeptidase mRNA is expressed in the anterior part of the midgut. The trypsinogen gene has an open reading frame of 759 bp and is constitutively expressed in adults 2- 24 h after the bloodmeal. Trypsin is activated by cleavage of chymotrypsin, a so far undescribed phenomenon in trypsin activation [1]. Cathepsin B- and cathepsin L-like activities in gut extracts of the bug Triatoma infestans are high at 5 and 10 days after feeding. Two cathepsin B-like cysteine proteinase genes and one cathepsin L-like proteinase gene have been cloned and sequenced, all containing the GCNGG motif and the three amino acid residues CHN of the catalytic domain. The ERFNIN motif is present only in the cathepsin L-like sequence. One of the cathepsin B-like genes is expressed at low, constitutive levels in unfed and fed T. infestans. In situ hybridizations on the gut show that cathepsin B mRNA is present in a distinct region of the anterior midgut and patchily distributed in the posterior midgut.

1 Kollien et al. (2003) Insect Mol. Biol. 12: in press

Anticoagulatory activities of saliva and fluids from the different salivary glands and stomach of triatomines Dimitrijevic, B. ~ t, Meiser, C. 1, Schaub, G. 1 ~Department of Special Zoology; Ruhr-University; Bochum; Germany

The salivary glands of blood-sucking triatomines contain many compounds, e.g. anticoagulants, platelet anti- aggregation factors and vasodilators. Anticoagulants are also secreted into the stomach to prevent clotting of the ingested blood. We have studied the anticoagulatory activities of saliva and contents/homogenates from the two or three pairs of salivary glands and the stomach of Dipetalogaster maxima, Rhodnius prolixus, Triatoma infestans, T. vitticeps, T. klugi, 7-. dimidiata and Panstrongylus megistus by measuring the effects on the coagulation time of human plasma. In all species, salivary gland contents/homogenates strongly prolonged the activated partial thromboplastin time (AP-I-I-) and also weakly that of the prothrombin time (PT). A strong inhibitory effect on the exogenous pathway of blood coagulation was evident for saliva of T. dimidiata, T. klugi and T. vitticeps, the contents of salivary gland 2 of D. maxima and the stomach contents of all species. The endogenous coagulation cascade was especially inhibited by saliva of T. dimidiata, the contents of salivary gland 2 of D. maxima, as well as the stomach fluids of all seven species. The salivary gland contents/homogenates of 7-. klugi and 7-. dimidiata and stomach contents of all species also strongly prolonged the coagulation time after addition of thrombin.

Trypsins of the human body louse, Pediculus h u m a n u s Waniek, P. j.1, Kollien, A. H. ~ t, Pr01s, F. 2, Habedank, B?, Schaub, G. A. 1 1Department of Special Zoology; Ruhr University; Bochum; Germany 2Institute of Anatomy II; Albert-Ludwig University; Freiburg; Germany 3Department of Parasitology and Tropical Veterinary Medicine; Free University; Berlin; Germany

Human sucking lice are a worldwide hygiene problem which has increased, in the last decade. The appearance of resistance against the classic insecticides has stimulated a search for new methods of louse control. Immunization of rabbits using faeces and midgut homogenates of lice increased larval mortality rates and reduced adult fertility. Since digestive enzymes could be a possible target in these immunizations, we screened a cDNA library of the whole insect with a trypsin-encoding cDNA fragment, and cloned and sequenced one trypsin-encoding cDNA (TRY1) of Pediculus humanus. The 1.0 kb genomic trypsin gene contained three introns of 102, 79 and 80 nucleotides. In situ-hybridisations with RNA probes on whole guts at different times after feeding, show regions of trypsin encoding gene expression in all of the midgut. In Northern blots, unfed first instar larvae have an identical or slightly lower level of trypsin mRNA than fed adult lice, and in adults 2 - 24h after the bloodmeal this gene shows a constitutive expression [1]. After in vitro transcription and translation, the activation peptide is cleaved by chymotrypsin, an previously undescribed phenomenon in trypsin activation.

49


Searching for this chymotrypsin gene and using degenerate oligonucleotide primers, a second trypsin-like serine proteinases encoding gene-fragment (TRY2) was found. The 5'- and 3'-end of the gene were identified by RACE PCR, and full mRNA sequence by RT-PCR. The deduced amino acid sequences of the two trypsins of P. humanus have an overall identity of about 36%. TRY1 shows 35-43% and TRY2 45-55% identity with the trypsins of several haematophagous Diptera.

1 Kollien et al. (2003) Insect Mol. Biol. 12: in press

Characterisation of Recombinant Immunoreactive Antigens from the scab mite Sarcoptes scabiei yon Witzendorff, C. 1 t, Matthes, H.-F. 1, Lucius, R. 1, Reich, B. ~, Kalinna, B. ~ ~Department of Molecular Parasitology, Institute for Biology; Humboldt-University; Berlin; Germany

Infestation with Sarcoptes scabiei causes an acute skin inflammation and affects humans and also causes losses in the livestock industry worldwide. The infection can be treated effectively with topically applied insecticides, however it can be difficult to diagnose. In general, the number of parasites on infested hosts is low, making direct detection of a sarcoptid mite quite difficult. Serological tests for the presence of circulating antibodies in the host is a sensitive and practicable method, and commercial ELfSA-based tests using whole- mite antigen extracts have been established for the definitive diagnosis of scabies infestations. In this context, a serological test using recombinant antigens of S. scabiei would be of advantage, as the breeding of the mites as a source of antigen on the host could be avoided. We constructed a cDNA phage library using the mRNA of S. scabiei var. suis. This library was screened with the serum of 10 S. scabiei infested humans. In this way we isolated 61 immunoreactive clones, 4 1 % of which have a common pattern of highly repetitive GA-nucleotide sequences, indicating that the genes are members of one family. These clones showed no reaction to the sera of human house dust mite allergics or healthy control persons in a Western Blot, suggesting their suitability as diagnostic antigens. The deduced proteins bear a central domain of degenerate repeats, flanked by non-repetitive 5" and 3" sequences. The role of the proteins coded by these clones is unknown. BALB/c mice were immunized with 3 synthetic peptides containing deduced sequences from these clones and these sera will be used in immunohistochemistry studies to localize the proteins within the mite. Additional novel genes identified in the screen are currently being cloned and expressed to test their suitablitly for serological diagnosis of scab mite infestation.

A new lure for host-seeking anthropophilic mosquitoes and a novel type of a simple, non-C02 mosquito trap. Geier, M. 1 t, Rose, A} , Eiras, A. E. 2

IBioGents GmbH; Regensburg; Germany 2Departamento de Parasitologia / Instituto de Ciencias Biologicas; Universidade Federal de Minas Gerais; Belo Horizonte; Brazil

Carbon dioxide is an unspecific attractant for blood-feeding arthropods and is widely used in conventional traps for host-seeking mosquitoes. These traps are used as monitoring tools, or to catch nuisance mosquitoes around human habitations. The CO2 is either liberated from dry ice, given off from pressurized gas bottles, or produced by burning propane or other explosive gases. We introduce a new attractant for a range of host-seeking mosquitoes that works without the unspecific and expensive CO2. The lure consist of a blend of volatiles that also emanate from human skin. The substances are set free from specially designed dispensers and are essentially non-toxic, cheap, easy to transport, and non- flammable. The attractiveness of the mixture strongly depends on the mixing ratio of the compounds. Release rates and blend ratio can be optimised to suit a number of anthropophilic mosquitoes. In dual-choice olfactometer studies, optimum mixtures have been developed for Aedes aegypti, Ae. albopictus (both vectors of yellow fever and dengue), Culex pipiens quinquefasciatus (vector of filariasis and ceGain arboviruses), and Anopheles stephensi (malaria). Although CO2 is not essentially necessary for the effectiveness of the new attractant, the blend can also be used to enhance the catching rate of carbon dioxide traps. Wind tunnel studies of the orientation mechanisms of flying mosquitoes in odour plumes led to the development of a novel mosquito trap. The design of the trap combines the generation of a specific plume shape for an optimum effectiveness of the attractants with visual cues to enhance the catch of the attracted mosquitoes. The trap is optimised for the new attractive blend, easy to produce and simple to use. I t can be applied indoors as well as outdoors and has been successfully tested in the laboratory and in the field in Brazil, Asia and Europe with a range of mosquito species.

50


Discrimination of bovid species by PCR-RFLP analysis: a possible technique for the identification of blood meals from haematophagous arthropods Steuber, S} t, Ab EI-Rady, A. 2, Clausen, P.-H. 2 ~BVL; Federal Office for Consumer Protection and Food Safety; 12277 Berlin; Germany 2Institute for Parasitology and International Animal Health; Freie UniversitBt; Berlin; Germany

Identification of the bloodmeal hosts of haematophagous arthropods is difficult because currently applied serological techniques are often hampered by advanced digestion of the bloodmeal and some cross reactivity between members of phylogenetically closely related species. Thus, bloodmeal surveys e.g. in tsetse flies have sometimes failed to identify the precise species taxon (e.g. Suidae: domestic pigs, bush-pigs, or warthogs) and samples could only be assigned to the family group (Bovidae, Suidae etc.) Recently, a polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP) has been developed to identify closely related animal species in meat products or animal feedstuffs. The aim of our study was to adapt this molecular biological technique to the identification of blood meal samples obtained from tsetse. The primers used were deduced from cattle mitochondrion DNA ( Bos taurus NCBI Acc. NC 00_1567) and are by and large complementary to the conserved region of the cytochrome b gene ( cyt b ) of vertebrates leading to a consistent but variable 359 bp-PCR product in all bovid species tested (n=10). The selection of appropriate restriction endonucleases sites was based on the comparison of mt DNA sequence data of bovids drawn from the search tool of the National Centre for Biotechnology Information (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide). Sites for all restriction enzymes that cut the amplified 359 bp sequence were identified by means of the free available programme NEB cutter V1.0 (http://tools.neb.com/NEBcutter/index.php3) designed by New England Biolab Incorporation (NEB Inc.). A theoretical flowchart of PCR-RFLP to allow unequivocal mapping and practical examples of endonuclease digest with selected restriction enzymes will be presented to demonstrate the accurate identification of important mammalian bovid species.

Identification of symbiotic actinomycetes and other bacteria in the intestinal tract of Triatoma klugi, T. brasiliensis and Panstrongylus megistus Hoffmann, T. 1, H0tt, S. 1 t Rogl, A. 1, Schaub, G} IDepartment of Special Zoology; Ruhr-University; Bochum; Germany

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted by blood-sucking bugs. Triatoma brasiliensis is one of the most important vectors in north-east Brazil and like Panstrongylus megistus it often colonizes houses, while 7-, klugi is a sylvatic species. Besides 7-, cruzi, different bacteria are present in the intestinal tract, but only actinomycete species seem to act as symbionts. So far, each triatomine species has its own specific symbiont In order to identify symbiotic bacteria of T. brasiliensis, P. megistus and 7-. klugi originating from field populations, the different regions of the intestinal tract were dissected under sterile conditions, homogenized and inoculated onto agar plates. The bacteria were characterized macro- and microscopically, then assigned to taxonomic groups by api-test-systems. Those bacteria which seemed to be actinomycetes were further classified according to their mycolic acids and the sequences of the 16S ribosomal DNA. In T. brasiliensis 18 different bacteria were isolated and four of them were actinomycetes: Corynebacterium sp., two different Gordonia terrae-isolates, and Gordonia amicalis. In P. megistus 16 different bacteria were separated, three of which were actinomycetes: different Brevibacterium spp. that did not possess mycolic acids in their cell membranes. In T. klugi, 21 different bacteria could be isolated, the only actinomycete present being Dietzia sp.. After feeding a mixture of blood and different isolates to aposymbiotic larvae of 7-. klugi and 7-. brasiliensis, Dietzia sp. did not show any symbiotic activity in T. klugi. In 7-. brasiliensis, Gordonia terrae and the non-actinomycete Citrobacter sp. also had no positive influence on vector development, moulting rate and mortality rate, while Gordonia amicalis partly increased the moulting rate of larvae and thereby could be classified as limited symbiotic.

New tools for monitoring gravid females of the mosquitoes Aedes aegypti and Aedes albopictus (Diptera: Culicidae), vectors of Dengue and other arboviral diseases. Eiras, A. E. 1, Rose, A. 2 t, Geier, M. 2 IDepartamento de Parasitologia / Instituto de Ciencias Biologicas; Universidade Federal de Minas Gerais; Belo Horizonte; Brazil 2BioGents GmbH; Regensburg; Germany

Vector surveillance (or monitoring) is used to determine changes in the population densitity of a disease vector. I t can be an efficient early early-warning system against disease outbreaks and provides valuabel information on when and where to take control measures. Methods currently used in monitoring Aedes mosquitoes include the collection of larvae and pupae from water containers in and around houses, determining landing or biting rates on humans, or using so-called ovitraps to collect mosquito eggs. All these methods are costly and time- consuming, and, in the case of the determination of landing/biting rates, ethically questionable because of the risk of infection. We have developed an improved monitoring system for Aedes mosquitoes that is more efficient and less costly. The system consists of a simple trap ("MosquiTRAP") that attracts gravid females through a combination of

51

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in WQrzburg

visual and olfactory stimuli. Because the MosquiTRAP catches adult mosquitoes, species identification is much easier and can normally be performed directly in the field during trap inspection. In contrast, larvae, pupae, or eggs collected in the conventional ways have to be identified in the lab, taking up to 10 days. The synthetic oviposition attractant ("AtrAedes") used in our system is based on substances that are released by hay infusions also used in ovitraps. Compared with a hay infusion, AtrAedes needs less maintanace and attracts more mosquitoes. Field experiments in urban areas in Brazil showed that catching rates correlate well with those from conventional surveillance techniques. An important additional feature of the new monitoring system is that trap inspectors are equipped with hand-held mini-computers for on-site data collection. These data are then loaded into a geographical information system (GIS) to generate maps with risk indices. These risk index maps are the basis for a quick, cost-effective and more environmentally friendly control of Dengue vectors.

Melanotic parasite encapsulation in the malaria mosquito Anopheles gambiae Volz, J.~, SteinbQchel, M}, Kafatos, F. C. ~, MQller, H.-M} t ~ European molecular biology laboratory; D-69117 Heidelberg; Germany

We study the mechanism of Plasmodium ookinete encapsulation in the midgut of refractory Anopheles gambiae mosquitoes. Melanotic encapsulation of pathogens plays an important role in insect innate immunity. Reactive quinones, which polymerize to melanin, are produced by phenoloxidase. This requires prior activation of prophenoloxidase (PPO) by specialised serine protease(s), which in turn are kept in check by serine protease inhibitors (serpins). Common features of all PPO-activating enzymes (PPAEs) identified so far from insects are the presence of at least one N-terminal cysteine motif (clip-domain), and their induction upon bacterial challenge to the organism. Genome analysis of Anopheles gambiae has revealed a subfamily of clip-domain serine proteases, which comprises 13 potential PPAEs. Expression profiling indicated that 4 genes are immune-responsive. In order to elucidate the candidate's potential function, RNA interference experiments were performed in adult mosquitoes. Knock-down results suggest that two of these genes are involved in the immune response towards gram- negative bacteria, and three are implicated in the regulation of parasite transmission. As only intermediate phenotypes could be observed, RNAi experiments with two selected candidate genes are being performed to determine whether these genes act synergistically. 9 PPO genes have been identified in the A.gambiae genome, in contrast to merely 3 PPO genes in Drosophila melanogaster. These 9 PPO genes are transcribed at different developmental stages, showing partly overlapping expression profiles, expression of several PPOs being again/further induced during blood meal digestion. The contribution of individual PPOs to ookinete encapsulation via RNAi is currently under analysis.

Miracidial host-finding: a neglected factor in trematode transmission. Hertel, J } t, Haberl, B}, Kedves, K. 1, Loy, C. 1, Haas, W. 1 iInstitute for Zoology I; University Erlangen-Nuremberg; Erlangen; Germany

Conflicting views on chemo-attraction of miracidia by snails are still subject for discussion. Former studies suggested a weak host-finding ability of miracidia and supposed non-specific small molecular weight components of snail conditioned water (SCW) to release miraicidial host-finding behaviour (increase of random turning: RCD; turn-back responses). In contrast, we recognised macromolecular glycoconjugates from SCW as substances releasing such behaviour and discovered the talent of some trematode species to identify the SCW from their host snail species specifically by these molecules. So far, the proof is missing, whether these findings effect miracidial transmission success under field conditions, just because many authors suggest snail susceptibility to be the main factor influencing transmission. Experiments on miracidial host-finding were employed exposing sentinel snails trapped in cages to miracidia in 500 I containers. As miracidial behaviour cannot be observed in such large environments, the number of snails being penetrated was analysed by PCR and snail penetration was rated as successful transmission. By changing experimental conditions we demonstrated, that miracidial transmission relies on the molecules released by snails to their surrounding area. Transmission seems to be influenced by the sensitivity to, as well as by the quality and amount of snail released molecules. Therefore miracidial host-finding behaviour seems to be an underestimated factor in the miracidial transmission process, together with snail susceptibility and habitat selection. Employed molecular and experimental methods can be implemented in field studies, avoiding difficulties of conventional microscopical determination techniques. Field experiments will contribute to the understanding of the general importance of molecules, releasing host-finding behaviour, for trematode transmission.

52


Following a parasite through its life cycle: how tough is it to cope with different immune systems? Hammerschmidt, K. 1 t, Kurtz, J.~ ~Department of Evolutionary Ecology; Max-Planck-Institute for Limnology; Plan; Germany

In host-parasite coevolution, continuing development is needed to maintain fitness. An arms race increasing parasite infectivity and host resistance can lead to various strategies on both sides. For parasites with a complex life cycle, that battle becomes even more complicated since more than one host is necessary to fulfil the life cycle. The tapeworm Schistocephalus solidus is confronted with the immune systems of a copepod, such as Macrocyclops albidus, the stickleback Gasterosteus aculeatus and, as final hosts, any species of fish eating bird. We focused on the two intermediate hosts to analyze the interactions between the parasite and the two different immune systems. Diverse genotypes of the tapeworm were not only expected to be variable in their infectivity and virulence towards both hosts but might also have to trade-off investment to different immune- evasive mechanisms in the two hosts. Using diverse tapeworm sibships, we found that infection success was highly variable among tapeworm genotypes in the copepod but less so in the stickleback host. Parasite sibships with high infectivity towards the copepod also grew better in this first host, whereas a negative correlation between infectivity and growth in the stickleback host was found. Genotypes, which were highly infective to copepods also elicited a lower innate immune response in the stickleback, and left the fish in better body condition. A possible explanation could be that genotypes are either adapted to cope with innate or adaptive immunity. The first step would be to avoid recognition by any part of the host immune system. Since surface sugars seem to play a major role for immune recognition, we started analyzing variation in such sugar components using fluorescent lectins, proteins that specifically recognize sugar residues.

Trematodes within host tissues: Diversity of orientation strategies Grabe, K. ~, Wulff, C. ~ t, Meyer, V. 1, Haas, W. ~ 1Dept. f. Parasitology, Zoological Institute I; Friedrich-Alexander-University; Erlangen; Germany

Schistosoma mansoni, Trichobilharzia ocellata and Diplostomum spathaceum invade vertebrate hosts (humans, birds and fish) and reach their final destination via complex pathways. However, there is no information about orientation mechanisms used in this migration. Using a choice-chamber (W-chamber) modelling the early events of infection, we comparatively characterized specific host cues used for localization of the appropriate microhabitats. Chemo-orientation responses of schistosomula were studied by exposing the parasites to chemical gradients of human and animal serum and its components. All species orientated towards low molecular weight fractions of their hosts' serum, and glucose and arginine were identified as the major stimulating cues. The responses of S. mansoni and T. ocellata were highly specific to the arrangement of hydroxyl groups of glucose. However, D. spathaceum responded with lower specificity; mannose and maltotriose were also attractive. Free arginine, in low concentrations as found in the epidermis, only produced orientation responses in the schistosomes, but in different concentration-sensitivities. The effect of bound arginine, present as a main component of proteins in the epidermis, was determined using tetrapeptides containing arginine in terminal or subterminal position. In fact, these also stimulated D. spathaceum and, in the schistosomes, even evoked more sensitive responses than free arginine. However, all species responded very differently to its position within the peptides. Our results demonstrate that the species examined respond to specific cues during migration within the host and suggest that the arginine and glucose content of the host's skin tissues are used as landmarks during the parasites' orientation towards deeper skin layers and blood vessels. Apparently, each trematode species has developed its own unique orientation strategy within the host. This might be a mechanism contributing to the host specificity of trematodes.

Effect of the nematode Camal lanus lacustr is on male traits and female mate choice in three-spined sticklebacks Skupch, K. C. i t , Kalbe, M. 1 IMPI for Limnology; Department of Evolutionary Ecology; PIoen; Germany

The three-spined stickleback (Gasterosteus aculeatus) is a fish species with paternal care, in which males exhibit a typical breeding colouration and courtship dance to attract females. Besides this, immunogenetic characteristics (MHC-class-II genes) were shown to be important for females' olfactory recognition of the mates. Theory predicts, that females base mating decision on these traits, which signal reliable information about heritable parasite resistance of the male. Parasites are known to affect a variety of changes in behaviour, colouration or odour of their hosts. Subsequently, females of several animal species avoid such parasitized males. In this study, we examined the effects of infection with the intestinal nematode Camallanus lacustris on visually and olfactory traits of male three-spined sticklebacks, and the resulting impact on female mate choice. C. lacustris is supposed to reduce its host's fitness being anquored in the intestine, feeding on blood and gut material. On account of this, infected fish could for example be less effective in brood-care or food uptake and may face high costs by permanent activation of the immune system. For the experiment, pairs of brothers with identical MHC-class-II genotypes were exposed/sham-exposed to infective C. lacustris (L3) in copepods. When both males had built a nest and were ready to mate, we tested

53


female preference in visual and olfactory set-ups. Finally, the fish were dissected to determine parasite load and compare physiological status by several morphological and immunological measurements. In current analysis we examine the impact of the parasite via male traits on female mate choice and thus expect females to choose the non-parasitized male to gain optimal fitness consequences for their offspring.

The pathogenic amoeba Balamuthia mandrillaris is a host for intracellular multipl ication of Legionella pneumophila bacteria Shadrach, W. S}, Laube, U. 2, Holland, G. 3, Ozel, M. 3, Kiderlen, A. F. 2, Flieger, A. 1 t 1NgS, Pathogenesis of Legionella Infections; Robert Koch-Institut; Berlin; Germany 2Department of Infectious Diseases; Robert Koch-Institut; Berlin; Germany 3ZBS4, Elektronenmikroskopie; Robert Koch-Institut; Berlin; Germany

Balamuthia mandrillaris is a free-living amoeba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other amoebae like Acanthamoeba, Hartmannella and Naegleria can provide a niche for intracellular survival of bacteria, including the bacterial pathogens Chlamydia, Helicobacter and Legionella. So far, the colonization of B. mandrillaris by bacteria has not been examined. In this study, we investigated whether the amoeba could host L. pneumophila bacteria. L. pneumophila is a facultative intracellular parasite of at least 13 different species of amoebae and the infectious agent of Legionnaires disease. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and, after an initial lag phase, could replicate within the amoeba about 4-5 log cycles from 24 to 72 hours after infection. Approaching completion of the intracellular cycle, L. pneumophila was able to destroy its host cell. Observations by light microscopy paralleled our quantitative data and revealed the rounding up, collapse, clumping and the complete destruction of the infected amoebae. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication and the ultrastructural features of a Legionella- infected amoeba resemble those described for other amoebae hosting Legionella bacteria such as A. castellanii and H. vermiformis. In summary, we have shown that L. pneumophila bacteria colonize B. mandrillaris amoebae. We hence speculate that the amoebae might serve as a host for bacteria in its natural environment.

The ameba Balamuthia mandrillaris infects immunodeficient mice also by cutaneous and oral routes Kiderlen, A. F. 1 *, Laube, U. 1, Radam, E. 1, Matzk, P}, Tata, P. S. ~ ZAbteilung fE/r Infektionskrankheiten; Robert Koch-Institut; Berlin; Germany

Balamuthia mandrillaris is a free-living ameba and opportunistic agent of granulomatous amebic encephalitis (GAE) in humans and other mammalian species. World-wide, Nl10 cases of Balamuthia GAE have been reported to date. Both natural source and route of infection remain speculative. In analogy to other free-living, potentially pathogenic ameba, B.mandrillaris may reach the brain after nasal/ oral uptake via the olfactory nerve (as Naegleria fowleri) or, after lung passage, via hematogenous dissemination (as Acanthamoeba spp.). Using highly susceptible immunodeficient mice, we previously provided data for both pathways. Furthermore, we have shown that B.mandrillaris also reaches the brain after subcutaneous, intravenous or ocular inoculations. We now show that B.mandrillaris ameba also infected immunodeficient mice through abraded, but not punctured, skin as well as after direct intra-esophageal inoculation. Groups of 12 female 11-weeks-old C57BL/6 rag -/- mice received lx104 B.mandrillaris (~10% cysts) in PBS either in 20 IJI into both nostrils (A) or in 20 IJI onto a depilated and abraded N100 mm 2 area on the back (B), or in two 100 pl portions by gavage into the esophagus (C). Morbidity was monitored in terms of weight, appearance and motility. On days 4, 10, 18, 24, 27, 32, 42 p.i. stool samples were collected and 2 mice group 1 sacrificed for macro-pathological, histological and flow-cytometric analysis. C57BL/6 rag -/- mice generally took N7 d longer to show signs of morbidity resp. to become moribund than the similarly T- and B-cell-deficient BALB/c sc~d/sc~ mice used previously. All three infected groups showed the typical signs of Balamuthia infection. Detailed histological analysis of all major organs will be provided. IF-microscopy revealed B.mandrillaris ameba in stool samples for group A and C mice. FACS-analysis showed an increase of inflammatory cells, including NK cells, coinciding with the appearance of B.mandrillaris organisms e.g. in the liver.

Intest inal parasites as biological indicators for environmental pollution Sures, B. 1 t 1Zoologie I, Abt. Parasitologie & C)kologie; Universit~t Karlsruhe; Karlsruhe; Germany

Intestinal helminths of vertebrates gain on increasing interest as potential bioindicators for heavy metal contamination in aquatic and terrestrial habitats. Among these parasites, particularly acanthocephalans have an enormous heavy metal accumulation capacity exceeding that of established free living sentinels. Metal concentrations several thousand times higher in acanthocephalans than in host tissues were described from field and laboratory studies. Whereas larval stages inside their intermediate hosts are not able to take up high amounts of metals, young worms immediately after infection of the final host begin to take up metals. After

54


four to five weeks of exposure, the parasites reach a steady state concentration orders of magnitudes higher than the ambient water level. Thus, acanthocephalans are not only very effective in taking up metals, but they can also respond very rapidly to changes in environmental exposure. Investigations on the environmental conditions affecting metal uptake have shown that the parasites are more consistent and reliable indicators for metal pollution than the host tissues as metal levels of the latter are much more dependent on environmental conditions. Thus, after a couple of years of research on the uptake of metals by acanthocephalans and on the factors affecting metal accumulation in intestinal parasites it should be asked if acanthocephalans meet the criteria commonly accepted for sentinels. I f parasites can be considered as promising sentinels, we need reasons for the establishment of "new" indicators. Therefore, this presentation summarises the knowledge about parasites as bioindicators and compares the accumulation properties between parasites and established free living indicators. Finally, this review presents possible answers to the question why it could be advantageous to have new and even more sensitive indicators for environmental monitoring purposes.

Parasites and automobile traffic: acanthocephalans accumulate platinum group metals (Pt, Pd, Rh) emitted from catalytic exhaust gas converters Zimmermann, S. ~ t, Thielen, F. ~, Sures, B. 1 iZoologisches Institut I, Parasitologie/Okologie; Universit~t Karlsruhe; Karlsruhe; Germany

The platinum group metals (PGM) platinum (Pt), palladium (Pd) and rhodium (Rh) are used in catalytic exhaust gas converters of cars to reduce the emission of hydrocarbons, carbon monoxide and nitrogen oxides. As a drawback the noble metals are emitted with exhaust gas into the environment. Consequently, elevated PGM levels were determined in road dusts, soils along heavily frequented roads and sediments of urban rivers. Recently, the biological availability of Pt, Pd and Rh has been verified in experimental studies. Thus, biomonitoring studies are greatly required to assess the distribution of PGM contamination in the biosphere and to evaluate the possible threat of the noble metals for the environment. In the last decade a number of studies revealed an exceptional heavy metal accumulation capacity of acanthocephalans parasitizing fish. Therefore, the use of intestinal fish parasites as sensitive indicators of environmental heavy metal contamination attained growing interest. Accordingly, we have performed exposure studies using two fish-parasite systems, European eel infected with Paratenuisentis ambiguus and barbel infected with Pomphorhynchus laevis, to test whether the acanthocephalan are also able to bioconcentrate Pt, Pd and Rh. The infected fish were exposed to tap water containing ground catalytic converter material. In all experiments the parasites demonstrated the highest PGM concentrations as compared with various fish tissues (e.g. muscle, liver, kidney). P. ambiguus contained for example 40 times higher Pt levels and 4 times higher Pd levels than eel liver. Also Rh was accumulated to a higher degree by P. ambiguus than by the eel tissues. In the exposure study with infected barbel the Pt-, Pd- and Rh-concentration was 22, 12 and 33 times higher in P. laevis than in fish liver, respectively. Similar experiments with the Zebra mussel (Dreissena polymorpha), which is well established as accumulation

indicator for heavy metals, revealed a significant lower PGM accumulation potential for the mussels than for the parasites. Thus, the acanthocephalans could be recognized as new promising accumulation indicator for the three automobile emitted PGM. Support by the program "Environmental Quality and its Security and Sustainment" (BW-PLUS) at the Forschungszentrum Karlsruhe with funds of the State of Baden-W~rttemberg is gratefully acknowledged.

Eel parasite diversity: A tool to monitor aquatic ecosystems? Thielen, F. 1 t, M~nderle, M. 1, Taraschewski, H}, Sures, B. ~ 1Zoologie I Abt. Okologie / Parasitologie; Universit~t Karlsruhe; 76131 Karlsruhe; Germany

In a long term field study European eels (Anguilla anguilla) were sampled at different locations in the river Rhine and at a reference site. The intestinal component community was analyzed using diversity indices (e.g. Shannon Index, Shannon Evenness, and Simpson Index).

Analysis of the diversity indices showed not only considerable distinctions between the different localities, but also a seasonal influence. At three of the four localities analyzed, the diversity increased form spring to summer and decreased again in autumn. These changes are due to seasonal patterns of prevalence and intensity of different parasite species such as Raphidascaris acus, Paraquimperia tenerrima, Anguillicola crassus and Bothriocephalus claviceps. Investigations on parasite communities are therefore not only useful in documenting food web structures but can also characterize the whole ecosystem, their respective hosts are part of. Long-term investigations on the parasite fauna of eels caught at the sampling site Karlsruhe for instance reveal conspicuous shifts in the component communities of intestinal parasites over the last few years. Our field data suggest that the massive spread of invasive invertebrates in the river Rhine after the inauguration of the Main-Danube-canal in 1992 has led to these changes in the parasite community. Especially changes in the amphipod fauna were significantly involved in this development as amphipod species act as intermediate hosts for numerous parasites of fish and water birds. This example clearly shows that the complex life cycles of parasites with free living larval stages, intermediate hosts - among them a huge assemblage of invasive species - and final hosts makes the parasite community vulnerable to ecosystem changes. Accordingly, environmental changes are reflected by alterations in parasite component communities.

55


A GIS-based study on the population dynamics of Ixodes ricinus in the Siebengebirge near Bonn Schwarz, A } t, Maier, W. A} , Kampen, H. ~ lInstitut f(~r Medizinische Parasitologie; Universit~t Bonn; Bonn; Germany

The Siebengebirge near Bonn is a forested hilly nature reserve which plays an important role for local recreation and tourism. Former studies carried out in the Siebengebirge have shown variable prevalences of Borrelia burgdorferi s.I. in Ixodes ricinus ticks depending on the tick densities. As tick abundances are known to be regulated by various biotic and abiotic factors, a study was performed to examine and edit the spatial distribution of the ticks by means of GIS (Geographic Information System). For this purpose, questing nymphal and adult ticks were collected from June to November 2003 by blanket dragging in five biotopes representing different plant communities. Tick abundances as well as (micro-)climatic factors and soil parameters were registered. The data were evaluated statistically and analyzed by using GIS. A total of 2767 ticks including 2595 nymphs and 172 adults were collected and exclusively identified as I. ricinus. On average, the seasonal activity of both developmental instars showed a bimodal course with maximum figures in June and September. However, with regard to the different biotopes examined the population dynamics varied considerably. A correlation appears to exist between tick density and the respective plant community. A given plant community in its turn is correlated with a certain microclimate and soil quality. The study shows that the environmental conditions favoured by questing ticks are reflected by the plant communities. These results can be displayed by GIS and, provided that they can be generalized, they may probably be used to predict 'tick risk areas' by GIS in unknown areas as well.

Borrelia burgdorfer i infection prevalences in questing Ixodes ricinus ticks in the city area and the outskirts of Bonn Maetzel, D. 1~, Maier, W. A. 1, Kampen, H. 1 ~Institut fElr Medizinische Parasitologie; Universit~t Bonn; Bonn; Germany

Recently, it was shown that the infection prevalence of Ixodes ricinus ticks with Borrelia burgdorferi s.I. in the Siebengebirge, a hilly nature reserve near Bonn, has more than doubled during the last decade. By contrast, in urban areas of Bonn comparable examinations have never been performed. In order to assess the risk of a Borrelia infection after a tick-bite in the city area of Bonn, questing nymphal and adult ticks were collected from April to October 2003 at 41 urban/suburban sites (17 private gardens, 9 public parks, 2 edges of fields and 13 fringes to forested outskirt areas of Bonn) along footpaths, in the underwoods beneath the paths and on lawns, meadows and beds. Infection with the various B. burgdorferi genospecies was determined by two different PCR approaches, a simple PCR for rapid screening of the ticks and a nested PCR for subsequent genotyping of borreliae. Out of 1320 ticks (744 nymphs, 576 adults) examined by the simple PCR, 235 (17.8 %) were found positive. The nested PCR products obtained from the positive ticks were processed by DNA hybridization with species- specific probes. All four genospecies of the B. burgdorferi complex known to occur in Germany have been identified: B. burgdorferi s.s., B. garinii, B, afzelii and B. valaisiana. In a few ticks mixed infections were detected, whereas in some other cases a DNA hybridization signal was absent. The PCR products of the latter as well as samples with unclear signals were sequenced. Unexpectedly, the average infection prevalence of ticks with B. burgdorferi s.I. turned out to be slightly higher in Bonn (17.8 %) than in the Siebengebirge (14 %) where contact between potential reservoir hosts and ticks is supposed to be more intense. It remains to be elucidated to what degree the specific tick host cenoses of rural and urban areas may influence the tick infection prevalence, as some B. burgdorferi genospecies are eliminated in certain hosts.

Different parasite communit ies in parapatric host populations of three-spined sticklebacks ( Gasterosteus aculeatus) Kalbe, M. ~t, Wegner, K. M. 1 1Evolutionary Ecology; Max-Planck-Institute for Limnology; PlOn; Germany

Parasitic infection is considered to be of profound importance as selective agent on populations of the host. Since parasite epidemics are not uniformly spread over space and time, selection pressure can also be expected to change. In three-spined sticklebacks, for example, it has been shown that macro-parasite communities are less complex in terms of species diversity in river habitats than in lake habitats. In turn there is also less immunogenetic variability of the host in river populations. However, comparison of adaptations in these populations are phylogeographicaily confounded because gene flow is absent between river and lake populations. To avoid such confounding effects, we compared macro-parasite community structure in a lake (Vierer See, Schleswig-Holstein, Germany) and one of its direct tributaries in three consecutive years. We found differences in species diversity between the two locations despite their close distance (< 300 m). Species diversity was lower on fish from the tributary than on those from the lake, which is consistent with the global pattern. While microhabitat parameters might primarily cause the different distributions of parasite species, the stage is nicely set for investigations on parasite induced selection on the host.

56

Scientific Program DGP, 21st Annual Meeting March 17 th -- 20 th, 2004 in W(Jrzburg

I s o l a t i o n and in vitro Cul t i va t ion of Nosema algerae f r o m Drosophila melanogaster Franzen, C. 1 t, Fischer, S. 2, Sch61merich, J}, Schneuwly, S. 2 ~Klinik und Poliklinik fDr Innere Medizin I; Universit~t Regensburg; Regensburg; Germany 2Lehrstuhl fDr Entwicklungsbiologie; Universit~t Regensburg; Regensburg; Germany

Background: Nosema algerae is a microsporidian known to infect mosquitoes. Under experimental conditions it infects a number of different insect hosts in addition to many invertebrate and vertebrate hosts. Recently N. algerae has been shown to occur in skeletal muscle and cornea of humans. Methods: In October 2003 a microsporidian infection occured spontaneously in the Drosophila colony of the Dep. for Biology at the Univ. of Regensburg. The species was determined as N. algerae by electron microscopy of spore and vegetative stages in infected flies. Spores were harvested from flies, purified, and added to monolayers of monkey kidney cells (Vero), human lung fibroblasts (MRC-5), and Drosophila $2 Schneider ceils. Cultures were maintained by 21°C ($2) or 31°C and 37°C (Vero, MRC-5). Cultures were monitored regularly and cells and supernatants were examined by light (Giemsa, chromotrope, Uvitex 2B) and electron microscopy. The DNA sequence of the SSU rRNA, the intergenic spacer region, and the LSU rRNA was determined by DNA sequencing after PCR amplification. Results: At 31°C the first vegetative stages were detected in MRC-5 cells 96 hours after adding the spores. At 120 hours ceils were filled with early (meronts, sporonts) and late stages of the life cycle (sporoblasts, spores). At 37°C development in MRC-5 cells was slower and the first spores appeared after 7 days. Development in Vero cells was poor and no development was seen in Schneider cells. The DNA sequence of the rRNA gene was nearly identical with published sequences of N. algerae. Conclusion: N. aigerae usually infects mosquitoes of the genus Anopheles but under experimental conditions the parasite has an extremely broad host range from different insect hosts to many invertebrate and vertebrate hosts. Here we report the first isolation and in vitro cultivation of N. algerae from a laboratory Drosophila colony. Parasites grow well in vertebrate ceils (MRC-5) at 31°C. Interestingly no growth was seen in Drosophila cells at 21°C.

Effects of t r e m a t o d e paras i tes on m o r t a l i t y in t h e c o m m o n cock le Cerastoderma edule Thieltges, D. W. 1 t 1Alfred Wegener Institute for Polar and Marine Research; Wadden Sea Sation Sylt; List; Germany

The common cockle Cerastoderma edule is one of the dominating benthic invertebrates of the tidal flats in the Wadden Sea. Large fluctuations in abundance have primarily been related to benthic predation and winter mortality. However, an additional potentially important factor has been largely neglected: parasitism. Cockles are a host to an array of digenean trematodes which mainly use the bivalve as a second intermediate host, and thus occur as metacercariae in the host tissue. Effects of these parasites on survival might be either direct (e.g. enhanced mortality) or indirect (i.e. only occurring in combination with other stressors like cold temperatures etc.). This poster will present results of field and laboratory experiments on the effects of trematode infections on direct and indirect mortality in uninfected and artificially infected cockles.

Paras i tes in n a t i v e and i n t r o d u c e d mol luscs of t h e W a d d e n Sea Krakau, M. ~ *, Thieltges, D. W. 1 1Wattenmeerstation Sylt; Stiftung Alfred-Wegener-Institut for Polar- und Meeresforschung; List; Germany

Recent research findings suggest that introduced species show a low or no parasite burden in their new habitats. This can resul t in an increased growth, better reproduction and consequently an advantage in competition with native species. Surveys on a local scale and investigations of other species are needed to confirm this general assumption. We here compare three native (Littorina littorea, Mytilus edulis and Cerastoderma edule) and three introduced mollusc species (Crepidula fornicata, Crassostrea gigas and Ensis americanus/directus) as intermediate hosts for parasites. Samples of 40-50 individuals of these species pairs (native-introduced) were taken at the same location and at the same time in two tidal basins in the north and south of the island of Sylt. Results show that the introduced species investigated are infected by the same native parasite species (mainly trematodes) as their native counterparts. However, prevalence and intensity are lower in the majority of cases. Non-native parasites imported with the introduced molluscs were not found.

57

Scientific Program DGP, 21st Annual Meeting March 17 th - 2 0 th, 2004 in WiJrzburg

Epidemiology

Risk assessment and prevention of alveolar echinococcosis: Activit ies of the ongoing EU-FP5 project EchinoRisk(QLK2-CT-2001-01995) . Kern, P.~ ~, Romig, T. ~ iUniversity of UIm; Universit~tsklinikum UIm; UIm; Germany

There are numerous indications that Echinococcus multilocularis transmission in Europe has intensified, that the parasite has spread to several European regions thought not to be endemic, and that transmission of the parasite is being established in urban areas. Against this background, in 2001 the 3-year project EchinoRisk was started to assess the status and the dynamics of transmission on a European scale, to identify risk factors for transmission, and to develop appropriate communication strategies which will enable the public to achieve a realistic risk perception in different European countries. The project is done in Austria, Czech Republic, France, Germany, Italy, the Netherlands, Poland, SIovakia, Switzerland and the UK. Four main areas of work are covered: to identify links between landscape parameters and endemicity levels, to obtain more information on the transmission in urban areas ('urban foxes'), to identify the genetic diversity of the parasite within Europe, and to develop communication tools for appropriate information of the public and public health authorities. Preliminary results of the project are presented in this paper.

Echinococcus mul t i locu la r is and fox biology Thoma, D. 1 t, Romig, T} , Heinel, S. ~, Janko, C. ~, Schreiber, T. ~, K6nig, A. 2, Dinkel, A. ~, Mackenstedt, U. t 1Department of Parasitology; University of Hohenheim; 70599 Stuttgart; Germany 2Department of Wildlife Biology and Wildlife Management; Technische Universit~t MOnchen; MOnchen; Germany

In many regions of central Europe, foxes have established stable and self-sustaining populations in urban areas. This is well documented for cities (e.g. Zurich), but less so for small towns and villages in rural areas. Since foxes are known to be frequently infected with Echinococcus multilocularis even in urban areas, attention needs to be focussed on these populations regarding the development of control strategies. While large-scale anthelmintic treatment with aircraft-distributed baits was evaluated in several studies, foxes in the immediate vicinity of human settlements appear to have limited access to such baits. Therefore, a study is in progress in a rural area in the highly endemic Swabian Jura on fox population densities, centers of activity and prevalence rates, to be used as baseline data for future anthelmintic treatment trials in and around rural villages. From March 2001 to September 2003, a total of 28 foxes were caught in traps and fitted with radio-collars for telemetric follow-up. 20 of them provided data of sufficient quality for further analysis with ArcGis (ESRI). Results to date include average home ranges of approximately 100 ha (95 % MCP), being somewhat intermediate in size between those of foxes from natural landscapes and those of strictly 'urban' foxes. The animals do not exhibit strict territoriality, frequently organising themselves in social groups and, therefore, achieving far higher population densities than assumed before. Most of the studied foxes foraged in the settlements only at night with a distinct focus on the peripheral area. Day rest sites were often far outside the villages, an obvious response to the local people's low acceptance for foxes in their vicinity. From the study area, 748 faecal samples were examined by PCR and coproantigen ELISA for E. multilocularis. Prevalence rates of more than 35 % (ELISA) indicate that such 'village foxes' have a high potential for transmission of this parasite.

Is the neozoan raccoon dog epidemiologically relevant as a definit ive host of Echinococcus m ul t i locular is? Tackmann, K. 1 t, Goretzki, j.2, Sutor, A. 3, Schwarz, S. 4, P~tzsch, C. t, Conraths, F. j.5 lInstitut for Epidemiologie; Bundesforschungsanstalt for Viruskrankheiten der Tiere; Wusterhausen; Germany 2Institut for Forst6kologie und Walderfassung; Bundesforschungsanstalt for Forst- und Holzwirtschaft; Eberswalde; Germany 3Wissenschaftszentrum Weihenstephan, Department: Okosystem und Landschaftsmanagement; Technische Universit~t MOnchen; MOnchen; Germany 4F6rderverein GroBtrappenschutz e.V.; Station Buckow; Buckow; Germany SInstitut for Epidemiologie; Bundesforschungsanstalt for Viruskrankheiten der Tiere; Wucherhausen; Germany

Recently the hunting bag of the raccoon dog (or Enok) has increased rapidly in the eastern part of Brandenburg and Mecklenburg West Pomerania, Germany. In some areas just as many raccoon dogs as foxes were shot. Although the hunting bag as a parameter for estimating the population density is not reliable, this clearly indicates that the raccoon dog has established a regional population, at the moment primarily in Eastern Germany, and there is an exponentially increasing population density. An influence of this neozoan predator population on regional biodiversity is not certain but likely. On the other hand the susceptibility of this canid species for E, multilocularis was only recently confirmed in Brandenburg (Thiess et al. 2001). The epidemiological consequences of the regional establishment of this new definitive host species are not known so far. There is some evidence for biological differences of epidemiological relevance between the regional fox and

58


raccoon dog populations. Raccoon dogs show a higher mobility (only during the invasion episode?), a higher reproduction rate, a different food strategy / preferences as well as different habitat preferences. Of special epidemiological interest are assumed differences between foxes and raccoon dogs in susceptibility (especially on quantitative parameters), individual infection intensities and their frequency on population level, duration and intensity of egg shedding as well as the duration of the infection itself. Also interferences between foxes and raccoon dogs on individual and population level or as definitive host populations are important. Investigations are planned to estimate the regional prevalence of E. multilocularis in raccoon dogs (incl. a comparison with the prevalence in the regional fox population) as a first step to answer the question whether the raccoon dog is epidemiological relevant for E, multilocularis or not.

Reference: Thiess A. et al. (2001). Berl. MQnch. Tier~rztl. Wochenschr. 114, 273-276

Strain character i za t ion of cystic echinococcosis in l ivestock in Sudan Omer, R. A} , Dinkel, A. a, Romig, T. a ~, Mackenstedt, U. 1, Aradaib, I. 2 aDept, of Parasitology; University of Hohenheim; Stuttgart; Germany 2Fac. of Veterinary Medicine; University of Khartoum; Khartoum; Sudan

Cystic echinococcosis (CE) is known to be caused by a variety of different genotypes of the Echinococcus granulosus complex, which apparently exhibit considerable differences concerning the susceptibility of humans and different livestock species. In central Sudan, CE in livestock is common, while human patients are rare. Although under-reporting of human cases is likely, the prevailing genotype of the parasite may also contribute to this situation, which is in contrast to countries in the Maghreb and in subsaharan East Africa. To obtain information on the genotypes present in central Sudan, 214 camels, 250 cattle and 400 sheep were examined for CE in the period from May 2001 to October 2002. The prevelance rates were found to be 56.6% in camels, 20.0% in cattle, and 2.5% in sheep. DNA characterization of cyst material showed that all samples belonged to the G6 genotype of E.granulosus (camel strain). In camels, the cysts were predominantly in the lungs (in 72% of all infected animals), which was less often the case with cattle and sheep (in 34% and 40% of infected animals). Fertility of the cysts was high in camels (75%) and cattle (100%), but in sheep all cysts were either sterile or calcified. While the presence of other genotypes in this region cannot be ruled out due to the comparatively low number of surveyed animals, G6 is certainly predominant. Although human CE caused by G6 has been reported from a few human cases in Mauretania, Kenya, Nepal, Iran and Argentina, it has never been associated with high prevalence situations of human CE. Further surveys, concentrating on human patients are urgently needed to clarify the relevance of this genotype for public health.

Helmintho log ica l -sero log ica l surveys as a tool of p revent ive medic ine - cost and benef i t Auer, H} t, AspOck, H. ~ 1Abt. f. Med. Parasitologie, Klin. Institut f. Hygiene u. Med. Mikrobiologie; Universit~t Wien; Wien; Austria

Alveolar echinococcosis (ae) and cystic echinococcosis (ce), caused by metacestodes of Echinococcus multilocularis (Em) and E. granulosus (Eg), respectively, are the most serious helminthic diseases in Central European countries. Both helminths as well as both forms of echinococcosis are prevalent in Austria with low incidence rates of 0,034/100.000/year (ae) and 0,458/100.000/year (ce). The dramatic severity of echinococcosis in general, and of alveolar echinococcosis in particular, results from the long-lasting asymptomatic course (ae: many years; ce: many months up to years) of infection so that the disease is often only diagnosed in an advanced stage of infection. Thus, early detection of echinococcosis, particularly of alveolar echinococcosis, represents a prerequisite for effective curative treatment. In order to assess the prevalence of echinococcosis (of the normal population as well as of particular occupational groups), in Austria several seroepidemiological surveys (based on enzyme-linked immunosorbent assay and westernblot technique) were carried out during the last decade. In 2002 a serological survey of hunters in the province "Burgenland" was carried out not (only) to assess the seroprevalence but to detect E. multilocularis infections in an early stage. The seroscreening was organised and supported financially by the hunting association, blood samples were drawn by medical officers of the provincial health authorities and the serological tests were done in the Clinical Institute of Hygiene and Medical Microbiology in Vienna. In total 891 serum samples were examined, in one serum specific antibodies against Echino-coccus antigens could be found by ELISA and westernblot. The hunter has been treated with albendazole during the past months. In order to establish seroscreenings for echinococcosis and other helminthoses (in particular toxocarosis) as a tool of preventive medicine, ethical, medical, social and - last but not least - economic aspects have to be considered.

59


B o v i n e Neospora caninum in fec t ion: p u t a t i v e r isk factors re la ted to t h e f a r m locat ion are not u n d e r t h e cont ro l of f a r m e r s Schares, G. 1 *, B~rwald, A. 1, Staubach, C. 1, Ziller, M. ~, KI6ss, D. ~, Schr6der, R. ~, Wurm, R. 2, Rauser, M?, Labohm, R?, Dr~ger, K?, Fasen, W. 4, He6, R. G?, Conraths, F. J.~ 1Institute of Epidemiology; Federal Research Centre for Virus Diseases of Animals; Wusterhausen; Germany 2Abteilung Serologie; Staatliches Veterin~runtersuchungsamt Arnsberg; Arnsberg; Germany 3Institut for Tiergesundheit; Landesuntersuchungsamt Rheinland-Pfalz; Koblenz; Germany 4Gesch~ftsfOhrung; Landeskontrollverband Rheinland-Pfalz e.V.; Bad Kreuznach; Germany

An ELISA to determine specific antibodies against a surface antigen of Neospora caninum was modified to examine bulk milk from cattle herds, With this ELISA it is possible to estimate an intra-herd prevalence and its application in an epidemiological study revealed a rapid overview over the distribution of bovine N. caninum infections in the German state of Rhineland-Palatinate. About 90% of the dairy herds of this state were examined. An overall prevalence of positive herds of 7.9% (95% confidence interval 7.0-8.9%) was determined. Prevalences were higher in regions with an increased degree of urbanisation. Logistic regression was applied to model the prevalence of positive herds on a district and city level. Variables describing the dog density, mean temperature in July, mean temperature in January and the total yearly precipitation in districts and cities were able to explain most of the observed variability in the regional prevalences. Among the 2421 members of a farmers' organization a questionnaire was distributed to obtain individual herd data on potential herd related risk factors. For 90% of the farms that responded, bulk milk results were available. Further data used for risk factor analysis were on the dog density and climatic factors of the municipality where the individual herds were located. A multiple stepwise logistic regression was performed to determine risk factors for herds likely to be bulk milk positive. In addition to a risk factor related to the individual farms (number of dogs on the farm) also regional factors related to the farm location were demonstrated as risk factors for individual herds, i.e. the dog density and the mean temperature in July in the municipality or city where the farm is localized. Our results provide evidence that, in addition to risk factors related to individual farms, also risk factors not under the farmers' control, i.e. factors related to the farm location, are important in the epidemioiogy of bovine neosporosis.

L e i s h m a n i a t rop ica in Jer icho ( A ' r i h a ) and its e n v i r o n s , a c lassical focus of Leishmania major AI-Jawabreh, A. 1 ~, Schoenian, G. 1 1Faculty of Medicine, Institut for Microbiology and Hygiene; Humboldt University; Dorotheenstr. 96; Germany

Between 1997 and 2002, 49 strains of Leishmania were isolated from Palestinians living in and around Jericho. A PCR amplifying the ribosomal internal transcribed spacer 1 (ITS1) was applied to their cultured promastigotes and to 207 skin scrapings spotted on filter-papers that proved positive for 109 of the subjects examined. Species identification was done by restriction of the ITS1 amplification product with HaeIII. Of the 49 cultures, 28 (57%) were L. major and 21 (43%) were L. tropica. Of 207 dermal samples tested directly, 109 (52.7%) were positive. Of these, 53 (49.5%) were infected with L. major and 52 (48.5%) with L. tropica. This is the first time L. tropica has been exposed in the population of the Jericho area and on such a large scale. The itinerant behaviour of some of this population precludes categorically declaring that L. tropica has recently become established in this classical focus of L, major. To do this, local infected sand fly vectors of L. tropica must be caught and identified and, if one exists, an animal reservoir host should also be exposed. Key words: Leishmania major, L. tropica, PCR-ITS1, filter papers, Jericho area

Babes ia in d o m e s t i c cats - an e p i d e m i o l o g i c a l s tudy in S o u t h Af r ica W0rth, S. ~, Zahler-Rinder, M. 1 * lInstitut for Vergleichende Tropenmedizin und Parasitologie; Ludwig-Maximilians-Universit~t MEmchen; MDnchen; Germany

Infections of wild and domestic felids with piroplasms have been described for Africa, Asia, America, and Europe. In spite of this nearly world-wide occurrence, knowledge about the species involved, the geographic distribution, host range and transmission is very limited or even lacking. In the Republic of South Africa, babesiosis of domestic cats is believed to be caused by Babesia fells. However, diagnosis is based on morphological features only which do not allow a differentiation from other small piroplasms. Therefore, domestic cats of a coastal region in South Africa were investigated to receive information about the range of species involved and also to determine the infection prevalence in this area. Species determination was based on morphology of the parasites in stained blood smears, serological reactions against a B. fells antigen in an indirect immunofluorescence antibody test (IFAT) and genotypic characterization by sequencing the 18S and ITS regions of the rDNA in the parasites from 13 cats. B. fells was diagnosed in 11 animals. In one cat, which was seropositive for the Feline Leukemia Virus, B. leo was found, a species previously described in lions. This represents the first detection of this species in a naturally infected cat. Furthermore, in one animal piroplasms were identified which could not be assigned to any known species. They were morphologically indistinguishable from B. fells and B. leo. However, antibodies against B. fells were not detected and the parasites were genotypically different from B. fells and B. leo, although closely related to both

60


species. The aspect of host specificity of feline Babesia is discussed especially in the context of concurrent infections with the Feline Leukemia Virus or the Feline Immunodeficiency Virus.

Based on the genotypic characterization of the feline piroplasms in this study, a real-time PCR for a specific detection of B. fells was developed. In an investigation of 206 blood samples originating from domestic cats of a small animal surgery in Port Elizabeth, South Africa, the results of this PCR test were compared with those of stained blood smears and an IFAT using B. fells as the antigen. Identical results of PCR and blood smears were found in 94.6 %, of blood smears and IFAT in 91.2 %, and of PCR and IFAT in 96 .1% of the cases. Piroplasms were diagnosed in 32.2 %, 35.3 % and 39.3 % of the cats examined by blood smears, PCR and IFAT, respectively. Specific antibodies were detected more frequently in cats older than 2 years. Clinical symptoms indicating feline babesiosis were observed in only about a quarter of the cats with a Babesia infection detected, and were seen most often in cats between 0.5 and 2 years of age.

Prevalence of Neospora caninum infections in dairy herds in Rhineland-Palatinate von Blumr6der, D } t, Schares, G. ~, Stambusch, R. 2, Labohm, R. 3, Klawonn, W. 3, Dr~ger, K?, Fasen, W. 4, Conraths, F. j.1 ~Institut for Epidemiologie; Bundesforschungsanstalt for Viruskrankheiten der Tiere; 16868 Wusterhausen; Germany 2Labor Thalfang; Landeskontrollverband Rheinland-Pfalz; 55543 Bad Kreuznach; Germany 3Fachbereich Tiermedizin; Landesuntersuchungsamt Rheinland-Pfalz; 55074 Koblenz; Germany 4Gesch~ftsstelle; Landeskontroflverband Rheinland-Pfalz; 55543 Bad Kreuznach; Germany

A cross sectional study was conducted in the German state of Rhineland Palatinate to determine the prevalence of N.caninum infections in dairy herds. The results will be compared with those of other regional or nation-wide cross-sectional studies conducted in the EU to investigate potentially existing regional influences. 100 dairy herds were randomly selected. They comprised of 4343 cows. The p38-Neospora ELISA (Schares et al., 2000) was modified and evaluated for the examination of milk samples. The overall prevalence as measured in the milk-ELISA was 3.96% (172 positive samples). Intra-herd prevalences varied between 0% to 42.86%. 35% (95% confidence interval, 25.88%-44.12%) of dairy herds showed an intra-herd prevalence of >= 5%. The prevalence was >= 10% in 15 of 100 herds (15%; 95% confidence interval, 8.17%-21.83%). In order to investigate the relationship between milk-positivity and potential risk factors, a questionnaire was filled in by the farmers. The presence of dogs was associated with an intra-herd prevalence >=5% in a univariate analysis (P = 0.02; chi-square test). Among all available dam-daughter pairs the association of their milk results was assessed. The p38-specific

antibody response of the dams was associated with that of their daughters (P = 0.002). The geographical distribution of dairy herds with a prevalence >= 5% tended to be uneven across the state. Prevalences were higher in regions with an increased degree of urbanisation [Rheinhessen/Pfalz (54.55%), Koblenz (39.02%)] than in less urbanised regions [Trier (27.08%)]. This is in accord with a study based on bulk-milk testing (Schares et al., 2003 in press).

References:

Schares,G., et. al. 2000 Int. J. Parasitol. 30, 1123-1130

Schares,G., et. al. 2003 Int. J. Parasitol., in press

A coproantigen survey of Echinococcus multilocularisin foxes in Baden-WOrttemberg Weible, A.-K}, Reule, M. 2, Pleydell, D. 3, Tourneux, F.-P. 4, Renner, C. 5, Thoma, D. 1, Mackenstedt, U. 1, Deplazes, p.6, Romig, T. 1 t 1Dept. of Parasitology; University of Hohenheim; Stuttgart; Germany 2Tollwut- und Epidemiologiezentrum; CVUA Freiburg; Freiburg; Germany 3Div. of Geography; University of Salford; Salford; United Kingdom 4CNRS; University of Franche-Comte; Besancon; France 5Abt, Tierseuchen; Ministerium f. Ern~hrung und I~ndlichen Raum Baden-WOrttemberg; Stuttgart; Germany 6Inst. of Parasitology; University of Zurich; Zurich; Switzerland

The occurrence of Echinococcus multilocularis in red foxes in Baden-W0rttemberg is comparatively well researched, and for parts of the country longitudinal data on the temporal development of the prevalence exist. However, such data were never obtained for the entire country within the same period of time, and most data are more than 8 years old. In view of the intense temporal dynamics of the parasite in central Europe, and to achieve planning data for intervention measures, the Ministry for Agriculture (MELR) of Baden-W0rttemberg launched in 2002 a state-wide prevalence survey with the administrative borders of the communes as sampling grid. Diagnosis was done by coproantigen detection in the rectal content of shot foxes, using a commercial ELISA kit (Bommeli AG). To date, 5,532 samples were tested, resulting in a total prevalence of 31.8%; an additional 5.7% gave borderline results. The sensitivity of the test system was established at 66% with a non- specificity of 10%, using dissection and the intestinal scraping method of 184 foxes from a high prevalence area. The spatial and seasonal prevalence distribution is presented. In addition, the presence of grassland

61


(calculated from CORINE data) as a putative risk factor for the parasite transmission is given for Baden- WOrttemberg as a ,risk map', and correlations between this factor and E. multilocularis prevalence rates is calculated for different spatial scales.

In tes t ina l parasi tes in Afghan residents employed in Camp Warehouse , Kabul Scheid, P. L. ~ t, Thoma, B. R. ~ ~Zentrales Institut des Sanit~tsdienstes der Bundeswehr Koblenz; Labor for Med. Parasitologie; Koblenz; Germany

Intestinal parasites continue to be a major cause of diarrheal diseases with 3.5 billion people estimated to be affected and 450 million diseased worldwide. Multiple parasitoses are endemic in Afghanistan. German soldiers serving in the ISAF mission are deployed mainly to Kabul. There are various kinds of exposition to endemic parasites, one of them being close contact to Afghan residents employed in the camp, who may spread parasites either directly, or indirectly via food, water or soil. In 2002 we examined 217 stool samples of presumably asymptomatic Afghan workers (kitchen aids, translators, housekeeping personnel). The specimens were taken routinely at the beginning of employment, sent to the Central Institute in Koblenz by using the Biosepar® transport medium and examined microscopically. 131 out of 217 samples were found to contain intestinal protozoa or helminth eggs, respectively. 76 samples (35%) contained more than one species adding to a total of 207 positive findings. Ascaris lumbricoides (48 cases), Entamoeba histolytica/dispar (11 cases) and Giardia lamblia (7 cases) were the most common pathogens found. 86 specimens (40%) also contained non- pathogenic protozoa (Entamoeba coil, Endolimax nana, Iodamoeba bOtschlii, Entamoeba hartmanni). After therapy of pathogens no re-infection occurred. We conclude that the routine parasitological examination of employed residents coming into close contact with our troops may contribute to the prevention of transmission and importation of parasites to Germany by infected soldiers.

Deve lopmenta l aspects of Echinococcus rnultilocularis metacestodes in the common vole (Microtus arvalis). Merli, M. 1 t, Romig, T. 1, Dinkel, A. ~, Mackenstedt, U. ~ ZFachgebiet Parasitologie; Institut for Zoologie; Universit~t Hohenheim; Stuttgart; Germany

While numerous data exist on the geographical range, host species and infection rates of E. multilocularis, the transmission dynamics of this parasite are still poorly understood. Epidemiological studies (including mathematical modelling) are often hampered by the lack of basic biological data (patency periods, the contribution of various host species to the biomass etc.). Relevant studies are difficult to conduct due to safety considerations (pathogenicity of Echinococcus eggs to humans) and/or the non-availability of the natural host species for experimental studies. E. multilocularis shows complex distribution patterns in rodents on a small spatial scale. A more reliable estimation of the infection dates of rodents sampled in the field would greatly help to understand the parasite's focal transmission. In the present paper we present data on the reproduction rate in the intermediate host Microtus arvalis, and give informations which could assist the estimate of infection age in rodents caught in the field. Laboratory-bred common voles were orally infected with E. multilocularis eggs from a naturally infected fox. In a first experiment, groups of five voles orally infected with 500 eggs each were necropsied every 2 weeks for a period of 16 weeks. Morphological aspects of the metacestodes were assessed macroscopically and histologically. Early stages (2-4 weeks) were additionally examined by PCR. In a second experiment, groups of seven voles were infected with 50, 200 and 2000 E. multilocularis eggs, respectively, and necropsied 4 months p. i. Size and weight of metacestodes were assessed and the number of protoscolices was determined. The correlation of the various parameter is presented and discussed.

The funct ion of wi ld nutr ia (Myocastor coypus) as in te rmedia te hosts for Echinococcus multilocularis in compar ison to wild muskrats (Ondatra zibethicus). Hartel, K. S. 1 t, Spittler, H.% Doering, H. 3, Winkelmann, j.4, Hoerauf, A. 1, Reiter-Owona, I. 1 i Institute for Medical Parasitology; University of Bonn; Bonn; Germany 2Forschungsstelle for 2agdkunde und WildschadensverhOtung; Landesanstalt for Okologie, Bodenordnung und Forsten NRW; Bonn; Germany 3Abt. W4: Oberirdische Gew~sser, Bau, Betrieb; Erftverband; Bergheim; Germany 4Tiergesundheitsdienst; Landwirtschaftskammer Rheinland; Bonn; Germany

The aim of this study was to compare the prevalence of of E. multilocularis in nutria and muskrats in two different areas in the southwest of North-Rhine Westphalia. One area (A) was a 60 km section along the banks of the river Sieg between Bonn and Windeck where the nutria is not established. The second area (B) was an appr. 60 km section of the river Erft, where both species share the same habitat. To our knowledge, data on the prevalence of E. multilocularis in wild nutria populations have not been published yet. The prevalence in muskrats in area A was recorded in 2001. Pre-examinations in area B showed that the prevalence of E. multilocularis in 114 foxes, shot in a hunting area nearby, was 36.8%. In area B 92 muskrats and 119 nutria were trapped between March 2002 and June 2003. Parasite detection in the rodents was

62


performed by examination of native tissue, in doubtful cases histologically or with nested PCR (University of Hohenheim). 13 out of 92 muskrats from area B (14.4%) contained metacestodes (larvae) of E. multilocularis and all of them were fertile. The prevalence in muskrats from area A had been recorded as 26.7% with a total of 20% fertile metacestodes. In contrast (p<0.05, Chi-Square), of the 119 nutrias examined, only 7 (5.8%) animals were infected with E. multilocularis and only one nutria (0.8%) contained fertile protoscoleces. From two other animals small metacestodes (1-2cm in diameter) without protoscoleces were isolated from the liver and confirmed histologically. The remaining 4 nutrias exhibited only small liver infiltrations (< lmm) which were confirmed by PCR as parasitic tissue. The results from our study suggest that the nutria is a less effective intermediate host for E. multilocularis than the muskrat. The lower prevalence in one of the muskrat populations might be influenced by the co-habitation of two competitive rodent species. This hypothesis has to be supported by further investigations, ideally by longitudinal studies in this area.

On the eradicabil i ty of f i larial diseases Duerr, H. p.1 t, Dietz, K. 1, Eichner, M. 1 ~University of TObingen; Department of Medical Biometry; TElbingen; Germany

The control of filarial diseases has been subject to past and current WHO activities. Whereas the Onchocerciasis Control Programme in West Africa has been terminated in 2002 without fulfilling the hope for eradication, the Global Programme to Eliminate Lymphatic Filariasis celebrates initial successes. This polarity raises the question whether filarial diseases can be eradicated. Reasons for optimism can be found in the existence of so-called breakpoints, i.e. thresholds in the infection intensity below which transmission fades without further efforts. Breakpoints can result from particular density-dependence, e.g. facilitated infection which has been suggested to occur in filarial diseases as a consequence of parasite-induced immunosuppression (Duerr et al., 2003). We investigate by mathematical models how the eradicability of filarial infections depends on density- dependent processes. The balance between the two types of density-dependence (facilitation and limitation) decides whether or not eradication is theoretically possible. Due to experimental accessibility, the processes in the vectors are better understood than the processes in the human host which have been investigated recently in the example of onchocerciasis (Duerr et al., 2004). The finding that facilitation processes imply breakpoints in the transmission cycle of filarial diseases provide a new theoretical basis for eradicability. However, the opposing influences of limitation processes are rather unknown and deserve attention. The design of future control activities and the predictability of intervention success highly depend on the level of our knowledge about parameters by which the infection dynamics are driven. We identify relevant parameters and provide information where future research should focus on.

Duerr, H. P., Dietz, K., Schulz-Key, H., BQttner, D. W., Eichner, M., 2003. Density-dependent parasite establishment suggests infection-associated immunosuppression as an important mechanism for parasite density regulation in onchocerciasis. Trans. R. Soc. Trop. Med. Hyg. 97, 242-250.

Duerr, H. P., Dietz, K., Schulz-Key, H., B0ttner, D. W., Eichner, M., 2004. The relationships between the burden of adult parasites, host age and the microfilarial density in human onchocerciasis. Int. J. Parasitol. in press.

63

Scientific Program DGP, 21st Annual Meeting March 17 t h 20 th, 2004 in Werzburg

Veterinary Parasitology

Development of a viabil i ty assay for Cryptosporidium parvum oocysts Najdrowski, M. ~*, Wackwitz, C}, Joachim, A. 2 ~Institut fDr Parasitologie; UniversitBt Leipzig; Leipzig; Germany 2Institut for Parasitologie und Zoologie; VeterinBrmedizinische Universit~t Wien; Wien; Austria

Cryptosporidium parvum is a coccidian infecting a broad range of mammals. The infectious stage is the oocyst which is resistant to most inactivation methods. I t is also difficult to assess viability and infectivity of oocysts found in the environment and there is no accepted method to determine both parameters. The approaches tried so far include excystation, vital staining and animal models. Excystation measures the ability of oocysts to release sporozoites, whereas vital staining determines the ability of cells to incorporate or exclude fluorescent dyes. Both methods measure viablity rather than infectivity. Animal testing has proven to be the most reliable. However, as it is laborious and cost intensive, efforts have been made to establish an in vitro assay for Cryptosporidium using cell culture and a variety of detection methods. We ha~/e used HCT-8 cells which were inoculated with 10-fold dilutions of bleached oocysts in cell culture medium containing a bile salt to enhance excystation. After 48 hours cells were tested for infection by PCR and IFA assays. DNA was purified and amplified and a specific band on agarose gel was obtained from monolayers inoculated with as few as 10 oocysts. The aim was to determine the efficacy of inactivation procedures by comparing the infectivity of desinfected with unchallenged oocysts. With IFA, the intracellular developmental stages were visualized by FITC labeled antibodies. The resulting fluorescing foci were analyzed with a computer-based picture analysis programme and the number of loci correlated to inoculum size. Thus, it was possible to rapidly assess the intensity of infection and the impact of inhibiting substances on in vitro development. The possible applications of the assays include testing of drugs and disinfecting compounds and procedures for their activity against Cryptosporidium intracellular stages and potentially infectious oocysts.

Occurrence of Neospora caninum and Hammondia heydorni oocysts in faeces collected from naturally infected dogs in Germany Schares, G} t, B~rwald, A. 1, Conraths, F. J}, Barutzki, D. 2 1Institute of Epidemiology; Federal Research Centre for Virus Diseases of Animals; Wusterhausen; Germany 2Tier~rztliches Labor; Freiburg; Germany

Among 4393 canine faecal samples submitted from March 2001 until June 2003 to a veterinary lab localized in Baden-WQrttemberg 20 samples were found to contain oocysts similar to those of Hammondia heydorni and Neospora caninum. The isolates of nine dogs were further examined by gerbil inoculation. Three induced a specific antibody response against western-blot antigens of N. caninum NC-1 tachyzoites in gerbils. No Toxoplasma gondii specific response was observed. The other isolates induced an antibody response neither against N. caninum nor against T. gondii. Morphological differences were observed between N. caninum positive and the other isolates. N. caninum positive oocysts revealed in sucrose solution (d = 1.3) a median length from 9.45-10.1 pm, a median width from 9.3-10.0 pm and a median length to width ratio of 1.02-1.04 pm. N. caninum negative oocysts had a median length from 10.6-12.8 pro, a median width from 9.66-11.46 pm and a median length to width ratio of 1.05-1.15 pm. For one of the latter isolates phylogenetically important DNA sequences were determined, i.e. sequences of the ITS-1 and the D2D3-region. Sequences were almost identical to those described for Hammondia-heydorni-Alabama-1. Consequently, the isolate was named Hammondia- heydorni-Freiburg-16112002. One of the three oocyst isolates that induced a N. caninum specific antibody response in gerbils showed DNA sequences almost identical to N, caninum isolates. This isolate was passaged via yIFN-KO mice into cell culture. The new cell culture isolate is called Neospora-caninum-Freiburg-06022002. The respective oocysts were obtained from a dog at the day of importation from Finland to Germany. Consequently the dog became not infected in Germany but in Finland. This strongly suggests that the respective isolate originates from the latter country. This is a first report of N. caninum in Finland and in addition shows one of the ways by which N. caninum is transmitted from one cattle population to another.

Identi f icat ion and characterization of Theileria lestoquardi proteins for use as diagnostic and immunizat ion tools Bakheit, M. A. 1, Seltzer, U}, Scholzen, T. 2, Ahmed, J. S. 1 t iGruppe: Veterinaer-Infektiologie und -Immunologie; Forschungszentrum Borstel, Abteilung: Immunologie und Zellbiologie; Borstel; Germany 2Gruppe: Tumorbiologie; Forschungszentrum Borstel, Abteilung: Immunologie und Zellbiologie; Borstel; Germany

Malignant ovine theileriosis caused by the intracellular parasite Theileria lestoquardi is a serious tick-borne disease affecting sheep and goats in many parts of the world. Mortalities to T. lestoquardi infection are always high even in the endemic populations. It is thus necessary to establish diagnostic tools which can be applied in

64

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in Wl3rzburg

epidemiological surveys to map out the distribution of the disease and to help in establishing strategies for vaccination and other disease control measures. To detect antigenic proteins of T. lestoquardi, a cDNA library was established from enriched parasite mRNA. Schizonts of the parasite were first isolated from the host lymphoblasts using nocodazole treatment and lysis with aerolysine, followed by gradient centrifugation. Ten randomly selected clones from the library were sequenced and compared to the GenBank data. Eight of these clones were of parasitic origin, which indicated the acceptable purity of the mRNA used in the library construction. The library was screened with field sera from infected sheep. Antibody screening of the library has led to the isolation of a parasite gene encoding a protein of 514 aa with an approximate size of 58 kDa. Proteomic data indicate that it is a hydrophilic protein (estimated pI = 4.7) without transmembranal helices or domains. The protein is Iocalised with great probability (78.3%) in the nucleus or the apical complex of the parasite. Three different fragments of the protein were expressed in pQE vectors; C- terminal and N-terminal fractions and the full length protein. Expressed proteins were purified on Ni-NTA columns and found to react with field sera in Western blots. In future, we will determine the suitability of the protein for diagnostic applications such as ELISA for the detection of antibodies against malignant theileriosis. The antigenic nature of the recombinant protein suggests its suitability as a part of a subunit vaccine for the control of malignant sheep theileriosis.

T cell proliferation and cytokine gene transcription in Eimeria bovis primary and reinfected calves Taubert, A. 1 t, S~Jhwold, A. ~, Hermosilla, C. 1, Zahner, H. 1 lInstitut for Parasitologie; Justus-Liebig-Universit~t GieBen; GieBen; Germany

Eimeria bovis is an important coccidian parasite of cattle causing severe haemorrhagic diarrhoea in primary infected calves. Reinfected animals develop protective immunity which is obviously independent of humoral immune reactions. Therefore we studied cellular immune reactions in primary and reinfected calves on the level of T cell proliferation and gene transcription (gt) of IFNy, IL-2, IL-4, IL-6, IL-8, IL-10, TNFa and TGFI~I using semiquantitative Real Time RT-PCR. Six calves were infected with 5 x 104 E. boris oocysts and reinfected on day 48 p.i.; 3 calves were left non-infected. T cell proliferation upon stimulation of peripheral blood mononuclear cells (PBMC) with merozoite antigen (MeroAg) and cytokine gt in whole blood probes was determined throughout the primary and reinfection period. T cell proliferation upon MeroAg stimulation was clearly enhanced on days 8-15 after primary infection, but was not increased after challenge infection. In cells of non-infected calves MeroAg induced considerable levels of IFNy, IL-2, IL-4, IL-6, IL-8 and TNFa gene transcripts. Primary infection of calves was followed by strongly enhanced IFNy, IL-2 and IL-4 cytokine gt on days 8-19 p.i. After challenge infection - except for day 8 and 12 p.chall, with moderately enhanced levels - IFNy and IL-2 gt were no longer stimulated by MeroAg whereas the IL-4 gt was elevated when compared with non-infected calves. IL-8 and TNFe gt were almost similarly induced in both, cells of infected and non-infected animals; the IL-6 gene was transcribed even at higher levels in non-infected calves. IL-10 and TGF~I gt were not induced by MeroAg at any time in any animal. In summary, we could not detect markedly enhanced MeroAg induced T cell reactions after challenge and suppose that the parasites had been eliminated very early after challenge.

Toxoplasma gondii oocyst uptake by mussels (Myt i lus galloprovincialis) Arkush, K. D. 1, Miller, M. A. 2, Leutenegger, C. M. 3, Conrad, P. A. 4, Tenter, A. M. st iBodega Marine Laboratory; University of California-Davis; Davis; United States 2California Department of Fish and Game; Marine Wildlife Veterinary Care and Research Center; Santa Cruz; United States 3Department of Medicine and Epidemiology; University of California-Davis; Davis; United States 4Department of Pathology, Microbiology and Immunology; University of California-Davis; Davis; United States 5Institut fElr Parasitologie; Tier~rztliche Hochschule Hannover; Hannover; Germany

In recent years, toxoplasmosis has been reported to be an emerging disease in marine mammals, including cetaceans, pinnipeds and sirenians. The sources of infection and routes of transmission in the marine environment are largely unknown, but it has been hypothesized that filter-feeding marine bivalve shellfish may serve as paratenic hosts by assimilation and concentration of infective oocysts of Toxoplasma gondii. We have studied the uptake of 7-. gondii oocysts by mussels (Mytilus galloprovincialis) that were exposed to the parasite under laboratory conditions. T. gondii-specific small subunit ribosomal RNA was detected in mussels as long as 21 days after exposure to T. gondii oocysts. Parasite small subunit ribosomal RNA was most often detected in digestive gland homogenate (31 of 35, i.e. 89%) compared with haemolymph or gill homogenates. Oocyst infectivity was confirmed using a mouse bioassay. Infections were detected in mice inoculated with any one of the mussel sample preparations (haemolymph, gill or digestive gland). These results show that mussels can harbour infective oocysts of T. gondii and that their predation by marine mammals may be one route of transmission of T. gondii in a marine environment. It is also likely that the consumption of T. gondii-infected bivalves is a new source of infection for humans, as has already been shown for Cryptosporidium spp. under natural conditions.

65

Scientific Program DGP, 21st Annual Meeting March 17 th -- 20 th, 2004 in Werzburg

The role of polar f i lament discharge in transmission of myxozoan actinospore stages Kallert, D. ~ *, Estzerbauer, E.% EI-Matbouli, M. 3, Haas, W. ~ ~Zoological Institute 1, Dept. f. Parasitology; University Erlangen- NElrnberg; Erlangen; Germany 2Hungarian Academy of Sciences; Veterinary Medical Research Institute; Budapest; Germany 3Institute for Zoology, Fish Biology and Fish Diseases; University of Munich, Faculty of Veterinary Medicine; M~nchen; Germany

Several myxozoan life cycles underlie obvious specificity towards teleost intermediate host species and tissues. To date it is unknown on which level during infection this specificity is generated and to which degree actinospore attachment participates. To date, there is no information on the involved host cues at all. But the requirement of both a chemical and a physical stimulus to discharge polar capsules was confirmed for M. cerebralis triactinomyxonspores (TAMs) so far. On the basis of the model organism Myxobolus cerebralis we analyzed the chemostimuli in fish mucus fractions. We compared the reactions in an in-vitro system to a salmonid infecting Henneguya and a cyprinid specific Myxobolus TAM species. Our results show congruence in the chemical characteristics of the unknown chemostimuli. For the initial attachment response by polar filament extrusion we found a lack of species specificity in the actinospores of the studied species. However, this reaction was found to be rather specific to fish mucus. We could exclude many substances triggering nematocyst discharge and other mucus components commonly used for fish recognition by other parasites using biochemical fractionation techniques and present a more detailed description of an amphipathic low molecular and lipophilic fraction causing TAMs to attach. Furthermore, experimental data indicate that shell valve opening and sporoplasm release of the actinosporean transmission stage is an invasion step that is independent from filament discharge and does not occur passively as previously suggested.

Isolat ion and initial characterization of a protein from Culicoides nubeculosus relevant to summer eczema in horses Langner, K. F. 1 *, Greiser-Wilke, I. 2, Schlote, S?, Heselhaus, J}, Leibold, W. 1 ~Immunology Unit; School of Veterinary Medicine Hannover; Hannover; Germany 2Institute for Virology; School of Veterinary Medicine Hannover; Hannover; Germany 3Section for Chemical Analytics and Endokrinology; School of Veterinary Medicine Hannover; Hannover; Germany

Summer eczema (SE), also known as sweet itch, is a seasonally recurrent type I allergic dermatitis affecting horses of many breeds. Biting insects, in particular midges of the genus Culicoides, are able to induce and maintain this allergy. Presently, effective treatment is restricted to symptomatic therapy or prevention of the midges. Trials of immunotherapy with extracts of whole midges were hardly effective or even aggravated the disease. Furthermore there is still a lack in understanding the relevant allergic mechanisms in horses. In order to obtain defined allergens we fractionated a whole body extract from a laboratory strain of Culicoides nubeculosus using different chromatographic methods such as ion exchange, hydrophobic interaction and gel filtration chromatography. The starting extract and the fractions were monitored by means of a functional in- vitro test (FIT) developed by S. Kaul (1998) for their properties to induce histamine release from suitably sensitized equine basophils. Moreover, Western Blotting is used to determine the binding of different equine isotypes (IgE, IgG1, IgG4) to the components of C. nubeculosus. The starting extract and several fractions were positive in the FIT using horses with SE plus some of the components were recognized by their IgE and IgG4. The results indicated that more than one protein is involved in the allergic reaction of SE. One of the positive fractions was further analyzed, resulting in the isolation of a highly purified protein selectively triggering basophils from most of the allergic horses and being recognized by their IgE and IgG4. This protein may serve the generation of a defined recombinant allergen from C. nubeculosus to study mechanisms regulating the outcome of SE and may help to develop a reliable immunotherapy in horses with SE.

This work is supported by a scholarship of the KarI-Enigk-Stiftung.

Diagnostic potential and l imitations of the PCR technique for the detection of trypanocidal failure in cattle Gall, y.1 ~, Clausen, P.-H. 1 ~Institut for Parasitologie und Intemationale Tiergesundheit; Freie Universit~t Berlin; Berlin; Germany

Drug resistant trypanosome infections in livestock have so far been reported from 14 sub-Saharan countries, in nine of them with multiple resistance. Since new trypanocides are unlikely to appear on the market in the near future, information about the occurrence, extent and impact of drug resistant trypanosome infections in livestock becomes increasingly important for the implementation of effective trypanosomosis and tsetse control strategies. In recent years, a number of in vivo and in vitro tests for the detection of drug resistance in trypanosomes have been developed. For various reasons, none of these assays is ideal for the investigation of field isolates. From previous studies, the PCR is known as a specific and sensitive diagnostic tool for the identification of trypanosomes in host blood as well as in the vector. It is superior to parasitological methods, particularly in chronically infected animals and in multiple infections. Furthermore, PCR seems to be an

66


appropriate tool to monitor the therapeutic efficacy, particularly in chronically infected animals. Yet, for the use of PCR as an indicator for trypanocide resistance in African animal trypanosomosis (AAT), a number of points should be considered: The choice of the primer pairs should be based on microscopic species identification. Correct trypanocidal treatment of all animals intended for examination is a prerequisite to exclude other reasons for treatment failure than drug resistance in trypanosome infections. Since T. brucei might invade the central nervous system which is inaccessible to drugs used in the treatment of AAT, PCR results might not be reliable in identifying drug resistance within this species. The combined use of PCR and isometamidium (or diminazene) -ELISA would allow the examination of animals without precisely knowing their treatment regime. Multiplex-PCR and/or PCR based on ITS-1 (Internal transcribed spacer) primers should be further developed as to reduce labour and costs. Standardisation of DNA extraction protocols and PCR protocols should be a future goal in order to determine the most suitable in terms of specificity, sensitivity, simplicity and costs and to facilitate comparisons of results.

Improving the management of trypanocide resistance in the cotton zone of West Africa: a coordinated regional study Clausen, P.-H. 1 t, Grace, D. ~, Diall, O. 2, Diallo, B?, Barry, M. 4, M0nstermann, S. 4, Bocoum, Z. s, Diarra, B. 6, Sidibe, I. 7, Affognon, H. 8, Waibel, H. 8, Randolph, T?, McDermott, j.9 1Institute for Parasitology and International Animal Health; Freie Universit~t Berlin; Berlin; Germany 2ILRI/BMZ project on chemoresistance; ICRISAT; Bamako; Mall 3Institut de Recherche Agrnomique de Guin~e; IRAG; Conakry; Guinea 4InternaUonal Trypanotolerance Center; ITC; Banjul; Gambia, The 5Laboratoire Central V@t~rinaire de Bamako; LCV; Bamako; Mall 6Unit@ Centrale de Lutte contre les Tsetse et la Trypanosomose; LCV; Bamako; Mall 7Centre International de Recherche-Developpement sur I "Elevage en zone Subhumide; CIRDES; Bobo Dioulasso; Burkina Faso 8Institute of Horticultural Economics; University of Hannover; Hannover; Germany 9International Livestock Research Institute; ILRI; Nairobi; Kenya

Trypanocidal drugs are the most commonly purchased and used livestock input by resource-poor farmers in sub-Saharan Africa. In West Africa, these drugs are critical in protecting the 17 million head of cattle from trypanosomosis. The effective use of trypanocidal drugs by smallholder farmers is threatened by the development of widespread resistance. This is a particular concern for smallholder crop-livestock farmers in the cotton-zone of West Africa. A BMZ-funded project has recently confirmed significant resistance to trypanocidal drugs in villages with high trypanosomosis risk in Burkina Faso, Mall and to a lesser extent in Guniea. Trypanocides were found to be widely used irrespective of trypanosomosis risk. They were purchased from a wide variety of suppliers without careful attention to quality and given without regard to standard doses and procedures. Preliminary analysis of animal health markets highlighted a great variation in prices of inputs between Mall and Burkina Faso due to differences in regulations and market information. Given the project findings, it was concluded, that: (1) the regional dimension of trypanocidal drug resistance, particularly as regards policy aspects and joint research needs must be taken into account; and (2) research at local level should focus on how to better deliver and ensure adoption of integrated control strategies in which drug use can be targeted and reduced.

Cyathostomin Recombinant Beta-Tubulin Expression Blackhall, W. 1 t, Pape, M}, yon Samson-Himmelstjerna, G. 1, Schnieder, T. 1 lInstitut fuer Parasitologie; Tieraerztliche Hochschule Hannover; Hannover; Germany

Small strongyles (Cyathostominae) are common nematode parasites of horses that have developed resistance to the benzimidazole anthelmintics used to control their populations. Some evidence suggests that the principal mechanism of resistance involves a phenylalanine to tyrosine mutation at codon 200 in the 2-tubulin proteins that are components of microtubules. As part of an ongoing project examining the role that this and other mutations may play in benzimidazole resistance, we have cloned both the phenylalanine- and tyrosine- containing alleles of the 2-tubulin isotype 1 gene from the small strongyle Cylicocyclus nassatus. Other work, however, suggests that alternative 2-tubulin residues or mechanisms may be involved in resistance. We have therefore also cloned a third allele that contains a phenylalanine to tyrosine mutation at codon 167, isolated from a population of C. nassatus resistant to benzimidazoles. Here we describe the construction of expression vectors containing these alleles and their expression in Escherichia coll.

67


Characterisation of susceptibility/resistance against Sarcocystis miescheriana in swine by phenotypic and genotypic variation of clinical and clinical-chemical traits in a F2-model, Hepp, S. 1, Hertrampf, B. ~, Daugschies, A. 2, Mackenstedt, U?, Zahner, H. 4, Reiner, G. ~ * iProfessur for Schweinekrankheiten; Justus-Liebig-Universit~t Giessen; Giessen; Germany 2Institut for Parasitologie; Universit~t Leipzig; Leipzig; Germany 3Institut for Parasitologie; Universit~t Hohenheim; Stuttgart; Germany 4Institut for Parasitologie; JLU Giessen; Giessen; Germany

Evidence for variation in resistance/susceptibility of farm animals against infectious diseases is known to occur within and between breeds. The best documented examples are those concerning gastrointestinal nematodes in sheep, coccidian parasites in chickens, and trypanosomes and tick infestation in cattle. These examples have been well exploited in breeding for resistance in subtropical and tropical farming systems. Knowledge about parasite resistance in pigs is less developed. I t is necessary to understand the genetic and immunological basis of resistance to parasitic infections for a comprehensive explanation of resistance to disease and its application in disease prophylaxis. Genomic approaches to detect and improve disease resistance in farm animals and molecular mechanisms involved in host-parasite interactions depend to a high degree on trait differences between founder breeds, i.e. on the animal model and on the heritability of those traits. Using Pietrain (PI) and Meishan (ME) pigs, we recently described a potential model for the investigation of susceptibility/resistance against Sarcocystis miescheriana. After oral infection with 5x104 sporocysts, clinical signs, loads of bradyzoites in muscle tissues and specific immune response could be used to discriminate between both breeds. Loads of bradyzoites in muscle tissues were 20 times higher in PI than in ME and correlated with the immune response and the bradyzoite load. Sarcocystis-specific breed-differences were in the range of 1 - 6 standard deviations, which layed the foundation for a genomic approach on the molecular basis of Sarcocystis-susceptibility. To provide animals for a genome-wide scan by QTL (quantitative trait loci)- analysis, we set up an infection-experiment based on F2-crosses between PI and ME. Our presentation describes the phenotypic and genotypic response of the F2-pigs on S. miescheriana infection and estimates heritability for susceptibility/resistance against the parasite.

Co-infection by Onchocerca-filariae, Dermatophilus-bacteria and bovine papular stomatitis virus (BPSV) in zebu-cattle of North-Cameroon, Lay, K. 1 t, Geiger, S. 2, Achu-Kwi, D.% B0ttner, M. 4, Renz, A. 1 I Institut for Tierphysiologe; Eberhard-Karls-Universit~t; TObingen ; Germany 2Institut for vergleichende Tropenmedizin und Parasitologie; Ludwig-Maximilian-Universit~t; MOnchen; Germany 3Institut de recherches zootechniques et veterinaires; Wakwa Center; Ngaound~r~; Cameroon 4Viruserkrankungen der Tiere; BFA; TObingen; Germany

In human onchocerciasis, cellular and humural immune reactions are stimulated as well as suppressed, leading to the well known diversity of the clinical manifestation. Co-infections with Mycobacteria were found associated with increased prevalence of onchocerciasis. The bovine model of highly endemic Onchocerca ochengi and O. gutturosa in African cattle prompts itself for an analysis of such interactions between concomitant infections by filariae, Actinomycetales and viruses in a natural host. I t also allows to quantitatively assess the immuno-modulatory effect of the saliva of the respective vectors or facilitators, e.g. in case of dermatophilosis, Amblyomma variegatum. Prevalences of the various parasites and infections in cattle at the local slaughterhouse in Ngaoundere were high, 22 of 31 cattle examined for O. ochengi (71%), and 27 for O. gutturosa (87%), 54 of 760 animals for severe dermatophilosis (7,1%), and 88% of 36 samples examined by EM for Bovine Papular Stomatitis viruses (BPSV). Skin biopsies from the udder of animals infected with dermatophilosis showed a significantly reduced microfilarial (mf) density compared to those without this infection: O. ochengi: 6,7 mf/mg vs. 15,4mf/mg (p<0,0001); O. gutturosa: 1,4 mf/mg vs 3,4mf/mg (p=0,05). The observed virus load of BPSV correlated negatively to the mf density of O. ochengi (p=0,0039) and O. gutturosa (p=0,05). The reproductive capacity of O. ochengi female worms in cattle with severe dermatophilosis (n=15) was significantly reduced as compared to non-infected animals (n= 14; p=0,002), and the proportion of pathologic developmental stages was increased (17% vs. 7%, p=0,023). This reduction in fertility was most evident in cattle co-infected with BPSV(n= 13). These results suggests that in bovine onchocerciasis, either the infection with dermatophilosis, BPSV or both inhibit the reproduction of the Onchocerca spp. This anti-filarial effect could be mediated through a switch from Th2 to Th l answer.

68


Cross-sectional survey on ectoparasite infestations in scavenging chickens in Bali, Indonesia Damriyasa, I.-M. 1, Ardana, I. B. 2, Prelezov, P?, Bauer, C. 3t ~Udayana University; Centre for Studies on Animal Diseases; Denpasar; Indonesia 2Trakia University; Department of Parasitology; Stara Zagora; Bulgaria 3Institute of Parasitology; Justus Liebig University; Giessen; Germany

Background: The chicken population in Bali consisted of 9.7 million birds including 5.1 million chickens of local breed and traditionally kept ('ayam kampung') according to the official veterinary census in 2000. Knowledge on the ectoparasite fauna in chickens of this tropical island is missing till now. Methods: 200 grower and adult free-range chickens of both sexes were purchased from owners of rural communities in two districts (Tabanan, Karagasem) with different climates in July-August 2002. Each bird was set alive into a plastic bag containing cotton wool saturated with chloroform for 5 rain; its feathers were then manually rubbed and the collected ectoparasites preserved with glycerine-ethanol for later identification. Results: All but one chicken were found to be infested with ectoparasites. The mite and Mallophaga species found and their prevalences were: Dermanyssus gallinae (Tabanan: 16%; Karangasem: 18%), Megninia cubitalis (82%; 63%), Menopon gallinae (99%; 98%), Uchida pallidula (syn. Menacanthus pallidulus) (48%; 65%), Lipeurus caponis (43%; 36%), Goniocotes gallinae (23%; 22%) and Goniodes dissimilis (6%; 33%). Additionally, one bird was infested with larvae of Haemaphysalis sp.

Defence Mechanisms & Immunology

Antimicrobial peptides of the arnoebapore superfamily identified in the soil nematode Caenorhabditis elegans Roeder, T. 1, Leippe, M. 2 ~ ~Research Center for Infectious Diseases; University of WfJrzburg; WDrzburg; Germany 2Zoological Institute; University of Kiel; Kiel; Germany

The nematode CaenorhabdiUs eleganshas been introduced as a model organism for the study of infectious diseases, i.e. as substitute for parasitic nematodes and as host model for bacterial infections. Although C. eleganscan cope with different types of bacteria, the structure of its immune system is still a matter of debate. Recently, we identified and cloned a family of five members characterized by substantial homology to the amoebapores, cytolytic and antimicrobial polypeptides of Entamoeba histolyticawhich are among the most important virulence factors of the the protozoan parasite. Four of them are located in a cluster within less than 5 kb on chromosome X of the C. elegansgenome. Cloning of the deduced cDNAs in combination with 5"- and 3"-RACE approaches disclosed the complete set of cDNAs for these amoebapore-like peptides. Evaluation of their deduced amino acid sequence revealed signal peptides at the N termini followed by the a SAPLIP domain, a structural feature characterized by a conserved cysteine motif and typical for amoebapores. RNAi-mediated gene-silencing of all members of this family revealed that one of them is necessary for survival and growth on E. coil, the food source of the worm in the laboratory. The corresponding worms grew extremely slowly and produced only negligible offspring. In addition, ingested E. coil were found to survive within the gut of the corresponding worms pointing to the inability of the gene-silenced animal to kill these bacteria. To further elucidate the physiological significance of these peptides, we monitored the fate of injected bacteria inside the worm and the expression patterns of the genes of the amoebapore-like peptides after various challenges. We are currently studying the target cell spectra of heterologously expressed peptides, the localization of amoebapore-like peptides in the worm by introducing GFP fusion constructs and are analyzing the effect of various organisms on the different worm mutants.

Molecular characterization of an invertebrate defense protein with broad-spectrum activity Bruhn, H. 1, Winkelmann, J.~, Leippe, M. TM

1Research Center for Infectious Diseases; University of WE~rzburg; W(Jrzburg; Germany 2Zoological Institute; University of Kiel; Kiel; Germany

Molecular systems in the body fluid of animals that lyses invaders are widespread in nature ranging from lower invertebrates to the vertebrates with their complement system. I t is known since decades that the body fluid of earthworms contains a variety of cytolytic and antibacterial activities to combat potential pathogens (including parasites) that may migrate from the environment into the body cavity . We purified a protein from the coelomic fluid of the annelid Eisenia fetida using column chromatography which kills eukaryotic cells presumably by forming pores in target cell membranes. By electron microscopy, such membrane lesions are represented as ring-like structures on erythrocyte membranes. Binding of the protein to artifical phospholipid membranes requires sphingomyelin and results in its oligomerization. The aforementioned properties resemble those of eiseniapore/lysenin, lytic proteins of Eisenia that has been described to bind specifically to sphingolipids. Whereas the lipid interaction of eiseniapore has been studied in detail, its molecular identity is

69


unknown. As the purified protein displays antibacterial activity towards gram-positives and gram-negatives, it appears that binding to target cells is not exclusively mediated by sphingomyelin and that other molecules serve as acceptors on bacteria. We characterized the protein structurally by N-terminal sequencing, mass spectrometry and molecular cloning. I t became evident that the protein indeed belongs to the lysenin-like protein family of the earthworm which contains numerous isoforms. We recently succeeded in expressing the major isoform lysenin 1 recombinantly in bacteria which allowed us for the first time to compare the biological activities of a single defined entity towards eukaryotic and prokaryotic target cells, to monitor the oligomerization behavior on target cell membranes and thereby to dissect between sphingomyelin-dependent and -independent mechanisms for killing foreign intruders. (Supported by the DFG)

The melanization response in Anopheles gambiae is controlled by SRPN2 Michel, K} t, Pelte, N. 2, Mueller, H. M. 3, Reichhart, J.-M. 2, Kafatos, F. C. ~ 1Additional Research; European Molecular Biology Laboratory; Heidelberg; Germany 2Institut de Biologie Moleculaire et Cellulaire; UPR9022; Strasbourg; France 3Additional Research; European Molecular Biology Laboratory; Strasbourg; Germany

The effects of mosquito immune responses towards Plasmodium have been known for some time, however the underlying mechanisms are still poorly understood. Integral part of the immune response is amplification and modulation of the initial pathogen recognition signal by protease cascades. One of these cascades leads to activation of prophenoloxidases (PPOs) and thus promotes melanization. Inferring from our knowledge of the Drosophila immune system, this cascade includes inhibitory serine protease inhibitors, serpins (SRPN), which fulfil the important functions of controlling accidental triggering and regulating the spread of the signal.

We report here our analysis of the SRPN2 gene in A. gambiae, which is the ortholog of serpin 27A in D. melanogaster. SRPN2 is expressed during all life stages of the mosquito and its expression is up-regulated during bacterial challenge and Plasmodium berghei infection. Adult mosquitoes lacking SRPN2 as a result of dsRNA treatment, had a significantly shortened life span, which was further decreased by bacterial challenge or injury. Strikingly, only two days post dsRNA injections melanotic spots were found on the body of SRPN2-KO females, which increased in size and number with age of the mosquitoes. In these mosquitoes, no PPO 4 or 6 proteins were detectable in the hemolymph and phenoloxidase activity was drastically reduced. SRPN2-KO mosquitoes were also refractory to P. berghei infection, as ookinetes were encapsulated once they penetrated the midgut.

Our results show that SRPN2, is indeed the functional homolog of Serpin27A of Drosophila. SRPN2 is likely to be the key regulator of melanization in adult A. gambiae. Furthermore, the severity of the KO phenotype emphasizes the important role that this cascade holds to overcome injury or infection in the mosquito.

Regulation of superoxide dismutase and catalase in hemocytes of schistosome intermediate host snails Zelck, U. i t , B~ssler, T. 1, Janje, B. 1 1Institute for Tropical Medicine / Molecular Parasitology Unit; University of T~bingen; TObingen; Germany

Snail hemocytes kill invading parasites by phagocytosis or encapsulation. During these processes the cells undergo an oxidative burst and produce a variety of reactive oxygen species. Of these, hydrogen peroxide was shown to be most toxic for trematodes which need to reproduce in snails as intermediate hosts. Hydrogen peroxide is generated by superoxide dismutase (SOD, EC 1.15.1.1) and detoxified by catalase (EC 1.11.1.6). We report here on the identification and regulation of both enzymes in the hemocytes of two trematode intermediate snail hosts, Lymnaea stagnalis and Biomphalaria glabrata. The hemocytes express both SOD and catalase. However, hemocytes of trematode-resistant snails constitutively express higher levels of SOD and lower levels of catalase compared to hemocytes from susceptible snails. This indicates a higher H202-based toxic potential for cells from resistant hosts. Expression of both enzymes is regulated during phagocytosis and encapsulation reactions at the transcriptional level. Phagocytosis of Zymosan A induced a 50 to70-fold excess for Cu/ZnSOD and a 3 to 8-fold excess for catalase expression after 45 min. This increase paralleled the increasing production of hydrogen peroxide by the hemocytes. Encapsulation of trematode larvae induced an up-regulation of Cu/ZnSOD during 2-48 hours upon stimulation, with the maximal induction at 24 hours. This increase paralleled the increasing production of hydrogen peroxide by the hemocytes and the increasing mortality of trematodes. Catalase expression increased after 12-48 hours with a peak at 36 hours indicating a stress-induced antioxidant response by the hemocytes. The relevance of our results for the compatibility/incompatibility outcomes in these host-parasite systems will be discussed.

70


Experimental serial passages of Sarcocystis singaporensis in intermediate hosts with different MHC class I background Reck, C. ~ t, J~kel, T} , Luz, J}, Kliemt, D}, Mackenstedt, U. ~ ~Institut f#r Zoologie, Fachgebiet Parasitologie; Universit~t Hohenheim; Stuttgart; Germany

Natural infections of S. singaporensis in rats - the parasite's intermediate host - show consistently moderate levels of parasite burden in the muscles, suggesting an optimization of virulence to achieve both, survival of the host and maximum propagation of the parasite. According to this hypothesis, highly virulent as well as non- virulent parasite lineages would have decreased reproductive success thus being eventually eliminated. Immunological studies have shown that the selecting force for such intermediate virulence levels could be linked to the immune system of the rat. In this experiment, the number of cystozoites in the rat was taken as the indicator for virulence. To study the influence of different immunological backgrounds of the intermediate host on the development of virulence, we performed eight serial rat-snake passages of isolate $5 in three different strains of laboratory rats (Lewis, Brown Norway, Wistar). These strains carry different alleles of MHC class I genes, a complex involved in cell-mediated immunity. Our data show that the different host strains have a quantitative influence on the production of cystozoites, indicating either adaptation of the parasite to the host strains during serial passages or selection of parasite genotypes by the different hosts. The virulence of S. singaporensis in Lewis rats increased continuously from passage one to five, followed by a decrease over the next three passages, until the level of passage one was reached again. With the Wistar rats, passages one to five showed a cyclic development, and increasing virulence levels from passage five onwards. In contrast, the parasites became completely avirulent after six passages in Brown Norway rats. These results provide a promising basis for further studies to understand the mechanisms underlying the obvious influence of the host's immune system on virulence levels.

Cell Invasion and Intracel lu lar Fate of the Microsporidium Encephalitozoon cunicufi Franzen, C. 1 t, M011er, A. 2, Hartmann, P}, Salzberger, B. 1 ~Klinik und Poliklinik for Innere Medizin I; Universit~t Regensburg; Regensburg; Germany 2Klinik for P~diatrie; Universit~t Bonn; Bonn; Germany

Background: Microsporidia are obligate intracellular protozoan parasites that have a unique mechanism to infect host cells. Microsporidian spores contain a long coiled polar tube that can be extruded from the spores and that can penetrate the membranes of new host cells. We studied the invasive process and intracellular fate of the microsporidium Encephalitozoon cuniculi in different cells. Methods'. E. cuniculi was grown in MRC-5 and Vero cells. Antiserum to E. cuniculi was produced in New Zealand white rabbits. Different cells (macrophages, MRC-5, Vero, Caco-2, 293) were infected with E. cuniculi spores and intracellular and extracellular spores were visualized at different time points by using a differential immunofluorescence staining technique. To study the effect of the inhibition of actin microfilament aggregation on invasion, cells were treated with cytochalasin D and internalisation of spores was compared with that of untreated cells. Characteristics of the phagosomes were determined by staining with lysosomal markers (LAMP- 1, LAMP-2, CD68). Results: Every examined cell line was able to internalise microsporidian spores. Only a few cells were infected by the polar tube from outside and a few spores seem to infect the cytoplasm from the vacuole after internalisation by the cell. The number of internalised spores was approximately 10-fold higher than the number of injected sporoplasms. Treatment of cells with cytochalasin D inhibited uptake of spores up to 92%. The membranes surrounding the internalised parasites stained positive for LAMP-l, LAMP-2, and CD68. Conclusions: We have established a system to measure the cell invasion of microsporidia into host cells. Entry of spores into cells was mediated by directed phagocytosis, as suggested by the inhibiting effect of cytochalasin D. Phagocytosed spores are transferred to a lysosomal compartment but some spores can escape from the maturing lysosomes and infect the cytoplasm by polar tube discharge.

Polymorphonuclear cell adhesion and adhesion molecule gene transcription in coccidia (Eimeria boris, Neospora caninum, Toxoplasma gondii) infected bovine endothel ial cells Hermosilla, C. 1 t, Zahner, H. 1, Taubert, A. 1 ZInstitut f(Jr Parasitologie; Justus-Liebig-Universit~t GieBen; GieBen; Germany

Eimeria bovis, Neospora caninum and Toxoplasma gondii are important coccidia but little is known about innate immune reactions against these parasites. They infect endothelial cells and develop within 2-3 days (T. gondii, N. caninum) or rather slowly within 18-20 days (E. bovis) into mature schizonts. To examine one aspect of innate reactions we tested polymorphous mononuclear neutrophil (PMN) adhesion on E. bovis, T. gondii and N. caninum infected bovine umbilical vein endothelial cells (BUVEC) under flow conditions. BUVEC were infected with 2,5 x 105 E. boris sporozoites or N. caninum and 7-. gondii merozoites and PMN adhesion was tested 0, 4, 8, 12, 16, 24, 48 and 72 hours p. i. in a parallel plate flow chamber. 7-. gondii infection caused strongest PMN adhesion and high PMN adhesion rates were induced by N. caninum infection, too. In contrast E. boris infection induced only weak PMN adhesion. Levels of gene transcription of the adhesion molecules E-selectin, P-selectin, VCAM-1 and ICAM-1 were determined using semiquantitative Real Time RT-PCR in BUVEC infected with 2,5 x

71

Scientific Program DGP, 21st Annual Meeting March 17 th - -20 th, 2004 in Werzburg

10 s E. bovis sporozoites or N. caninum and T. gondii merozoites. TotaI-RNA was isolated 0, 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hours p. i. and examined for E-selectin, P-selectin, VCAM-1 and ICAM-1 gene transcription. 7-. gondii infection clearly induced the strongest upregulation of E-selectin, P-selectin, VCAM-1 and ICAM-1 gene transcription followed by N. caninum infection. Again, the weakest reactions were caused by E. bovis sporozoites. We hypothesize that E. bovis sporozoites - in contrast to T. gondii or N. caninum merozoites - may render BUVEC less responsive to prevent immunological attacks and to enable their long lasting development within the host cell.

NK cells contribute to the control of Trypanosoma cruzi infection by killing free parasites by Perfor in- independent mechanisms Lieke, T. i t, Graefe, S}, Gaworski, I. ~, Fleischer, B. l, Jacobs, T. 1 lImmunologie; Bernhard-Nocht-Institut; Hamburg; Germany

The protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas'disease in man, is a major health in Latin America. In infected host, trypomastigotes circulate in the blood and invade a variety of cells in which they multiply intracellularly as amastigotes. The acute phase leads to an immune response that restricts the proliferation of the parasite. However, parasites are able to persist in different tissues, which causes the pathology of Chagas'disease. Natural killer (NK) cells play an important role in innate resistance to a variety of pathogens. In the present study we analyzed whether NK cells participate in the control of experimental T. cruzi infection. Our results demonstrate that lytic pathways were activated using chromium labelled YAC-cells as target. To further analyze the role of NK cells in vivo they were depleted from C57/BL6 mice by anti-asialo antibodies. This leads to an increased parasitemia during the acute phase, but tissue parasite burdens were not signifcantly altered according to quantitative real-time PCR. Further experiments indicate that the antiparasitic activity of NK cells was contact-dependent and was accompanied by membrane leakage. However, cells from perforin-deficient mice were as active as cells from wildtype mice, which indicates that lysis was not mediated by perforin. Thus we demonstrate that NK cell limit the propagation of the parasite by acting on circulating T. cruzi trypomastigotes.

Invo lvement of natural kil ler T cells and CDld in Leishmania major-induced murine leishmaniasis Mai, B. 1 t, Moll, H. ~ ~ Insitut fDr Molekulare Infektionsbiologie; Universit~t WDrzburg; RSntgenring 11; Germany

Natural killer (NK) T cells coexpress T and NK cell receptors and recognize glycolipid antigens in the context of CDld. Upon stimulation, NK T cells rapidly produce large amounts of interleukin 4 (IL-4) and interferon-y (IFN- y) and, therefore, have been suggested to exert critical immunoregulatory functions. In this study, we analyzed the NK T cell response to Leishmania major lipophosphoglycan (LPG), a major parasite surface molecule that has been shown to mediate protection against murine leishmaniasis. Increased levels of IFN-y could be detected in the sera of mice already four hours after intravenous injection of LPG, indicating a rapid cellular response. Dendritic cells (DC) that had been loaded with LPG in vitro induce a vigorous proliferative response of NK T cells. In contrast, DC from CDld-deficient mice as well as wild-type DC treated with anti-CDld antibody were unable to stimulate NK T cell proliferation in response to LPG. To examine the relevance of the CDld- mediated NK T cell stimulation for pathogenesis, we infected CDld-deficient mice with L. major and compared the course of disease with that of wild-type mice. The results showed that CDld deficiency is associated with increased susceptibility. Upon polyclonal restimulation in vitro, CD4 ÷ T cells from L. major-infected CDld- deficient mice secreted significantly higher amounts of IL-4 and less IFN-y than CD4 + T cells from infected wild- type mice, suggesting that CDld-restricted NK T cells contribute to the development of a protective Th l response. The findings demonstrate that CDld-restricted NK T cells, activated by the non-protein antigen L. major LPG presented by DC, are involved in the immune response to Leishmania parasites. This provides the first evidence for CDl-mediated recognition of a parasite-derived glycolipid antigen by NK T cells.

Modulation of dendrit ic cells by Echinococcus multilocularis antigen H~rter, G. l, Kern, p.1 ~ Manfras, B. ].1 ~Universit~tsklinikum UIm, Robert-Koch-StraBe 8; University of UIm, Department of Internal Medicine, Division of Infectious Diseases and Clinical Immunology; 89081 UIm; Germany

Infection of humans with Echinococcus multilocularis (E.multilocularis) causes alveolar echinococcosis (AE). It has been unknown to what extent cellular immunity can be induced by E. multilocularis infection. We have previously demonstrated that in chronic AE the frequencies of E. multilocularis antigen-specific cells committed to Thl-cytokine production were low (mean 0.5% of CD4+ T cells) in comparison to recall responses to viral or bacterial pathogens. We investigated the effect of E. multilocularis antigen (E.m. Ag) on human dendritic cells (DC) compared to antigens from yeasts (zymosan) and soluble Toxoplasma gondii antigen (STAG). DC were generated from monocytes or isolated directly from blood. Activation of DC was assessed by detection of TNF- o, IL-6, IL-10 and IL-12. Cytokines were detected by intracellular cytokine staining, ELISA in the cell supernatant, and quantification of specific mRNA expression by TaqMan-PCR. E.m. Ag did not significantly alter

72

Scientific Program DGP, 21st Annual Meeting March 17 th -- 20 th, 2004 in WQrzburg

the expression of cell surface molecules like HLA-DR, CD80, CD86 or CD83. Moreover, the IL-12 production following priming dendritic cells with E.m. Ag was lower compared to DC primed by STAG (IL-12 52,19% versus 88,18% in intracellular cytokine staining, 611 pg/ml versus 1482 pg/ml (ELISA), mRNA expression 2 fold versus 7.5 fold compared to control). IL-10 expression on mRNA level and production in intracellular cytokine staining was decreased in DC primed with E.m. Ag compared to DC primed with zymosan. E.m Ag does not alter the phenotype of human DC but reveals an impairment of cytokine production. These findings provide a possible explanation how the E. multilocularis evades the host's cellular immune system in alveolar echinococcosis.

Isolation and characterization of a secretory component of Echinococcus m u l t i l o c u l a r i s metacestodes potentially involved in modulating the host-parasite interface Walker, M. ~, Dematteis, S. 2, Baz, A. 2, Gottstein, B}, Hemphill, A. 1 t ~Institute of Parasitology; University of Bern; Bern; Switzerland 2Immunology Department, School of Sciences; University of Montevideo; Montevideo; Uruguay

Echinococcus multilocularis metacestodes are fluid filled, vesicle-like organisms, which are characterized by continuous asexual proliferation via external budding of daughter vesicles, predominantly in the liver of infected individuals. Tumour-like growth eventually leads to the disease alveolar echinococcosis (AE). We employed the monoclonal antibody mAb E492/G1, previously shown to be directed against a carbohydrate-rich, immunomodulatory fraction of Echinococcus granulosus, to characterize potentially related components in E. multilocularis. Immunofluorescence studies demonstrated that mAbE492/G1-reactive epitopes were found predominantly on the laminated layer and in the periphery of developing brood capsules. Respective molecules were continuously released into the exterior medium and were also found in the parasite vesicle fluid. The mAbE492/G1-reactive fraction in E. multilocularis, named Em492-antigen, was isolated by immuno-affinity chromatography. Em492-antigen had a protein : carbohydrate ratio of 0.25, reacted with a series of lectins, and also recognized the laminated-layer-associated Em2(G11) antigen. The epitope recognized by mAbE492/G1 was sensitive to sodium-periodate, but not affected by protease treatment. Anti-Em492-IgG1 and -IgG2, and lower levels of IgG3, were found in sera of mice suffering from experimentally induced secondary, but not primary, AE. However, with regard to cellular immunity, a suppressive effect on Con-A- or crude parasite extract-induced splenocyte proliferation in these mice was observed upon addition of Em492-antigen, but Trypan-blue exclusion tests and TEM failed to reveal any cytotoxic effect in Em492-antigen-treated spleen cells. This indicated that Em492-antigen could be modulating the peri-parasitic cellular environment during E. multilocularis infection through as yet unidentified mechanisms, and could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface. Current studies are focussed on elucidating those factors and mechanisms involved in the potential immunosuppressive action of En492.

Modulation of heterologous immune responses by a glycoprotein of Ascar is s u u m Hartmann, S. 1., Mueller, M. I, Adam, R. ~, Lucius, R. 1 1Molekulare Parasitologie; Humboldt Universit~t zu Berlin; Berlin; Germany

Immunomodulatory mechanisms are a characteristic of helminth infections leading to long lasting chronic infections. It has been shown that helminth infections modify responsiveness to nonparasite antigens and other infectious agents in infected animals. We produced a monoclonal antibody against a secretory antigen of the rodent filaria Acanthocheilonema viteae which showed cross-reacting epitopes in the hog roundworm Ascaris suum. The native target protein in A. suum, a 60 kD glycoprotein (As60) was purified by affinity chromatography and used in vivo for the immunization of jirds which were challenged with A. viteae infective larvae. Two independent immunization trials showed a significant increase in the worm burden of 30% and an upregulation of the microfilarial load of 60%. The corresponding gene of As60 was cloned from an A. suum uterus cDNA library using primers derived from the protein data. The As60 cDNA was expressed in E. coil and in insect cells using a baculovirus expression system. The native and both recombinant As60 proteins were tested in vitro in a polyclonally-stimulated and an antigen-specific T cell proliferation assay using ovalbumin TCR- transgenic mice. A marked suppression of the cellular proliferation was found in presence of the native and the eukariotically-expressed As60. In contrast, the E. coil-derived As60 had no effect on the cellular proliferation which might be due to the lack of post-transcriptional modifications. Especially glycosylation seems to be of major importance as the As60 amino acid sequence reveals 21 potential glycosylation sites. In conclusion, a glycoprotein of A. suum showed a marked potential to suppress T cell reactivity in mice in vitro and to inhibit the heterologous immune response against a filarial infection in vivo.

73


Diverse mechanisms of Toxoplasma gondii to interfere with the IFN-y- induced MHC class I I expression in murine macrophages Lang, C. ~ t, Algner, M. ~, Gross, U}, LOder, C. G. K. 1 1Department of Bacteriology; University of G6ttingen; GOttingen; Germany

The obligate intracellular parasite Toxoplasma gondii is able to establish persistent infections in its immunocompetent host . This may be facilitated by different immune evasion mechanisms of T. gondii including the inhibition of IFN-y-induced MHC class I I expression by its host cell and antigen presentation to CD4+ T cells. In the present study, the prerequisites of the parasite that are necessary to mediate the inhibition of MHC class I I expression in murine macrophages were analysed. The results indicate, that the host cell invasion, but not the intracellular replication plays a critical role in the interference with MHC class I I expression. Treatment with cycloheximide showed, that de novo protein biosynthesis by the parasite seems not to be required. Furthermore, isolated parasites that were separated from the host cells by a microporous membrane also inhibited the MHC class I I expression in murine macrophages. A T. gondii lysate (TL), but not excretory/secretory antigens of the parasite mimicked the inhibitory effect of viable parasites on MHC class I I expression. Comparison of the mechanisms underlying the inhibition of MHC class I I expression by viable 7-. gondii and its lysate showed that the mRNA levels of MHC class II and the transcription factor 'class I I transactivator' (CIITA) were diminished by both viable parasites and TL. However, analysis of the DNA-binding activity of the 'signal transducer and activator of transcription' 1 (STAT 1) after activation with IFN-y revealed differential effects of viable T. gondii or TL on Jak/STAT signaling. Furthermore, only TL, but not viable parasites strongly induced the secretion of interleukin 10 by murine macrophages, a cytokine that is known to inhibit MHC class I I expression. In conclusion, T. gondii may employ different mechanisms to inhibit MHC class II expression suggesting a complex regulation of this immune evasion strategy.

Identi f icat ion and characterisation of protective immune cells from Eimeria falciformis infected mice Schelzke, K. 1 *, Lucius, R}, Kretschmer, U. 2, Pogonka, T. ~ ~Institut for Biologie, Lehrstuhl for Molekulare Parasitologie; Humboldt Universit~t Berlin; Berlin; Germany; 2Experimentelle Rheumatologie; Deutsches Rheumaforschungszentrum; Berlin; Germany

Protection against infections with the obligate intracellular parasites of the genus Eimeria is mainly based on cellular immunity. Data from related Eimeria species showed that CD4 ÷ T cells limit primary infection, while CD8 ÷ T cells mediate protection in secondary infections. The exact effector mechanisms are not known so far and are being investigated. In order to identify and characterise protective cells and their effector functions, mesenterial lymph node cells (MLNC) of Eimeria falciformis infected BALB/c mice were used in adoptive transfer experiments. MLNC of infected donors were prepared on different days post infection (p.i.) and transferred intravenously into naive mice. Recipients were subsequently challenged with 100 oocysts per mouse to evaluate the protective potential of the transferred cells. The mice showed a 50% and 75% reduction in oocysts excretion following transfer with donor MLNC from day 4 p.i. and 10 p.i., respectively. Lymphocyte transformation tests were used for in vitro analysis of MLNC proliferation by stimulation with parasite antigen. A significant stimulation of MLNC was observed already on day 4 p.i. and was maintained till day 12 p.i., indicating the presence of parasite-specific cells. To analyse the participating cells we made additional attempts to identify the cell population associated with protection. Therefore we performed transfer experiments with separated CD8 ÷ and CD8 cells. Data of these experiments will be presented and discussed.

Endemically exposed asymptomatic individuals show no increase in the specific Leishmania (Viannia) panamensis-Thl immune response in comparison to patients with localized cutaneous Leishmaniasis Trujillo-Vargas, C. M. 1 *, Robledo, S. M. 2, Velez, I. D. 2, Patino, P. J?, Erb, K. J.1 1Center for Infectious Diseases; University of Wuerzburg; Wuerzburg; Germany 2Programa de Estudio y Control en Enfermedades Tropicales; Universidad de Antioquia; Colombia; Germany 3Grupo de Inmunodeficiencias Primarias; Universidad de Antioquia; Colombia; Germany

In Colombia, most cases of human Cutaneous Leishmaniasis are caused by Leishmania (Viannia) panamensis. Interestingly, up to 30% of the exposed population do not suffer Leishmaniasis although it is likely that they are continuously infected with Leishmania parasites. Since it is believed that the induction of efficient Th l immune responses protects against Leishmania infections both in humans and animal models, we determined if endemically exposed asymptomatics show stronger Leishmania-specific Th l immune responses than patients with active localized cutaneous Leishmaniasis (LCL). We found that Montenegro skin test was slightly higher among asymptomatic individuals compared to patients suffering from LCL. However, PBMC from patients with LCL showed similar Leishmania-specific proliferative responses compared to PBMC from asymptomatic individuals. Furthermore, PBMC from both groups also secreted similar amounts of IFN-y, IL-12p40 and IL-IO after in vitro exposure to L. panamensis. No IL-4 was detected in the supernatants. Taken together our results suggest that lack of LCL development in endemically exposed asymptomatics cannot be explained by stronger anti-Leishmania Th l immune responses or decreased Th2 responses in these individuals in comparison to

74


individuals who develop LCL. It may be posible that other mechanisms are responsible for resistance in endemically exposed asymptomatics to cutaneous Leishmaniasis in Colombia.

The role of B- and T-cell- immunity in Toltrazuril-treated C57BL/6 WT, pMT and nude mice experimentally infected with Neospora caninum Gottstein, B. 1 t, Ammann, P.~, Mueller, N. 1 ~Institute of Parasitology; University of Bern; Bern; Switzerland

Neospora caninum is a protozoan parasite known for causing predominantly abortion in cattle and neuromuscular disease in dogs. So far, no efficient metaphylactic chemotherapy has been developed. In preliminary studies, toltrazuril had been successfully used against experimental neosporosis in mice and calves. In the present study, we used immunocompetent and immunodeficient mouse strains to address the role of immunity in supporting chemotherapy of experimental N. caninum infection. WT, MMT and athymic nude mice were intraperitoneally inoculated with 1 x 106 Nc-1 tachyzoites. The drug was administered in the drinking water for six consecutive days such as to obtain a daily dose of approximately 20 mg toltrazuril per kg body weight. The infection course was monitored by clinical, histological and immunohistochemical means as well as by the search for parasite DNA upon PCR-analyses of various organs. In immunocompetent WT mice, treatment proved high efficacy by abrogation of any lesion formation or PCR-positivity in medicated C57BL/6 mice and signicant reduction of lesion formation or PCR-positivity in BALB/C animals. Similarly, treated MMT mice exhibited a significant reduction in cerebral lesion formation as well as in parasite DNA detectability by PCR, when compared to non-treated animals. Athymic nude mice, however, did not respond to treatment in that only a delay of the parasite dissemination was achieved, and nude mice still diseased by neosporosis, although at a later time point than non-treated animals. We thus conclude from our results that treatment with toltrazuril appears to act parasitostatic rather than parasitocidic. This was supported by the fact that (i) although the lack of B-cells didn't impair the effect of toltrazuril, (ii) the lack of T-cells did not allow full efficacy of treatment anymore. Therefore, chemotherapy with toltrazuril against experimental infections with N. caninum requires the support of T-cell immunity to become successfull.

Protective and allergenic properties of Acanthocheilonema viteae tropomyosin, Sereda, M. J.1 t, Lucius, R. ~, Kranich, S}, Stirati, R. ~, Volkmer-Engert, R. 2, Hartmann, S. ~ IDepartment of Molecular Parasitology; Humboldt University; Berlin; Germany 2Department of Medical Immunology; Charite; Berlin; Germany

Nematode tropomyosin is a potential vaccine candidate as it was shown in several studies that it induces significant protection against challenge infections. However, the protective mechanisms induced by this protein are still unclear. Therefore, we used tropomyosin of the rodent filariae A, viteae to study the effects of different immunization schedules. Jirds were immunized with E. coli expressed recombinant tropomyosin or native tropomyosin, both together with different adjuvants (Alum, STP) or tropomyosin as a DNA vaccine. Protection against challenge infection was about 30% in the experimental groups where a Th l response was stimulated by the adjuvant STP or by intramuscular DNA immunization. In contrast, immunizing jirds with A. viteae tropomyosin emulsified in Alum, a Th2 stimulating adjuvant, led to a protection against challenge infection of 5% only. High titers of IgG1 and IgG3 were detected in vaccinated jirds. In addition, invertebrate tropomyosins are described as pan allergens. Investigating the allergenic function of A. viteae tropomyosin showed that immunization with A. viteae tropomyosin led to the production of reactive IgE antibodies. Especially native A. viteae tropomyosin showed a high allergenic potential. The allergenic properties of IgE-epitopes, originally described in shrimps tropomyosin, were tested using synthetic peptides from A. viteae tropomyosin in a biological allergen assay. Some peptides induced IgE production and stimulated the degranulation of cells. In conclusion, filarial tropomyosin induced low levels of protection regardless whether a native or a recombinant protein was used. In addition, the primarily Th l inducing adjuvant STP was more sufficient to stimulate protection in comparison to Alum which induces primarily Th2 responses. This is of particular interest as filarial tropomyosin is additionally shown to be a potential allergen with probably Th2 inducing properties in vivo.

Chemokines, GM-SCF, COX-2 and iNOS gene transcription in coccidia (Eimeria bovi$, Neospora caninum, Toxoplasma gondii) infected bovine endothelial cells Taubert, A. 1 *, Zahner, H. 1, Hermosilla, C. ~ l Institut fear Parasitologie; Justus-Liebig-Universit~t GieBen; GieBen; Germany

Eimeria bovis, Neospora caninum and Toxoplasma gondii are important coccidian parasites. Sporozoites of E. bovis and merozoites of N. caninum and 7-. gondii, respectively, all can invade endothelial cells and develop to mature schizonts either rapidly within 3 days as in case of N. caninum and 7-. gondii or slowly within 18-20 days as in case of E. boris. Endothelial cells are very potent cells producing a broad range of adhesion molecules, cytokines, chemokines and other molecules upon activation in order to attract leucocytes and to initiate inflammatory responses. In the present study reactions of bovine umbilical vein endothelial cells

75


(BUVEC) upon parasite infection were investigated on the level of chemokine (GROo, IL-8, IP-10, MCP-1, RANTES), GM-CSF, COX-2 and iNOS gene transcription. BUVEC were infected with 2.5 x 10 s E. boris sporozoites and T. gondii and N. caninum merozoites, respectively. Gene transcription of IL-8, GROG, GM-CSF, IP-10, MCP-1, RANTES, COX-2 and iNOS was determined 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hours p. i. using semiquantitative Real Time RT-PCR. Infection with E. boris sporozoites induced low levels of GM-CSF, RANTES, MCP-1 and iNOS gene transcripts within the first two days of infection whereas gene transcription of IL-8, GROo, IP-10 and COX-2 was almost not affected. In comparison, infection with N. caninum and 7-. gondii merozoites induced much stronger IL-8, GROo, MCP-1, RANTES, GM-CSF, IP-10, and COX-2 gene transcription. The iNOS gene transcription of was very low in general. We suggest that E. bovis sporozoites in contrast to 7-. gondii or N. caninum merozoites may render BUVEC less responsive to prevent immunological attacks and to enable the parasite's long lasting development within the host cell.

Immunomodulatory activity of antigens of the rodent filariae Acanthocheilonema viteae Rausch, S } ~, Lucius, R. 2, Sonnenburg, B. 2, MQIler, M. 2, Hartmann, S. 2 iDepartment of Chemistry and Biosciences; University Karlsruhe; Karlsruhe; Germany 2Department of Molecular Parasitology; Humboldt-University; Berlin; Germany

Filarial worms persist in their hosts for years by undergoing and evading immune responses. It has been shown, that especially secreted molecules of adult worms are potential immunomodulators. We used the rodent filariae A. viteae as a model to identify new immunomodulatory components. Analyzing the reactivity of spleen cells in the early and chronic phase of infection showed a massive suppression of T cells from infected jirds in the chronic phase of infection in comparison to the T cells of the early phase of infection. This effect was observed in response to a polyclonal stimulus as well as to A. viteae-antigen specific stimulation of spleen cells of infected jirds. To analyze components responsible for this effect we separated antigen extracts of adult worms and culture supernatant of female worms by gel chromatography. The separated antigen fractions were tested in antigen specific T cell proliferation assays using A. viteae-infected jirds and ovalbumin TCR transgenic mice. Three fractions showed an effect on the antigen-specific cellular proliferation. The cellular proliferation was suppressed by about 47, 48 and 43 %. These fractions contain antigens with a molecular weight of 27-11 kDa, 6-4 kDa and <2 kDa. Similar results were found with fractionated culture supernatant of A. viteae worms. Secreted proteins with immunosuppressive properties were detected in fractions which correspond to proteins found in the separated antigen extract. The immunosuppressive activity of one fraction could be due to Av17, a cysteine protease inhibitor with strong immunomodulatory properties. ELISA and Blot analysis showed that the fractions contain Av17. So far, our results show that unspecific and specific T cell responses are suppressed by A. viteae in the chronic phase of infection. Components wich are involved in the cellular immunosuppression are shown to be secreted by worms.

Nitric oxide production in spleen and liver of Calomys callosus infected with Trypanosoma cruzi. Dost, C. K. i t , Saraiva, J?, Zentgraf, U. 1, Engels, W. 3, Albuquerque, S. 2 1Allgemeine Genetik; Universit~t TElbingen; TElbingen; Germany 2FCFRP; University of Sao Paulo; Ribeirao Preto; Brazil ~Lehrstuhl for Entwicklungsphysiologie; Universit~t T~bingen; TObingen; Germany

Nitric oxide (NO) has a major role in macrophage defense against intracellular microorganism, including parasites such as Trypanosoma cruzi. On the other hand, NO is also known as a double edged sword, generated at high levels it causes intense tissue necrosis. The aim of this work was to determine the NO-production in Calomys callosus infected with two different strains of T. cruzi. Male C.callosus were i.p. infected with 4x103 blood derived trypomastigote forms of the Bolivia (BOL, group 1) and the Boliva-sobrenadante (BOL-SB, group 2) strain. At day 5, 7, 9, 12, 14 and 45 after infection, hepatocytes and splenocytes were prepared from whole organs and cultured for 42h in appropriate medium either with LPS ( lmg/ml) or without. Nitrite levels of the supernatants were measured by the Griess-reaction. Parasitemia curves were obtained at the same days after infection. Splenocytes of C, callosus were unable to produce NO during the infection with BoI-SB, whereas Bol infected cells showed enhanced values at day 9 and 12 compared to controls without infection. Stimulation with LPS leads to an earlier and higher production of NO, being most severe on day 14 in both infection models. In contrast, hepatocytes infected with Bol showed no significant differences during infection compared with non- infected controls. Remarkably, stimulation with LPS led to a significant decrease at day 12, followed by an increase to the highest values at day 14. BoI-SB strain caused a decrease of NO in hepatocytes already during an early stage after infection reaching control levels at day 12 and 14. This decrease was even more pronounced after LPS stimulation. The T. cruzi strain Bol, with a predominance of broad structured forms, caused significant higher values of NO in splenocytes as the slender BoI-SB strain according to the different morphology and pathology described in literature, which is more severe in Bol-infected animals. In hepatocytes increased levels of NO were not observed as they were detected in splenocytes. During the course of infection with T. cruzi it seems that the production of NO is decreased in liver-cells. Since the NO-system has to be triggered very accurately, increased levels of NO already detected in non-infected cells compared to splenocytes, indicate that further stimulation, provoked by the parasites, may turn on other mechanism to prevent an overproduction of NO in these highly specialized metabolic cells. This could be one reason for the observed decrease in NO production during infection with two different strains of 7-. cruzi. Further experiments are carried out to analyze the triggering of NO production in this resistant animal model.

76


In vitro-screening of anti-inflammatory compounds released by parasitic worms HQbner, M. P.~ t, Herrmann, K.-U. 1, Hoffmann, W. H. t, Soboslay, P. T. 1, Schumacher, S. ~, Laufer, S. 2, Kalbacher, H. 3, Schulz-Key, H. z IInstitute for Tropical Medicine; University of TObingen; TDbingen; Germany 2Institute of Pharmacy; University of TfJbingen; TEIbingen; Germany 3Medical and Natural Sciences Research Center; University of TfJbingen; TElbingen; Germany

Parasitic worms are releasing anti-inflammatory substances in their host in order to establish an anergic hyporeactive immune environment substantial for a long-term survival. Based upon this hypothesis we developed an in vitro-system for the screening of helminth-derived compounds with anti-inflammatory properties. Female adult worms of the rodent filaria Litomosoides sigmodontis as well as metacestode tissue of Echinococcus multilocularis were cultivated in vitro for several weeks and culture supernatants were collected at regular intervals. The following protocol was used for the induction of pro-inflammatory cytokines by human immune cells: Peripheral blood mononuclear cells (PBMC) were isolated from the buffy coat fraction of donor blood and stimulated in vitro with E.coli Lipopolysaccharid (LPS). Worm culture supernatant was added to the PBMC similarly or prior to LPS-stimulation. Pure cell culture medium was added in control assays. Aliquots of the cell cultures were isolated at daily intervals for 96 hours and their content of pro-inflammatory cytokines II- 113, TNFa, IFNy was quantified by ELISA. Supernatants of female L. sigmodontis drastically reduced the production of IL-I~, IFNy and TNFa of LPS-stimulated PBMCs. Pre-incubation of these cells with worm culture supernatants prior to LPS-stimulation further reduced the release of pro-inflammatory cytokines. This effect was strictly dose-dependant. Crude fractionation of the filarial worm culture supernatant showed the molecular mass of the active compound being less than 30kDa. Similar anti-inflammatory effects were obtained with vesicles or metacestode tissue of E. multilocularis. The identification and molecular characterisation of helminth-derived anti-inflammatory substances may be useful for the development of new approaches for the therapy of immune disorders such as auto-immune diseases or exaggerating immune responses as being typical in sepsis.

Identification of immunomodulatory proteins of the gastrointestinal nematode Heligmosomoides polygyrus Rzepecka, j.1 t, Lucius, R. 1, Doligalska, M. 2, Rausch, S. 1, Hartmann, S. 1 1Department of Molecular Parasitology; Humboldt-University; Berlin; Germany 2Department of Parasitology; Warsaw University; Warsaw; Poland

Heligmosomoides polygyrus is a parasitic nematode which persists in the mouse intestine for more than 8 months during a primary infection. The long persistence of the parasites is due to immunomodulation of effective immune responses of the host. Parasite-mediated immunomodulation is also shown to downregulate the immune responses raised against other nematodes such as Trichinella spiralis or to reduce helicobacter- induced gastric atrophy. The aim of this study was to characterize immunomodulatory factors of H. polygyrus which are involved in the parasite-induced immune suppression. Therefore, antigen extracts of adult H. polygyrus were separated by gel chromatography and the immunomodulatory activity of each fraction was analysed. Four fractions, containing proteins of the molecular weight between 83 and 7 kDa showed immunosuppressive activity in a antigen- specific proliferation assay of spleen cells isolated from ovalbumin TCR-transgenic mice. In particular one fraction which contained proteins from 83 - 42 kDa interfered with the cellular proliferation by 35%. The reduced cellular reactivity coincided with an effect on the inducible nitric oxide (NO) production of IFN-g stimulated mouse macrophages. The NO production was downregulated by 65% in presence of proteins from this reactive fraction. Interestingly, we found in parallel that this reactive fraction showed allergenic properties as it induced a strong degranulation of a rat basophil leukaemia cell line sensitized with anti-H, polygyrus IgE antibodies. In conclusion, our results indicate that proteins of H. polygyrus which induce cellular hyporeactivity are potential allergens.

Eimeria bovis infection reduces adhesion of polymorphous polynuclear neutrophils to infected endothelial cells by downregulating adhesion molecule gene transcription Taubert, A. 1 *, Zahnert, H. I, Hermosilla, C. 1 l Inst i tut f[Jr Parasitologie; Justus-Liebig-Universit~t GieBen; GieBen; Germany

Eimeria bovis represents an important parasite of cattle leading to severe diarrhoea in calves. Its first and long lasting schizogony takes place in lymphatic endothelial cells of the ileum. The developing macroschizonts may represent potential targets for immune attacks and we described recently polymorphous mononuclear cell (PMN) adhesion to infected bovine umbilical vein endothelial cells (BUVEC). Compared with Toxoplasma gondii and Neospora caninum - which develop much faster in BUVEC than E. boris - PMN adhesion was clearly lower. This rises the question whether E. boris actively inhibits PMN adhesion to protect itself from elimination by early innate immune reactions and to allow its long lasting development. In this work we tested whether E. boris infected BUVEC react in the same manner to TNFa stimulation as non infected cells. In a parallel plate

77


flow chamber bovine PMN were tested for adhesion to uninfected BUVEC stimulated with TNFa and to BUVEC infected with E. bovis (2.5 x 105 sporozoites) 6 days before stimulation. It turned out that E. bovis infection downregulated TNFQ mediated PMN adhesion (58% reduction). To learn more about the molecular basis of this effect we studied adhesion molecule (E-selectin, P-selectin, VCAM-1 and ICAM-1) gene transcription using semiquantitative Real Time RT-PCR. Non infected and E. boris infected BUVEC were stimulated with TNFo. Total RNA was isotated and gene transcripts were quantified. Transcripts of all four genes were markedly reduced in infected cells: VCAM-1 by 59%, ICAM-1 by 67%, E-selectin by 74% and P-selectin by 82%. In conclusion we hypothesize that the parasite renders infected BUVEC less reactive, thereby protecting the host cell from immunological attacks and enabling its long lasting development within the cell.

Chemokine and chemok ine receptor dynamics in dendri t ic cells exposed to Leishmania major Steigerwald, M. 1 t, Moll, H. 1 IInstitut fElr molekulare Infektionsbiolgie; Universit~t W~rzburg; W~rzburg; Germany

Dendritic cells (DC) are bone marrow-derived professional antigen-presenting cells and the most potent inducers of T cell-mediated immune responses. After taking up invading pathogens, DC differentiate from a "processing" into a "presenting" stage, while migrating to the secondary lymphoid organs in order to present microbial antigens to specific T cells. This migratory capacity of DC is tightly regulated by their response to chemokines. In the present work, we examined the differential expression of chemokines and chemokine receptors (CCR) in bone marrow-derived DC from mice that are either susceptible or resistant to infection with Leishmania major. Using RNase protection assays and RT-PCR, we demonstrated that the expression levels of CCR2 and CCR5 are down-regulated, whereas that of CCR7 is up-regulated after DC exposure to L. major. These alterations were observed with DC from both resistant and susceptible mice. On the other hand, expression of the chemokine CXCL10 was increased only in L. major-infected DC from resistant mice. To evaluate whether the parasite-induced modulations in CCR expression correlate with changes in the responsiveness to the corresponding chemokines, DC were tested for their migratory activity. The results showed that exposure of DC to L, major caused a reduced chemotactic response to CCL2 and CCL3, the ligands of CCR2 and CCR5, and an increased migration in response to CCL21, the ligand of CCR7. Thus, L. major induces a decrease in the responsiveness of DC to chemokines promoting their migration to the site of infection (CCL2 and CCL3) and an enhanced reactivity of DC to CCL21 which directs their homing to lymph nodes. The findings indicate that Leishmania parasites selectively modulate the migratory activities of DC, which are critical for their subsequent immunological functions.

Membrane -permeab i l i za t ion by saposin- l ike proteins - var ia t ions on a common fold Bruhn, H. 1 ~, Hecht, 0. 2, Gr~tzinger, 3. 2, Leippe, M. 3 1Research Center for Infectious Diseases; University of WOrzburg; WElrzburg; Germany 2Biochemical Institute; University of Kiel; Kiel; Germany 3Zoological Institute; University of Kiel; Kiel; Germany

The recently solved solution structure of amoebapore A, a major pathogenicity factor of Entamoeba histolytica, represents not only the first tertiary structure of a pore-forming toxin of an eukaryotic parasite but also allows an up-dated view on the mechanisms of saposin-like proteins with membrane-permeabilizing function. For amoebapore A, a histidine-mediated, pH-dependent dimerization appears to be the essential step for further oligomerization in the membrane environment eventually leading to the pore. Notably, the crucial electrostatic interactions of particular residues are conserved throughout the amoebapore isoforms but their slight differences in biological activities are mirrored also in the structures. Remarkably, a kind of amoebapore-like protein already evolved in prokaryotes. The antibiotic peptide bacteriocin AS-48 from Enterococcus is devoid of disulfide bridges and circular but nevertheless has adopted the SAPLIP-fold and, interestingly, forms a dimer in solution. In this case, a rotation of the single subunits is postulated to trigger the destruction of bacterial membranes. Dimerization is frequently found with saposin-like proteins. Saposin B, a cofactor for lysosomal enzymes, encloses lipids in a hydrophobic cavity between the two monomers, demonstrating that other structural scenarios of lipid interaction are possible for SAPLIPs, presumably also in the case of membrane permeabilization. By contrast, the fairly identical and well superimposable structures of NK-lysin and granulysin, the mammalian relatives of amoebapore found in cytolytic lymphocytes, are those of soluble, globular, monomeric and highly charged proteins, which are able to perforate a membrane without the need of strong protein-protein interactions. Apparently, similar folded proteins can use different modes of action to achieve the same goal: killing of microbial and/or eukaryotic cells by membrane permeabilization. (Supported by the DFG)

78


Molecular cloning of Acanthocheilonerna viteae serpin: a potential immunomodulator in filarial infections. Hermann, A.-J. ~ t, Lucius, R}, M~ller, M. 1, Sonnenburg, B}, Hartmann, S. 1 ~Department of Molecular Parasitology; Humboldt-University; Berlin; Germany

Filarial parasites are constantly exposed to an array of immune effector mechanisms of the host. Their survival depends on the evasion of harmful immune responses or the modulation of the immune responses. One strategy to cope with the immune responses is the release of immunomodulatory components that block effector mechanisms. Among the secreted molecules serpins have been described with functions in immune evasion. Serpins belong to the serine protease inhibitors and exist in vertebrates and invertebrates where they exert several functions. They are involved in proteolytic cascades, cell migration and blood pressure. In this study we are interested to analyse the immunomodulatory capacity of Acanthocheilonema viteae serpin in comparison to a homologous protein of the free-living nematode Caenorhabditis elegans. The aim of the project is to investigate whether serpins of the rodent filarae A. viteae are specifically used by the parasite to evade immune responses of the host or whether serpins of the free-living nematode C. elegans have similar immunomodulatory activities. By PCR we amplified a sequence of A. viteae serpin which contains a highly conserved region, the so called serpin signature. The labelled PCR product was then used to screen a cDNA library which led to the isolation of one clone with homologies to serpin. In parallel we have cloned one serpin of C. elegans which was subsequently expressed in E. coll. The recombinant proteins will be tested regarding their specificity towards different serine proteases and their immunomodulatory properties will be analysed. Results of this study will give insights in the evolutionary arm race between hosts and parasites on the molecular level. In addition, we might reveal characteristics of parasite molecules which reflect an adaption to their parasitic life style.

Immune response against primary and secondary alveolar echinococcosis in the mouse model Sch6nfelder, K. 1 *, Merli, M. 1, Traub, K. 1, Mackenstedt, U. ~ iFachgebiet Parasitologie; Universit~t Hohenheim; Stuttgart; Germany

Most experimental studies on immune responses against alveolar echinococcosis (AE) in the mouse model are based on infections with secondary AE, where metacestode tissue is directly inoculated into the peritoneal cavity. Animals infected orally with Echinococcus eggs (primary AE) are rarely used due to safety considerations and difficulties to obtain infective material. However, recent studies on E. muldlocularis immunity showed a variety of differences in the induced immune response between the primary and secondary model (e. g. splenic proliferation, antibody response). The principal differences of secondary AE are the absence of the 'early antigen phase' when the oncosphere and the early developing metacestode are presented to the immune system, and the atypical Iocalisation of the metacestode (peritoneal cavity). Aim of the present study was to characterise the immunological differences between the two models during the course of infection. Twelve week old female Balb/c mice were orally infected with 2000 E. multilocularis eggs, or were infected intraperitoneally with homogenized metacestode tissue. The animals were necropsied two and six weeks p. i. (early stage of infection) and four months p. i, (chronic stage). We compared the expression of various cytokines (IL2, IFNy, IL4, IL l0 and TNFo) by CD4+ T-helper ceils on a single cell level with flow-cytometric techniques, and the proliferative response of spleen cells after stimulation with ConA and E. multilocularis-crude antigen. In addition, we determined the level of specific antibodies (IgG and isotypes, IgM).

79


Vaccines, Chemotherapy, Antiinfectives (Wirkstoffseminar)

Identification of Eimeria tenella secretory antigens for the development of a subunit vaccine against coccidiosis Klotz, C} t, Lucius, R. ~, Pogonka, T. ~ 1Department of Molecular Parasitology; Humboldt University Berlin; Berlin; Germany

In order to identify new immunoprotecive proteins as part of a subunit vaccine against chicken coccidiosis, we screened secretory proteins of Eimeria tenella in a DNA based immunisation strategy. A cDNA library was constructed from E. tenella sporozoites and generated in the yeast cell strain YTK12. Cells of this mutant strain allow the growth of recombinant yeast containing E. tenella secretory proteins only. The selection procedure gave 191 independent clones, which finally represented 25 unique genes coding for deduced proteins with a typical N-terminal signal sequence of secreted proteins; 14 of these were chosen for an immunisation trial. Sequences of the appropriate genes were subcloned into the DNA vaccination plasmid pCDNA3. Expression of the heterologous genes was tested in vitro by analysing transfected COS7 cells in immunoblot experiments. For the immunisation experiment, groups of three-week old White Leghorn chickens were immunised three times intramuscularly with 100 pg each of the respective DNA-constructs. Blood samples were collected to monitor the antigen-specific IgG antibody response. One week after the last booster immunised chickens were challenged orally with 500 E. tenella oocysts each. The total oocyst output and weight gain were used to estimate protection levels. Results of this trial will be presented and discussed.

Vaccination of mice against experimental Neospora caninum infection using NcMICl-recombinant antigen and DNA-vaccination Alaeddine, F. ~, Hemphill, A. ~ t 1Institute of Parasitology; University of Bern; Bern; Switzerland

NcMIC1, a microneme-associated protein identified earlier in N. caninum tachyzoites and putatively involved in adhesion and invasion of host cells in vitro, was investigated for its potential as vaccine candidate in mice. Recombinant recNcMICl was expressed in Escherichia coil as poly-histidine-tagged fusion protein. Separate groups of mice (10 animals each) were immunised with adjuvants (control), purified recNcMIC1 resuspended in adjuvants, the DNA-vaccination plasmid pcDNA3.1, pcDNA3.1 containing NcMIC1 cDNA insert, or a combination of DNA-vaccine and recombinant antigen. Immunisation with a crude somatic antigen (NCl-extract) was included in the experiments. Following intraperitoneal challenge with N. caninum tachyzoites, the presence of the parasite in the different organs was assessed by a N. caninum specific PCR, while the parasite burden in infected brain tissue was assessed by quantitative real-time PCR. While in the infection control group 6 out of 10 animals succumbed to the infection 8-20 days p.i., and few animals were exhibiting clinical signs of neosporosis in the other control groups, the vaccinated animals remained free of symptoms until 21 days p.i. However, parasite DNA was detected by PCR in the brain of all animals of all groups, controls and vaccinated, but the parasite burden was drastically reduced in the group vaccinated with recombinant recNcMIC1. Thus, vaccination with recNcMIC1 prevents the occurrence of clinical signs of N. caninum infection, and reduces the parasite burden in the brain.

Secondary and primary murine alveolar echinococcosis: combined albendazole/nitazoxanide chemotherapy exhibits profound anti-parasitic activity

Stettler, M. 1, Rossignol, J.-F. 2, Walker, M. 1, Gottstein, B. ~, Hemphill, A. 1 t IInstitute of Parasitology; University of Bern; Bern; Switzerland 2Romark Research Laboratories; Tampa. FL; United States

In this study, the efficacy of chemotherapy employing nitazoxanide (NTZ), albendazole (ABZ), and a NTZ / ABZ-combination against alveolar echinococcosis (AE) were investigated in an experimental murine model. Following secondary infection, meaning intraperitoneal injection of 20 Echinococcus multilocularis metacestodes, the drugs were administered by intragastric inoculation on a daily bases for a period of 5 weeks. Treatment was started either immediately on the day of infection, or at 2 months post infection (pJ.), respectively. Application of the NTZ / ABZ-combination starting at 2 months p.i. was proven to be most effective in terms of reducing parasite weight (from 4.3 + 0.9 g to 1 + 0.05 g; p = 0.001). Inspection of treated parasites by transmission electron microscopy (TEM) showed that ABZ- and NTZ-treated metacestode tissues, respectively, were heterogenous in that both largely intact parasites as well as severely altered metacestodes could be observed. NTZ / ABZ-combination treatment induced the most severe ultrastructural alterations, including massive reduction in length and number of microtriches, severely damaged tegumental architecture, and progressive loss of viability of the germinal layer, associated with encapsulation by host connective tissue. A comparative pharmacokinetic study in mice revealed that the application of ABZ and NTZ in combination resulted in a 2- to 4-fold increase of albendazole sulfoxide (ABZSO) serum levels for the period of 4~8 h following drug uptake compared to application of ABZ alone. In a third experiment, mice were orally

80


infected with Echinococcus multilocularis eggs, and treated with NTZ starting at 2 months p.i.. This resulted in a significantly lower lesion number in treated versus untreated mice (p = 0.01). This investigation indicates the potential value for NTZ and/or a combined ABZ/NTZ chemotherapy against AE.

Differences in the genetics of benzimidazole resistance between sheep tr ichostrongyles and cyathostomins yon Samson-Himmelstjerna, G. ~ t, Buschbaum, S. ~, Dr~gem011er, M. 2, Schnieder, T } ~Institut fDr Parasitologie; Tier~rztliche Hochschule Hannover; Hannover; Germany 2InsUtut fDr Parasitologie; Universit~t Leipzig; Leipzig; Germany

The mechanism of benzimidazole (BZ) resistance in trichostrongyles from sheep has repeatedly been shown to be closely linked with a single nucleotide polymorpism (-FrC to TAC) at the beta-tubulin codon 200. While susceptible populations contain almost exclusively individuals homozygous for the phenylalanine coding -I-I-C, highly resistant ones present only the TAC allele. We have shown that the same polymorphism is present also in cyathostomins and that the respective -I-I-C/TAC allele frequencies also differ between susceptible and resistant populations. However, in contrast to the situtation in sheep nematodes, phenotypically highly resistant cyathostomin populations do only show low percentages of homozygous TAC individuals. We have established a real-time PCR test to analyse the frequency of the two beta-tubulin codon 200 alleles using TaqMan minor groove binder probes. The high specificity and sensitivity of the assays allowed the reliable genotyping of single larval or adult worms. Here we found only less than 10% of the larvae from highly resistant field populations to be homozygous TAC. In further investigations we analysed the effect of continued selection by Iongterm bi- weekly BZ high dosage treatment on the phenotype and genotype of the experimentally established BZ resistant cyathostomin population. It was found that neither the egg hatch test LD50 values nor the codon 200 allele frequencies changed significantly during the course of the study. I t may be concluded that apart from the polymorphism at codon 200 other sites contribute to the mechanism of BZ resistance in cyathostomins. We therefore analysed the complete beta-tubulin coding sequences obtained from several individual worms isolated of a highly BZ-resistant cyathostomin population. I t was found that in all worms a TTC/TAC polymporphism at the beta-tubulin codon 167 could be detected. Previously this polymorphism has already been correlated to the mechanism of BZ resistance in different organisms. Further studies will be required to determine the importance of this site for the mechanism of BZ resistance in cyathostomins.

Ident i f icat ion of secreted molecules of Eimeria tenella by bioinformatic approaches Klotz, C. 1, Lucius, R. 1, Marh~fer, R. 2, Seizer, p.2, Pogonka, T. ~ t ~Molekulare ParasitoIogie; Humboldt Universit~t Berlin; Berlin; Germany 2Drug Discovery/BioChemInformatics; Intervet Innovation; Schwabenheim; Germany

Protozoa of the genus Eimeria are intracellular parasites within the gut of many vertebrates causing coccidiosis, a serve infection of poultry. Infections lead to extraordinary losses in livestock husbandry. To identify molecules involved in the host-parasite interaction, in particular excretory/secretory (E/S) and surface proteins, we used a bioinformatic approach for the analysis of E. tenella EST databases. An EST database of E. tenella with 12187 entries was clustered on the basis of similarities, resulting in 2997 unique assemblies. Using the BlastX algorithm, a significant number of assemblies showed homology with entries in public databases. Resulting homologous amino acid sequences were analysed for the presence of an N-terminal signal sequence using an improved SignaIP algorithm. The signal sequence indicates that molecules are directed to the secretory pathway. A resulting list contained 114 entries, with 53 assemblies having similarities to proteins of related apicomplexa parasites and 43 with phylogenetically more distant organisms. A classified list will be presented in details and discussed. Some assemblies showed homologies to genes of proteins Iocalised to the mitochondrion, which do not normally have the N-terminal signal sequences. To verify that we identified putative secreted Eimeria proteins, we examined the secretion of six proteins in yeast cells. Yeast ceils, transfected with the Eimeria sequences, have grown on selective media, indicating that the selected Eimeria molecules are indeed secreted proteins and our approach represents an adequate method to identify E/S or surface molecules in databases.

Effect of ant iretroviral protease inhibitors alone and in combination with paromomycin on the excystation, invasion, and in vitro-development of Cryptosporidium parvurn Hommer, V. 1, Eichholz, J.1, Petry, F. ~ t 1Institute of Medical Microbiology & Hygiene; Johannes Gutenberg-University; Mainz; Germany

With the spread of the HIV in the early 1980s cryptosporidiosis was regarded as an AIDS-defining disease. As an opportunistic pathogen the intestinal parasite C. parvum became an important cause of chronic diarrhoea leading to high morbidity and mortality in immunocompromised patients. With the introduction of protease inhibitors (PIs) in highly active antiretroviral therapy (HAART) the incidence of cryptosporidiosis in AIDS patients has declined substantially in western countries. We have therefore tested the effect of five PIs which are used in HAART on the excystation, the invasion and the development of the parasite in a cell culture system. The human ileocecal adenocarcinoma cell line HCT-8 served as a host cell. None of the PIs had an

81

Scientific Program DGP, 21st Annual Meeting March 17 th -- 20 th, 2004 in W0rzburg

effect on the excystation rate and only nelfinavir showed a moderate but statistically significant inhibition on the host cell invasion during a time period of 2 h (43% at 33 IJg/ml). More pronounced inhibitory effects could be found when PIs were present over the total time of intracellular development (48 h). Indinavir, nelfinavir and ritonavir led to statistically significant inhibition of parasite development (means: 51%, 23% and 34%, at 200, 10, and 10 IJg/ml, respectively). In combination with the aminoglycoside paromomycin, the PIs indinavir, ritonavir and to a lesser extent saquinavir showed an increased inhibitory effect (94%, 88%, and 56%, respectively) compared to the PIs or paromomycin alone. Preliminary in vivo experiments support the in vitro results. IL-12p40 knock-out mice were infected with C. parvum and treated with indinavir, paromomycin and a combination of both. Parasite shedding was reduced by up to 80% compared to non treated control mice. The data presented here suggest a direct action of certain PIs on C. parvum. Furthermore, an additive inhibitory effect of PIs in combination with paromomycin was seen which supports the clinical and microbiological findings in patients where such combinations are used.

Effects of b isphosphonates on the growth of Entamoeba histolytica in vitro and in vivo Bruchhaus, I. 1 t, Nozaki, T. 2, Meints, G. A?, Oldfield, E. 3 1Molecular Parasitology; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 2National Institute of Infectious Diseases; Department of Parasitology; Tokyo; Japan 3Department of Chemistry; University of Illinois at Urbana-Champaign; Illinois; United States

The effects of a series of 102 bisphosphonates on the inhibition of growth of Entamoeba histolytica have been determined, and selected compounds were further investigated for their in vivo activity. 47 compounds tested were active (IC50 < 200 IJM) versus E. histolytica growth in vitro. Several of the most promising compounds in in vitro inhibition appear to be those with biphenyl, n-alkyl, arylalkyl, and phenoxyalkyl sidechains. The most active compounds (IC50 N4-9 IJM) were nitrogen-containing bisphosphonates with relatively large aromatic sidechains. We also found an unusual pattern of reactivity with the bisphosphonates in that many potent nitrogen containing species (such as pamidronate and risedronate) used as farnesylpyrophosphate synthase (FPPS) inhibitors in bone resorption therapy had relatively little activity against E. histolytica proliferation, in vitro, while most of the active compounds had low activity versus FPPS. The activity against E. histolytica is dependent on its chain length, with optimal activity being found for a ~C9-C10 chain length. Several pyridine- derived bisphosphonates were quite active (IC50 N10-20 IJM), and this activity was shown to correlate with the basicity of the aromatic group. The activities of all compounds were tested versus a human nasopharyngeal carcinoma (KB) cell line to enable an estimate of a therapeutic index (T.I.). Five bisphosphonates were selected and then screened for their ability to delay the development of amebic liver abscess formation in an E. histolytica infected hamster model. Two compounds were found to decrease liver abscess formation at 10 mg/kg i.p. with little or no effect on normal liver mass. Taken together, these results show that bisphosphonates appear to be useful lead compounds for the development of novel anti-amoebic drugs.

The T r y p a n o t h i o n e - d e p e n d e n t glyoxalase I I as drug target in afr ican Trypanosomes Irsch, T. 1 t, Krauth-Siegel, R. L. ~ ~Biochemie-Zentrum Heidelberg; Universit~t Heidelberg; Heidelberg; Germany

The glyoxalase system, composed of glyoxalase (GLX) I and II, catalyzes the glutathione-dependent conversion of 2-oxoaldehydes into the corresponding 2-hydroxyacids. The predominant function of the ubiquitous pathway is the detoxication of the cytotoxic and mutagenic methylglyoxal that is mainly formed as a by-product of glycolysis. Inhibitors of the glyoxalase system have been designed as potential drugs against tumors and malaria. The dependence of bloodstream African trypanosomes on glycolysis as sole energy source prompted us to characterize the parasite glyoxalases as probable new targets for an antitrypanosomal drug development. A gene has been cloned from T. brucei that encodes a protein of 296 amino acid residues. The deduced protein sequence shows the highest degree of similarity with putative proteins from T. cruzi and L. major where 66% and 51%, respectively of all residues are identical. In the functionally characterized human and A. thaliana GLX II 36% and 31% of the residues are conserved. The parasite enzyme catalyzes the hydrolysis of lactoylglutathione, generated by the GLX I reaction, at about 10% of the rate observed with bovine liver GLX II. In contrast to the mammalian enzyme, T. brucei GLX II does not show saturation kinetics up to 5 mM lactoylglutathione. Trypanosomatids have an unparalleled thiol metabolism based on trypanothione [bis-(glutathionyl)spermidine] instead of the ubiquitous glutathione. Trypanothione is the donor of reducing equivalents for the synthesis of DNA precursors as well as the detoxication of organic hydroperoxides. To elucidate if the parasite specific dithiol is also involved in the deto×ication of methylglyoxal, lactoyltrypanothione has been synthesized. T. brucei glyoxalase I I - but not the mammalian enzyme - readily hydrolyses the thioester yielding D-lactate and regenerating trypanothione. Silencing of the gene by RNAi is in progress to reveal if the enzyme is essential for T. brucei.

82


I r revers ib le inhibit ion of t rypanothione reductase by unsaturated mannich bases Lee, B}, Bauer, H. 1, Davioud-Charvet, E. ~, Krauth-Siegel, R. L} t 2Biochemie-Zentrum Heidelberg; Universit~t Heidelberg; Heidelberg; Germany

Trypanothione reductase (TR) is a flavoenzyme unique to trypanosomal parasites. By maintaining trypanothione, T(SH)2, in its reduced form, TR plays a crucial role in the thiol redox metabolism of the parasites. Decreased TR levels cause oxidative stress and with less than 10% of the wild type TR activity, trypanosomes are unable to grow. Due to these findings and the structural differences between the parasite and host enzymes, TR is a promising target for drug development against African sleeping sickness and Chagas' disease. The trypanothione disulfide binding site of TR is negatively charged and contains three key residues: the redox active Cys53-Cys58 couple and His461 which acts as a proton donor/acceptor. Based on this knowledge and the crystal structure, a series of unsaturated Mannich bases were designed and studied as potential inhibitors of Trypanosoma cruzi TR. Incubation of the enzyme with the compounds in the presence of NADPH caused an irreversible inhibition indicating that one of the active site cysteines had been modified. Surprisingly, the inhibitory potency of the Mannich bases increased upon storage of the DMSO stock solution. To study this phenomenon in more detail, probable common intermediates were synthesized. A cyclopentenone derivative proved not to be an inhibitor of the enzyme. In contrast, a divinylketone was a highly reactive irreversible inhibitor of TR. These results, together with spectroscopic data, MS analysis and HPLC experiments suggest that the divinylketone derivative which is generated as a common product is responsible for the inhibition of TR by different Mannich bases.

DNA topoisomerase inhibitors as ant i - trypanosomal drugs Steverding, D. ~ t, Deterding, A. ~, Dungey, F. A. l, Thompson, K.-A. ~ ~School of Biological Sciences; University of Bristol; Bristol; United Kingdom

DNA topoisomerases are relevant targets for anti-tumour and anti-bacterial agents. However, drugs developed as potential anti-cancer agents could also be of use against African sleeping sickness. Therefore, the trypanocidal activities of 14 different DNA topoisomerase inhibitors, including drugs currently used in chemotherapy, were investigated against in vitro-cultured bloodstream forms of Trypanosoma brucei. All compounds exhibited anti-trypanosomal activities with 50% effect dose (EDso) and minimum inhibitory concentration (MIC) values varying from 3 nM to 30 pM and from 100 nM to >100 pM, respectively. The most trypanocidal DNA topoisomerase inhibitors were aclarubicin and mitoxantrone with EDso values of 3.5 nM and 3 nM and MIC values of 100 nM and 10 pM, respectively. On the other hand, the DNA topoisomerase inhibitors exhibited varying cytotoxicities towards human HL-60 cells and therefore less favourable selectivity indices compared to commercially available drugs. However, as the human HL-60 cells are malignant cells, the cytotoxicity of DNA topoisomerase inhibitors is probably overestimated in comparison with normal mammalian cells. In conclusion, the data support the potential of DNA topoisomerase inhibitors for rational antitrypanosomal drug development.

Targeting enzymes involved in spermidine metabolism of parasitic protozoa-a possible new strategy for antiparasit ic t reatment Andrea, G. 1, Hamels, I } , H6rauf, A. 1, Kaiser, A. 1 t ~Institut for Medical Parasitology; UNI BONN; BONN; Germany

Sequencing data obtained from the Plasmodium, Anopheles gambiae and human genome projects provide a new basis for drug and vaccine development. One of the most characteristic features in the process of drug development against parasitic protozoa is target identification in a biological pathway. The polyamine pathway might be an important resource for new drug targets. Here, we present data that pinpoint the existence of two enzymes of the polyamine pathway involved in spermidine metabolism in P. falciparum, i.e. deoxyhypusine synthase (DHS; EC 1.1.1.249) and homospermidine synthase (HSS; EC 2.5.1.45). Recent data obtained from the malaria genome databases showed that at least a putative gene encoding DHS is present in the parasite. Sequencing data from the P. falciparum genome project prove that the eukaryotic initiation factor eIF5A (the substrate for DHS) exists in P. falciparum. Here, we present the amino acid sequence of eIF5A from P. vivax, which causes tertiary malaria. EIF5A from P. vivax shows 82% nucleic acid and 97% amino acid identity to its homologue from P. falciparum. GC/MS data and inhibitor studies with agmatine prove that the triamine homospermidine occurs in the parasite. These data suggest a separate locus encoding HSS in P. falciparum. The hss gene recruits from the dhs gene in eukaryotes. Here, we present genomic DNA fragments obtained by amplification with primers of a conserved region (amino acid positions 550-1,043) between the putative P. falciparum DHS gene (dhs) and the HSS gene (hss) from the plant Senecio vulgaris (Asteraceae). The amplification product from different P. falciparum strains reveals differences in sequence identity, compared with the putative dhs gene from P. falciparum strain 3D7. Expression of the full-length clone and determination of HSS-specific activity will finally prove whether a separate region encoding HSS exists.

83


Virtual screening for novel kinase inhibitors as potential drugs for the t reatment of parasitic diseases Beyer, C. 1 ~, Cramer, 3.1, Seizer, P. M~ 1 1BioChemInformatics; Intervet Innovation GmbH; Schwabenheim; Germany

The rapid development of high performance computing systems and the decreasing costs of hardware components allow the combination of these technologies in structure based drug design. A promising approach for this application is the networked deployment of common personal computers resembling a virtual supercomputer controlled by grid computing software. An example of a computationally intensive application is the molecular docking used for structure based rational drug design. Common to all molecular docking approaches is the prediction of protein-ligand interactions, for example the binding of a small chemical compound to a target protein. In virtual screening, however, multiple dockings of large compound libraries are performed against a target protein. The aim of this project is to establish and utilize a grid computing environment for the virtual screening of parasitic target proteins. Cyclin-dependent kinases (CDKs) are key molecules in the cell cycle. Therefore, they are prominent target proteins in the field of cancer and parasitic diseases. CDKs of the protozoan parasite Eimeria tenella, which causes coccidiosis in chickens, have been identified from genomic and EST sequence data. Based on these sequences, homology protein models were built using various X-ray crystal structures of human CDK 2 as templates. Virtual screening of a compound library on these models was then performed using grid computing as described above. The result of this screening was a set of novel potential chemical inhibitors of Eimeria tenella CDKs.

Interact ions of methylene blue with the glutathione redox system of Plasmodium falciparum in vitro Eubel, J.~, Coulibaly, B. 2, Davioud-Charvet, E. 1, Becker, K. 3, Schirmer, R. H} t 1Biochemie-Zentrum Heidelberg; Universit~t Heidelberg; Heidelberg; Germany 2Centre de Recherche en Sant# de Nouna; CRSN; Nouna; Burkina Faso 3Interdisziplin~res Forschungszentrum; Universit~t Giessen; Giessen; Germany

In the form of BlueCQ (methylene blue + chloroquine), methylene blue (MB, ref.1) is currently tested as an affordable antimalarial drug Combination in clinical trials that are conducted at Nouna, Burkina Faso (2). One target of MB is P. falciparurn glutathione reductase which is inhibited non-competitively. Glutathione reductase catalyzes the reaction NADPH + GSSG + H + = NADP + + 2 GSH, where GSSG and GSH denote glutathione disulfide and glutathione monothiol, respectively. The binding site of MB is probably the drug-binding pocket between the two subunits of the enzyme (3). In addition, MB is a subversive substrate of glutathione reductase: it is reduced to leukoMB at the expense of NADPH. The reaction between MB and NADPH also occurs spontaneously, the second order rate constant being 6.5 M-1 s-1 at 37°C and pH 7.3. NADH was found to have a similar reactivity. As reported by Kelner and Alexander (1985) MB can be reduced by GSH (4) but so far we could not reproduce this addition-replacement reaction at a measurable rate. Discussed are advantages of pleiotropic agents as antiparasitic drugs.

(1) Guttmann P, Ehrlich P (1891) 0ber die Wirkung von Methylenblau bei Malaria. Berl Kiln Wochenschr 28, 953-956

(2) Schirmer RH, Coulibaly B, Stich A, Scheiwein M, Merkle H, Eubel J, Becker K, Becher H, MQller O, Zich T, Schiek W, Kouyat6 B (2003). Methylene blue as an antimalarial agent. Redox Report 8, 272-275

(3) Sarma GN, Savvides SN, Becker K, Schirmer M, Schirmer RH, Karplus PA (2003) Glutathione reductase of the malarial parasite Plasmodium falciparum: Crystal structure and inhibitor development. J Mol Biol 328, 893- 907

(4) Kelner MJ , Alexander NM (1985) Methylene blue directly oxidizes glutathione without the intermediate formation of hydrogen peroxide. J Biol Chem 260, 15168-15171

The threedimensional structure of a Mu-class related glutathione S-transferase from Plasmodium falciparum Perbandt, M. 1, Burmeister, C. 2, Walter, R. D. 2, Betzel, C. 1, Liebau, E. 2 t 1DESY; University of Hamburg; Hamburg; Germany 2Biochemistry; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany

Glutathione S-transferases (GSTs, EC 2.5.1.18) catalyse the nudeophilic addition of glutathione to electrophilic substrates, thereby detoxifying a wide variety of both endobiotic and xenobiotic compounds. Furthermore, different members of this superfamily have peroxidase and isomerase activity, bind endogenous and exogenous ligands non-catalytically and inhibit the Jun N-terminal kinase and thereby protect the cell against H202-induced cell death. Although the utility of the glutathione S-transferase from P. falciparurn (Pf-GST1) is yet to be established, the critical role played by Pf-GST1 in detoxification makes it a viable drug target against malaria. The structure of the Pf-GST1 without glutathione and in complex with its inhibitor S-hexyl-glutathione has been determined by X-ray diffraction analysis. The resolved structure shows that the Pf-GST1 has several structural

84


features incommon with the Mu-class GSTs and is therefore closely related to this class, even though alignments with its members display identities of less than 35 %. The comparison of the unligated with the

ligated enzym revealed that the active centre remains unchanged upon S-hexyl-glutathione binding. A detailed comparison of the parasitic enzyme with the human host Mu-class demonstrates that, although the overall structure is similar, the shape of the hydrophobic binding pocket differs substantially. Whereas the space for voluminous substrates is limited in the human enzymes, the additional space between the Pf-GST1 Mu-loop and the C-terminus may be exploited in the design of alternative parasite-specific selective inhibitors.

The proteasome inhibitor MLN-273 inhibits cell cycle progression in Plasmodium falciparum parasites Lindenthal, C. ~, Weich, N. 2, Klinkert, M. Q.3, ZInstitute for Tropical Medicine; University of TObingen; TObingen; Germany 2Inflammation; Millenium Pharmaceuticals Incorporation; Cambridge, Massachusetts; United States 3Department of Molecular Medicine; Bernhard-Nocht Institute for Tropical Medicine; Hamburg; Germany

Protein degradation is regulated during the cell cycle of all eucaryotic cells and is mediated by the ubiquitin- proteasome pathway. Specific and reversible inhibitors of the 20S proteasome have been developed recently as anti-cancer agents, since they induce apoptosis in rapidly dividing cells. We have tested MLN-273, a dipeptidyl boronic acid proteasome inibitor, belonging to this potent and highly specific class of molecules on in vitro cultures of Plasmodium falciparum laboratory-adapted strains. MLN-273 was found to be an effective inhibitor of P. falciparum growth in vitro, blocking the development of the erythrocytic forms of P. falciparum at an early ring stage. Importantly, uninfected erythrocytes were not affected by the drug.

A chloroquine eff lux pump is genetical ly linked with pfcrt and chloroquine resistance in Plasmodium falciparum McLean, J.1 t, Sanchez, C. ~, Stein, W. 2, Lanzer, M. ~ ZAbteilung Parasitologie, Hygiene-Institut; Universit~tsklinikum Heidelberg; Heidelberg; Germany 2Biological Chemistry, Silberman Institute of Life Sciences; Hebrew university of Jerusalem; Jerusalem; Israel

The rapid spread of resistance to chloroquine in Plasmodium species was one of the major factors that led to the current upsurge in malaria morbidity and mortality. Despite the central importance of chloroquine, there is still no consensus on the parasite's mechanism of resistance to it. Through observation of chloroquine accumulation under trans-acceleration conditions we have found evidence for a pump that expels chloroquine from the parasite. This pump is both energy-dependent and saturable. We have further linked the activity of the pump to polymorphisms within the gene pfcrt, a key determinant of chloroquine resistance. Experiments with parasites that have had their pfcrt locus allelically exchanged show that the presence of an active pump is a necessary requirement for resistance. Polymorphisms in another gene weakly linked to chloroquine resistance, pfmdrl, have no effect on the action of the pump suggesting that factors other than a pump may play roles in determining the extent of chloroquine resistance.

Functional analysis of the P. falciparum chloroquine resistance determinant pfcrt in Xenopus oocytes Nessler, S. i t , Friedrich, 0. 2, Planelles, G. 3, Sanchez, C. 1, Lanzer, M. 1 IAbteilung Parasitologie, Hygiene-Institut; Universit~tsklinikum Heidelberg; Heidelberg; Germany 2Medizinische Biophysik, Institut for Physiologie und Pathophysiologie; Universit~t Heidelberg; Heidelberg; Germany 3Institut National de la Sante et de la Recherche Medicale, Unite 467; Universite Paris V; Paris; France

Chloroquine was the first line antimalarial drug for more than three decades. Widespread chloroquine resistance (CQR) in the malarial parasite Plasmodium falciparum and the absence of other drugs suitable for application in developing countries has contributed to dramatic increases in malaria mortality. Point mutations in the novel membrane protein PfCRT are genetically linked with CQR. Epidemiological surveys were also able to associate pfcrt polymorphisms with clinical treatment failure. The PfCRT protein is localized to the digestive vacuole membrane of the intraerythrocytic parasite and contains ten predicted transmembrane domains.

To study the biological function of this protein we have expressed both a wildtype pfcrt allele and a chloroquine resistance associated pfcrt allele in Xenopus laevis oocytes. As the high AT content of malarial genes prevents stable cloning and heterologous expression, we have synthesized both alleles following the yeast codon usage. The expression of PfCRT in the membrane of Xenopus oocytes was demonstrated by Western analysis and immunofluorescence. Our data show that pfcrt expressing oocytes have an increased Ca 2+ extrusion activity. This leads to a dramatic reduction in free intracellular Ca 2+ and the subsequent activation of several Ca 2+- sensitive endogenous transport systems including a non-selective cationic conductance and an Na+/H + exchanger. The rate of Ca 2+ extrusion differed depending on the pfcrt allele expressed and was highest in oocytes expressing the pfcrt allele associated with chloroquine resistance. We propose that the pfcrt polymorphisms affect intracellular Ca 2+ homeostasis in P. falciparum. This discovery has major implications for overcoming chloroquine resistance in malaria.

8G


Carboxylic acid derivatives of menadione of Plasmodium falciparum glutathione reductase inhibitors and prodrugs Bauer, H}, Blot, C. 2, Schirmer, R. H}, Davioud-Charvet, E} t 1Biochemie-Zentrum Heidelberg; Heidelberg University; Heidelberg; Germany 2Ing~nierie Mol~culaire et Biomol~culaire; Universit# Libre de Bruxelles; Brussels; Belgium

Due to rising resistances, new drugs against malaria are needed continuously. Plasmodium parasites are exposed to elevated fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of thiols which are regenerated by disulfide reductases; these include the glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and of human erythrocytes as well as the thioredoxin reductase of P. falciparum. The aim of our interdisciplinary research is to substantiate inhibitors of the glutathione reductases from parasite and host erythrocyte as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in the parasites. Reversal of drug resistance by GR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine. The development of menadione chemistry has led to the selection of a series of carboxylic acids - the most potent inhibitor of the parasitic enzyme being the 6-(3-methyl- l ,4-dioxo-l,4-dihydro-naphthalen-2-yl)- hexanoic acid M5 - and to the preparation of double-headed prodrugs which were found to be active also in vivo. The weak antimalarial activity observed with M5 was correlated with low membrane permeation due to the presence of the carboxylate function which is, however, essential for recognition by GR. Bioisosteric displacement of the carboxylic acid with tetrazole to provide improved bioavailability and comparable acidity led to increased antimalarial properties, but only with the cyanoethyl-protected tetrazoles. This suggests a pro- drug effect occurring in infected red blood cells. Mechanistic studies on the hexanoic acid derivative M5 as enzyme inhibitor revealed an uncompetitive inhibition type with respect both to NADPH and to glutathione disulfide. The potential binding site of M5 will be discussed in light of the recently resolved 3D structure of P. falciparum GR. Moreover, a recent finding which shows that M5 is reduced in the absence of the disulfide substrate prompted us to prepare bioreductive alkylating agents based on the M5 structure as suicide- substrates. Such compounds are useful to identify the site where M5 reduction takes place and to improve glutathione depletion in vivo.

Series of novel polyamine synthesis inhibitors show an antiprol i ferat ing effect on Plasmodium falciparum Das Gupta, R. 1 t, Krause, T. 1, Khomutov, A. 2, M011er, S, 3, L0ersen, K. ~, Walter, R. D.1 ~Biochemistry; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 2Engelhardt Institute of Molecular Biology; Russian Academy of Sciences; Moscow; Russia 3Division of Biological Chemistry and Molecular Microbiology; University of Dundee; Dundee; United Kingdom

Eukaryotic cells have the ability to synthesize the natural polycations putrescine, spermidine and spermine. Their intracellular levels are closely related to important processes like cell growth, cell proliferation and stabilization of DNA and RNA. It is assumed that inhibition of polyamine synthesis is a suitable therapeutic strategy against parasitic infections. In Plasmodium falciparum, the key enzymes of the polyamine synthetic pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are combined in a bifunctional protein. This unique bifunctional property differs from their monofunctional counterparts in other organisms including man and therefore offers a promising target for chemotherapeutic treatment. We investigated the effect of a number of new inhibitors against both - ODC and AdoMetDC - on enzyme activity, growth rate and polyamine pattern of P. falciparum in culture. The most effective ODC inhibitor 1-aminooxy-3- aminopropane (APA) inhibits ODC activity with a Ki value of 8 nM and blocks parasite proliferation with an IC50 value of 2 MM. Reversed phase HPLC analysis revealed that this compound affects the polyamine levels of the parasites remarkably.

U I S 2 , a new Plasmodium candidate for mult istage drug target ing Sayed Ibrahim Aly, A. 1 t Janse, C. 2, Waters, A. p.2, Matuschewski, K. 1 1Department of Parasitology; Heidelberg University School of Medicine; Heidelberg; Germany 2Malaria Group, Department of Parasitology; Leiden University Medical Centre; Leiden; Netherlands

UIS2 encodes a metallo-phosphatase with a putative signal sequence and is conserved among all Plasmodium species. A structurally related family of acid phosphatase ectozymes is discussed as a virulence factor for pathogenic bacteria, such as Francisella tularensis, and for other eukaryotic parasites, for instance Leishmania donovani. The presence of the metallo-phosphatase domain in numerous pathogenic microorganisms suggests that UIS2 may provide an attractive target for rational drug design. We identified UIS2 initially as a transcript that is highly upregulated in infectious salivary gland sporozoites of Plasmodium berghei. To study the UIS2 gene function we employed a reverse genetics approach. A series of knock out experiments using alternative targeting strategies revealed that UIS2 most likely performs vital functions during erythrocytic schizogony. These findings were supported by expression studies: UIS2 transcripts can still be detected in the erythrocytic stages and appear upregulated in late schizonts, albeit at much lower level compared to sporozoites. These findings raise the possibility that UIS2 functions at different mammalian

86


cell invasive stages during the Plasmodium life cycle. Currently we are attempting additional gene targeting strategies to verify that UIS2 is an essential gene.

I n vitro effect mannosamine on glycosylphosphatidylinositol synthesis in Plasmodium falciparum Santos de Macedo, C}, Schwarz, R. T. ~ t, Azzouz, N. ~ qnstitut for Virologie - Med. Zentrum for Hygiene; Philipps-Universit~t Marburg; Marburg; Germany

In vivo effects of sugar analogues (2-deoxy-2-fluoro-D-glucose, 2-deoxy-D-glucose and D-mannosamine) on GPI biosynthesis in P. falciparum were recently described (Santos de Macedo et al., 2001). In contrast to mammalian cells and other parasitic protozoa where mannosamine was shown to inhibit GPI biosynthesis by being incorporated into the trimannosyl core glycan of GPIs leading to the accumulation of an aberrant GPI biosynthetic intermediate (mannosamine-mannose-glucosamine-phosphatidylinositol), it is not able to inhibit GPI biosynthesis in P. falciparum. We also observed that mannosamine is converted irreversibly at a high rate into glucosamine in vivo. However mannosamine was incorporated in lipids no GPIs. As mannosamine seemed not to be incorporated in vivo into GPIs in P. falciparum, we hypothesize that ManN would be incorporated into dolichol to form DoI-P-ManN and then be transfered into lipids. Therefore, we developed a cell free system to investigate in vitro the incorporation of radiolabelled mannosamine into glycolipids in P. falciparum. In this system, parasite membranes were incubated in the presence of radiolabelled mannosamine, DoI-P, ATP and GTP. After incubation, lipids were extracted and analysed by thin layer chromatography (TLC) and DEAE- cellulose anion exchange chromatography. The analyses showed labelled hydrophobic glycolipids with

mannosamine and that the glycolipids possess a phosphate group. Chemical (nitrous acid deamination, mild acid hydrolysis and alkaline hydrolysis) and enzymatical (PI-PLC) treatments of tritiated mannosamine-labelled glycolipids synthesized in vitro showed the presence of lipids with the chemical behaviour of GPIs. Further analyses by Bio-Gel P4 size-exclusion chromatography and high-pH anion exchange chromatography (HPAEC) suggested the presence of a mannosamine-containing Man3 GPI-like structure. Further analysis showed that mannosamine was incorporated in the place of glucosamine in the GPI structure.

Santos de Macedo, C., Gerold, P., Jung, N., Azzouz, N., Kimmel, J. and Schwarz RT. (2001) Eur. J. Biochem. 268, 6221-6228.

Ultrastructural studies on Wolbachia-endobacteria in Onchocerca ochengi after tetracycl ine t rea tment Plappert, S. 1 t, AIfons, R. 2, Trees, S?, Mackenstedt, U. 1 1Fachgebiet Parasitologie; Universit~t Hohenheim; Stuttgart; Germany 2InsUtut for Zoophysiologie, AG Parasitologie; Universit~t TObingen; TObingen; Germany 3Liverpool School of Tropical Medicine and Faculty of Veterinary Science; University of Liverpool; Liverpool; United Kingdom

The African bovine parasite Onchocerca ochengi is a nodule-dwelling filarial nematode. Due to the similarities of this parasite with O, volvulus, the causative agent of river blindness, O. ochengi has been used in numerous chemotherapy trials. Recent studies on O. ochengi showed that the antibiotic oxytetracycline has promising macrofilaricidal properties, due to its effects on endobacteria of the genus Wolbachia. Wolbachia bacteria belong to the alpha-proteobacteria and are very common endosymbionts of filarial nematodes, insects, mites and crustaceans. The bacteria are thought to play an essential role in the metabolism of most filarial nematodes and may be the responsible cause for the immunopathology of river blindness.

The aim of this study was to investigate the effect of oxytetracycline-treatment on the endobacteria as well as the effect of the elimination of the endosymbionts on the worm tissues. Adult O. ochengi were removed from bovines at several points of time during prolonged chemotherapy and were subsequently examined using electron-microscopy techniques. In this poster we show the ongoing degeneration and elimination of the endobacteria and the resulting effect of the treatment on the hypodermis and the intra-uterine developmental stages of adult O. ochengi.

I n vitro efficacy of antimicrobials against Balamuthia mandril laris, free- l iving ameba and opportunistic agent of encephalit is Tata, P. S. 1, Radam, E. 1, Laube, U. ~, Kayser, 0. 2, Kiderlen, A. F. ~ t 1Abteilung for Infektionskrankheiten; Robert Koch-Institut; Berlin; Germany 2Institut for Pharmazie - Pharmazeutische Technologie; Freie Universit~t Berlin; Berlin; Germany

Balamuthia mandrillaris was first described 1990 as an opportunistic causative agent of lethal granulomatous amebic encephalitis (GAE) in man and other mammalian species. The mechanisms of pathogenesis, criteria for an early diagnosis and a reliable therapy remain unsolved. The in vitro data from Schuster [1] reveal sensitivity of B.mandrillaris ameba for some pharmaceuticals in terms of proliferation over long incubation periods. However, little is known regarding the amebacidal effect of drug action in the first 24 hours. As most of the

87


drugs are rapidly metabolized in the body to functionally inactive substances, the amebacidal drugs seem preferable over amebastatic ones. In our study, a panel of more than 40 established and novel pharmaceutical agents were tested for inhibition of the cytolytic activity of B.mandrillaris. The assay proved useful as a highly sensitive, reliable and time-saving means of screening drugs for effects on this ameba. The assay is based on the release of bacterial 6- galactosidase (6-gal) as reporter enzyme from stably transfected murine mastocytoma P815 target cells. In intact cells, 6-gal accumulates in the cytoplasm. Elevated concentrations of 6-gal in the supernatant, measured as conversion of a luminescence substrate, indicate lysis and death of the cells. Balamuthia ameba are spontaneously cytopathic for eukaryotic cells. This can now easily be measured as 6-gal release by affected P815(6-gal) target cells. A drug that is toxic for B.mandrillaris is very likely to affect its cytolytic potency, either by direct kill or by physiological inhibition. - Some of the drugs (e.g. amphotericin B, pentamidine isethionate, emetine, an aphidicoline-derivative) were highly active, even at concentrations below 5 IJM - Amphotericin B formulated as nanosuspension was the most active, also in comparison to other AraB formulations (liposomes, colloidal dispersions)

[1] Sch~fer H et al. (1990) J Immunol Methods 204:89

[2] Schuster FL, Visvesvara GS (1998) J Euk Microbiol 45:612

A screen for genes t h a t can confer resistance to mi l te fos ine in Leishmania Choudhury, K. 1 t, Clos, J.~ ZParasitology Section; Institute for Tropical Medicine; Hamburg; Germany

Miltefosine was first developed as an anti-cancer drug and has proven to be an effective, alternative drug against visceral leishmaniasis (VL).It was recently approved for treatment of VL in India. Resistance to miltefosine has been observed in carcinoma cell lines that express multidrug resistance genes. Future use of miltefosine against VL raises the risk of clinical resistance in leishmaniasis. Therefore, it is important to understand possible mechanisms of resistance to miltefosine in Leishmania . Our aim is to identify gene(s) that mediate miltefosine resistance by using a genetic complementation strategy. L. donovani and L. infantum promastigotes were transfected with cosmid DNA from L. donovani and L. infantum genomic DNA libraries. Recombinant promastigotes were selected using miltefosine. Cosmids from resistant parasite clones were isolated, transformed into E. coil and identified according to their restriction fragment patterns. Four cosmids each from the L. donovani (pcosLdM1-M4) and from the L. infantum selections (pcosLiM1-M4) were found dominant in the survivors, pcosLiM1-M4 were retransfected into L. infantum and selected under miltefosine a second time to verify their ability to confer resistance. Subsequent analysis of the cosmid DNA revealed that only pcosLiM1 and pcosLiM2 were dominant in the reisolates after two rounds of selection. These two cosmids overlap in part, indicating that they share the same gene(s) that confer resistance. Parasites harbouring these cosmids show significantly increased IC50 for miltefosine. Sequence analysis of pcosLiM1 and pcosLiM2 and of the cosmids derived from L. donovani is under way.

T r y p a n o t h i o n e - d e p e d e n t perox idases in afr ican T r y p a n o s o m e s Schlecker, T. 1 t, Voncken, F. 2, Clayton, C. E. 2, Krauth-Siegel, R. L. 1 IBiochemie-Zentrum Heidelberg; Universit~t Heidelberg; 69120 Heidelberg; Germany 2Zentrum f~r Molekularbiologie Heidelberg; Universit~t Heidelberg; 69120 Heidelberg; Germany

Glutathione peroxidases are selenoproteins which catalyze the reduction of hydroperoxides at the expense of glutathione. In Trypanosoma brucei, a genomic locus encodes three nearly identical cysteine homologues of these classical glutathione peroxidases. One of the proteins, PXIII, carries a mitochondrial as well as a glycosomal targeting signal. Polyclonal rabbit antibodies against PXIII detect a single protein band in extracts of both the mammalian and the insect stage and localize the protein in the cytosolic, mitochondrial and glycosomal fraction of procyclic 7". brucei. Recombinant PXIII uses the trypanothione/tryparedoxin couple instead of glutathione as electron donor for the reduction of H2Oz, t-butyl hydroperoxide and cumene hydroperoxide (Hillebrand et al. 2003). Recently we could show that thymine hydroperoxide is a probable physiological substrate of the parasite enzyme. In contrast, phosphatidylcholine hydroperoxide which is reduced by the related T. cruzi peroxidase I is not a substrate but inhibits the T. brucei enzyme. RNAi experiments were performed to elucidate if the peroxidases are essential for the parasites. Bloodstream and procyclic T. brucei were transfected with the vector pHD678 which contained two copies of the gpx gene in opposite directions under control of a tetracycline-inducible PARP promotor. Northern blot analysis yields a prominent mRNA of about 900 bl5 and a faint band of about 1300 bp in bloodstream and procyclic cells. In the presence of tetracycline both mRNAs are degraded, expression of the proteins is down-regulated to less than 5% and cell growth is significantly impaired in bloodstream as well as procyclic 7-. brucei. These data clearly show that the peroxidases play an essential role in both developmental stages of the parasite. Work is in progress to crystallize T. brucei PXIII and to identify other physiological substrates of the enzyme.

88


Identification of cysteine proteases of intraerythrocytic life stages of Plasmodium falciparum by a biotinylated small-molecule probe Schirmeister, T. ~ t, Gelhaus, C}, Vicik, R. ~, Leippe, M. 2 ~Pharmazie und LMC; Universit~t WElrzburg; WOrzburg; Germany 2Zentrum for Infektionsforschung; Universit~t WOrzburg; WElrzburg; Germany

Cysteine proteases of Plasmodium falciparum have been shown to be essential for survival of the malaria parasite. They have been implicated in several cellular functions, including hemoglobin degradation, host cell rupture with concomitant release of parasites from the parasitophorous vacuole, and host cell invasion. Hence, these proteases offer potential new chemotherapeutic targets. Plasmodium falciparum expresses three papain- family cysteine proteases, known as falcipains. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites appears to be larger than was previously realized. Epoxysuccinylpeptides and analogous aziridine derivatives are well-known inhibitors of cysteine proteases. They irreversibly block enzyme activity by alkylation of the cysteine residue within the active site. To identify cysteine proteases of intraerythrocytic life stages of P. falciparum, we synthesized a biotin-labeled cysteine protease inhibitor containing the electrophilic aziridine-2,3-dicarboxylic acid unit. The inhibitor showed antiplasmodial activity. We could show that the target proteases are localized within the food vacuole of the trophozoite and in the host cell cytosol. Accumulation of undigested materia, presumably globin, is observed. 2D-PAGE showed several affinity- labeled spots which are being characterized by mass spectrometry. One of them already has been identified as falcipain 2.

Genomics & Proteomics

Developmental regulation of mitochondrial targeted proteins in I.eishmania donovani Harder, S} t, Bente, M. ~, Krause, E}, Bruchhaus, I. ~ 1Molecular Parasitology; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 2Institute of Molecular Pharmacology; Berlin; Germany

Parasites of the genus Leishmania appear in two distinct life cycle stages: amastigotes and promastigotes. Promastigotes, the developmental and infective stage are present only in the insect vector, phlebotomine flies. They are inoculated into the vertebrate host during a bloodmeal and convert into the amastigote form, replicating within the phagolysosomes. With the help of a proteomic approach we analysed this transformation which is essential for the parasite's survival. By using 2D-gel electrophoresis we observed defined changes in the proteome pattern. Altogether 44 different proteins were shown to be amastigote-specific. 34% of them contain a mitochondrial targeting sequence, as e.g. a peroxiredoxin, a Rieske iron-sulfur protein, an ATPase 6- subunit and several unknown proteins. Therefore, it is likely that the Leishmania mitochondrion is involved in the differentiation process. Peroxiredoxins are found in different organisms, dedicated diverse functions, including general cell detoxification and signalling in proliferation as well as differentiation processes. Furthermore they are crucial for defence against oxidative stress. Whereas, the differential expression for this protein was confirmed by Western blot analysis using a specific antibody raised against the recombinant protein, the mRNA amount is not increased during transformation. Electron microscopic studies show that the peroxiredoxin, as predicted from the deduced amino acid sequence, is Iocalised in the mitochondrion. Low amounts of the protein are found in promastigotes in the kinetoplast area whereas in amastigotes the amount increases and is distributed over the whole mitochondrion. An increase in protein synthesis is thus accompanied by a change of Iocalisation within the organelle. Generating deletion mutants and investigating how the lack of this specific protein effects the parasites shall allow further characterisation of the mechanisms of stage differentiation and the involvement of the mitochondrion in this process.

Identification of stage-specific kinases in Leishmania donovani using a proteomic approach Bente, M. 1 t, Harder, S. 1, Krause, E. 2, Bruchhaus, I } ~Molecular Parasitology; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 2Institute of Molecular Pharmacology, Berlin; Berlin; Germany

To pass through its life cycle protozoan parasites of the genus Leishmania have to develop from the flagellated promastigote vector stage to the intracellular amastigote stage present in mammals. This transformation is a complex differentiation process requiring the controlled expression and regulation of various genes. Using a proteomic approach (a combination of 2-D gel electrophoresis and MALDI TOF MS) the in vitro induced stage differentiation of L. donovani was investigated. In this context we identified promastigote-specific as well as amastigote-specific proteins involved in phosphorylation of proteins/nucleotides: adenylate kinase, aldolase-epimerase related protein, P-type ATPase, thymidine kinase, arginine kinase and nucleoside diphosphate kinase B. Western blot analyses using specific antibodies raised against the purified recombinant proteins confirmed this changed expression for the thymidine kinase and the arginine kinase. For both proteins the increase in expression occurs directly after heat shock of the in vitro stage differentiation. The stage specific expression

89


seen on the protein level for both proteins could not be confirmed by Northern blot analyses, because no signal was obtained for the arginine kinase as well as thymidine kinase in promastigotes and amastigotes, respectively. The involvement of these kinases (thymidine kinase, arginine kinase) in stage differentiation and pathogenicity is currently studied in detail using gene deletion mutants. For gene knock-out it is important to determine the number of gene copies present within the Leishmania genome. Southern blot analyses indicate, that both genes are present as single-copy genes within the genome. First the phenotype, growth and differentiation of the deletion mutants should be analysed. In a second step we focus on the changes of the proteome pattern of the deletion mutants as well as on changes in the proteome pattern of phosphorylated proteins during stage differentiation.

Target identification and rational drug design based on ESTs from parasitic invertebrates Krasky, A. 1 ~, Rohwer, A} , Schroeder, J.1, Seizer, P. M} 1Drug Discovery BioChemInformatics; Intervet Innovation GmbH; Schwabenheim; Germany

A modern concept for the development of new drugs against parasitic diseases of humans, livestock and companion animals (e.g. filadasis, malaria, leishmaniasis) is the target-based approach. This involves the identification of target proteins in parasites which could serve as a molecular point of attack for antiparasitic drugs. The target protein must play an essential role in the life cycle of the respective parasite. The inhibition of such a target protein should result in an eradication of the parasite. To prevent toxicity problems for vertebrate host organisms, it is advantageous that these proteins show significant differences to their vertebrate counterparts. In the present work we identified potential target proteins in parasitic nematodes (Ascarls suum, Brugia malayi and Haemonchus contortus) and arthropods (Rhipicephalus sanguineus and other acarids) using

sequence comparison methods on expressed sequence tags (ESTs). Interesting target proteins (e.g. major sperm protein, ATP-synthetase OSCP subunit, calponin and S-adenosyI-L-methionine synthetase) were characterised in detail by subjecting them to in-depth bioinformatic analysis. The latter protein was used to conduct homology modeling and preliminary docking experiments. The results demonstrated that the model structure is suitable for rational drug design.

Structural and evolutionary analysis of the transcribed sequence of Boudicca, a Schistosoma mansoni retrotra nsposon Copeland, C. S. 1, Heyers, 0. 2, Kalinna, B. H. 3 ~, Bachmair, A. 4, Stadler, P. F. s, Hofacker, I. L. 6, Brindley, P. j.1 1Department of Tropical Medicine; Tulane Unversity; New Orleans; United States 2Department of Molecular Parasitology; Humboldt University Berlin; Berlin; Germany 3Humboldt University Berlin; Department of Molecular Parasitology; Berlin; Germany 4Max Planck Institute for Plant Breeding Research; Cologne; Germany 5Bioinformatics, Department of Computer Science; University of Leipzig; Leipzig; Germany 6Institute for Theoretical Chemistry and Structural Biology; University of Vienna; Vienna; Austria

Boudicca is a gypsy-like, long terminal repeat (LTR) retrotransposon that has colonized the genome of the human blood fluke, Schistosoma mansonL Previous studies have indicated that more than 1000 copies of Boudicca reside within the S. mansoni genome, although many of them may be degenerate and inactive. Messenger RNAs transcribed from genomic copies of Boudicca were investigated by reverse-transcription PCR. Overlapping RT-PCR products corresponding to the gag and pol polyproteins of Boudicca, along with relevant sequences of genomic fragments of Boudicca, were assembled into contigs. Consensus sequences from these contigs were used to predict the sequence and structure of transpositionally active copies of the Boudicca retrotransposon. They verified that Boudicca has a kabuki-like Cys-His box motif at the active site of its gag protein, a classic DTG motif as the active site of the protease domain of the pol ORF2, and indicated a contiguous integrase domain at the C-terminus of pol with strong identity to integrase from the LTR retrotransposons CsRnl and kabuki, as well as to the conserved integrase core domain. Models of the secondary structure of the Boudicca transcript suggested that the first AUG was occluded by a stem loop structure, which in turn suggested a method of regulation of expression, at the level of translation, of Boudicca proteins. In addition, phylogenetic analysis targeting discrete domains of Boudicca revealed a generalized radiation in sequences among the multiple copies of Boudicca resident in the schistosome genome.

Schistosoma mansoni miracidia transformed by particle bombardment infect Biomphalaria glabrata snails and develop into transgenic sporocysts Heyers, 0.1, Walduck, A. K. 2, Brindley, P. j.3, Blei6, W. 1, Lucius, R. 1, Dorbic, T. 4, Wittig, B. 4, Kalinna, B. H. ~ ~ 1Department of Molecular Parasitology; Humboldt University Berlin; Berlin; Germany 2Department of Molecular Biology; Max Planck Institute for Infection Biology; Berlin; Germany 3Department of Tropical Medicine; Tulane University; New Orleans; United States 4Centre for Somatic Genetherapy; Free University Berlin; Berlin; Germany

To date studies towards a transfection system for schistosomes focussed on the adult and sporocyst stages for particle bombardment. This poses the inherent problem of how to introduce these transgenic parasite stages back into the lifecycle. We have now shown reporter gene expresion in the miracidial stage of Schistosoma mansoni which had been subjected to particle bombardment with a plasmid DNA construct encoding enhanced

90


green fluorescent protein (EGFP) under control of the S. mansoni heat shock protein 70 (HSP 70) promoter and termination elements. Bombarded miracidia were also able to penetrate and establish in Biomphalaria glabrata the intermediate host snail. Gold particles could be detected in the germ balls of parasites in paraffin-sections of snail tissue. The bombarded miracidia were able to develop normally and to transform into mother sporocysts. Reporter gene activity could be determined at 10 days post infection by RT-PCR from snail tissues, but not by microscopy or Western Blot which probably reflected sub-optimal expression levels of the constructs. Our findings indicate that it is feasible to return transgenic miracidia to the life cycle, a crucial step for the establishment of a transgenesis system for schistosomes.

Analyses of t issue-specific gene activity in transgenic schistosomes Wippersteg, V. 1 t, Liedtke, S. ~, EI-Bahay, A. 0.1, M0nnich, M. 1, Philipp, C. 1, Ribeiro, F. 2, Kusel, J. R. 2, Rossi, A. 3, Klinkert, M. Q?, Grevelding, C. G. 1 iInsUtut for Genetik; Heinrich-Heine Universit~t; DOsseldorf; Germany 2Division of Biochemistry & Molecular Biology; University of Glasgow; Glasgow; United Kingdom 3Institut for Tropenmedizin; Universit~t T~bingen; T~bingen; Germany

With the rapid progress being made on sequencing the genome of Schistosoma mansoni, there are now increasing requirements for reverse genetic methods. Their development is essential to promote functional gene analyses in this parasite. For this purpose, we have developed a gene-transfer system based on the particle bombardment technology to generate transgenic schistosomes. To prove the reproducibility and reliability of this ballistic approach we tested different promoter-reporter-gene constructs, which revealed tissue-specific expression patterns following transformation. With the help of a recently introduced staining method we were able to localize the activity of two genes, the cysteine protease ER60 and calcineurin A, in the excretory system of living adult male schistosomes by confocal laser scanning microscopy. This improves existing methods, because Iocalisation studies were limited to sections of fixed specimens so far. To identify the regulatory elements of genes predicted to be ubiquitously expressed in schistosomes, we started a genome-walking approach and cloned the 5"UTR's of several genes. Data base analyses revealed in most cases the presence of conserved promoter elements. To test their functionality as promoters, GFP reporter- gene constructs were made containing these 5"UTRs and used for both the transfection of heterologous cell cultures and for transformation experiments with schistosomes. The characterization of these promoters will be useful for future experiments designed to investigate the consequences of ectopic gene expression as well as for the development of strategies towards germ-line transformation.

Ten dif ferent mRNAs arising from trans- and alternative-spl icing mechanisms code for the Onchocerca volvulus glutathione S-transferase 3 (Ov-GST3) H6ppner, j.1, Walter, R. D. 1, Liebau, E. 1 t 1Biochemistry; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany

The glutathione S-transferases (GSTs) comprise a complex and widespread enzyme superfamily that plays a key role in phase II detoxification by catalysing the conjugation of reduced glutathione to a large variety of electrophilic toxic molecules. The novel GST-class Omega carries out functions not originally associated with GSTs. The proteins are upregulated during oxidative stress conditions and are involved in the removal of reactive oxygen species and in the regeneration of S-thiolated proteins. Previously we have demonstrated that the Omega-class GST of Onchocerca volvulus (Ov-GST3) is dramatically upregulated in response to environmental oxidative stress, suggestive of its important role in the protection of the parasite against reactive oxygen species derived from the host 's immune system. Here we investigate the genomic organisation of the Ov-GST3; the coding region of the gene consists of 8 exons and 7 introns. Alternative splicing of exon 4 and 5, including a splice form that causes early translational termination, results in four different gene product variants. Furthermore, 5 unique transcription start sites, each trans-spliced at the 5"-end, were detected. This alternative initiation results in the removal of a potential signal peptide, perhaps leading to a differential Iocalisation of the proteins in the cell. Investigations are on the way to determine whether the resulting variants lead to proteins with limited changes in function or code for completely different proteins with varying biological functions.

Caenorhabditis elegans as a model for overexpression of filarial antigens Pillai, S. 1 t, Hartmann, 5.1, Lucius, R. 1, Kalinna, B. H. 1 1Department of Molecular Parasitology; Humboldt University; Berlin; Germany

Onchocerciasis (the cause of River Blindness) is endemic in over 80 countries and affects over a 120 million people worldwide. A vaccine would be an efficient and affordable method to control this debilitating disease. Acanthocheilonema viteae infection in jirds is the accepted model to study this disease. Immunization of jirds with native A. viteae antigen has been shown to provide protection against challenge, whereas only partial immunity was seen when they were vaccinated with recombinant antigens expressed in bacteria. This suggests that conformational epitopes, glycosylation and/or enzyme activity of vaccine antigens are required to elicit an effective protective immune response. We propose therefore that expression of potential vaccine candidates/antigens should be done in an eukaryotic system to obtain the appropriate post-translational modification as in native antigens.

91


We use C. elegans as a system for overexpression of the A. viteae antigen cystatin, an immunomodulatory protein and candidate vaccine antigen. Genomic and cDNA sequences of cystatin were cloned downstream of C. elegans promoters hsp16/41, let854, and mec7 in the vectors pPD49.83, pPD103.05 and pPD118.20, respectively. Transgenic C. elegans containing cystatin as extrachromosomal arrays were obtained by microinjection of phaI mutants. Transcription of A. viteae cystatin in C. elegans was analysed by RT-PCR. The expressed cystatin was purified by affinity chromatography. In order to increase the relatively low expression levels of cystatin, we plan to utilize its endogenous promotor. To this end the 5" sequence of cystatin was cloned upstream of a GFP reporter plasmid and promoter studies in C. elegans are in progress.

Invest igat ion of differential ly transcribed genes in hypobiosis induced and not induced third-stage Dictyocaulus viviparus larvae by quanti tat ive real- t ime PCR Strube, C}, von Samson-Himmelstjerna, G. ~, Schnieder, T } t IInstitute of Parasitology; Hannover School of Veterinary Medicine; Hannover; Germany

Hypobiosis represents a life cycle adaptation to ensure parasite survival in adverse extrinsic and intrinsic environments. In lungworm inhibition of development is induced by exposure of third-stage larvae to low temperatures. Hypopbiotic Dictyocaulus viviparus larvae persist in the lung of infected cattle for several months before they mature to egg-laying adults. To identify hypobiosis regulating genes and their transcription in D. viviparus Suppression Subtractive Hybridization was performed to enrich differentially transcribed genes in hypobiosis induced and not induced third-stage lungworm larvae. Following construction of subtracted cDNA libraries of the two larval populations differential screening was used to identify false positive cDNA clones. We detected 58 clones of hypobiosis induced and 44 clones of not induced larvae to be differentially transcribed. Verification using cDNA dot blot confirmed 26 clones and 22 clones, respectively. These gene transcripts were sequenced and compared with published sequences of other organisms. 3"- and 5"-RACE were performed to obtain full length cDNA of gene transcripts showing significant homologies. By using quantitative real-time PCR we investigated the transcription level of two differentially transcribed genes homologous to a N- methyltransferase of Caenorhabditis elegans and the superoxide dismutase of Haemonchus contortus, respectively. For N-methyltransferase, in hypobiosis induced larvae we obtained a tenfold higher transcription rate normalized to the housekeeping gene (elongation factor 1(]) compared to the not induced larvae. Concerning the superoxide dismutase homologue no significant differences in copy number could be detected. Functional implications of the genes found to be differentially transcribed will be discussed.

Novel saposin-l ike proteins of Entamoeba histolyticadifferent from the amoebapores Winkelmann, j.1, Bruhn, H. 1, Leippe, M. 2 ~ 1Research Center for Infectious Diseases; University of W#rzburg; W#rzburg; Germany 2Zoological Institute; University of Kiel; Kiel; Germany

Amoebapores, the pore-forming polypeptides of Entamoeba histolytica, have been shown to play a pivotal role in the pathogenicity of the protozoan parasite. They belong to the functionally diverse family of saposin-like proteins (SAPLIPs). The members of this family are characterized by a conserved pattern of six cysteine residues and the ability to interact with lipids. The data set analysis of the recently completed genome project of E. histolyticarevealed 16 genes encoding SAPLIPs in addition to and highly divergent from the amoebapores. Transcriptional analysis indicated that all of these genes are transcribed in amoebae at the same time. Despite a considerable size variation of the putative gene products (77 - 1009 amino acid residues) they always contain a single, C-terminally located SAPLIP domain and are virtually all preceded by a signal peptide suggesting that they are secretory products. Notably, other functional or structural domains were not identified. These multiple sequences represent a remarkably rich source of natural variants of SAPLIPs within a unicellular organism. Their functional characterization may provide information about general structure-function correlations for amoebapores and the entire protein family. Those members that resemble amoebapores in size and molecular architecture but are clearly diverse with regard to charge distribution and hydrophobicity are of particular interest as they may attribute to the versatility of the arsenal of an archaic phagocytozing cell in order to interact with a variety of target cells. Therefore, we expressed several, potentially membrane- disrupting members of this protein family recombinantly in E.coliand performed comparative functional assays to analyze their biological activity.

Proteomic approach towards the characterization of the parasitophorous vacuole in Plasmodium falciparum- infected erythrocytes, Nyalwidhe, j.1, Baumeister, S. 1, Lingelbach, K. ~ t 1Parasitologie; Philipps Universit~t; Marburg; Germany

After invasion of erythrocytes, the human malaria parasite P, falciparum resides and completes its asexual stage of development within a parasitophorous vacuole. The parasitophorous vacuole membrane (PVM) which forms a barrier between the host cell cytosol and the parasite surface has the dual function of protecting the parasite from potentially harmful substances and facilitating nutrient access from the external milieu. The parasitophorous vacuole is a unique compartment between the parasite and the host cell because it contains specific proteins that are believed to be involved in cell biological functions essential for parasite survival. These proteins can be postulated to be involved in several processes including nutrient acquisition from the

92


host cytosol, protein sorting within the vacuole and release of merozoites at the end of the intraerythrocytic development. To characterize the vacuolar proteome we have developed an experimental strategy that allows the selective biotinylation of soluble vacuolar proteins using a non-permeant biotin derivative that is introduced into infected erythrocytes after their permeabilization with the pore-forming protein Streptolysin O (1). The derivatives access the vacuolar space through the non-selective pores within the PVM (2) and selectively biotin label the vacuolar proteins (3). The soluble vacuolar proteins that are biotin labeled are isolated by affinity chromatography, separated by 2D gel electrophoresis and analyzed by MALDI-TOF-MS to identify novel vacuolar proteins.

(1) Ansorge et al. Biochem J. (1996) 315, 307-314.

(2) Desai and Rosenberg PNAS (1997) 94, 2045-2049.

(3) Nyalwidhe et al. 2002 JBC 277,40005-40011.

Using cysteine protease inhibitors to target proteins involved in survival and egression of intraerythrocytic stages of Plasmodiurn falciparum Gelhaus, C. 1 t, Vicik, R. 2, Schirmeister, T. 2, Leippe, M. ~ 1Zoological Institute; University of Kiel; Kiel; Germany 2Institute of Pharmacy and Food Chemistry; University of WElrzburg; WElrzburg; Germany

Cysteine proteases are known to play an essential role in the survival of the intraerythrocytic malaria parasite Plasmodium falciparum. Multiple cellular functions of malarial cysteine proteases have been investigated, i. e. degradation of hemoglobin, cleavage of host cell proteins, and release of invasive merozoites from the parasitophorous vacuole (PVM). The parasitophorous vacuole membrane (PVM) shields the parasite throughout its intraerythrocytic life cycle and is involved in complex processes for bidirectional transport of proteins, nutrients, and metabolites. We used a biotinylated aziridine dicarboxylic acid as a covalent inhibitor of cysteine proteases to localize and affinity-purify potential target proteins of the compound. Our results demonstrate that some inhibitor-reactive proteins are transported to the host cell cytoplasm across the vacuolar membrane in early stages of the parasite life cycle giving further evidence for the role of cysteine proteases in degradation of host cell cytoplasm. Affinity chromatography yielded several protein entities including falcipain 2 and 3. The inhibitor decreased viability and the reinvasion rate of parasites and led to the formation of PVM-enclosed merozoite structures (PEMS) as previously described for the common cysteine protease inhibitor E-64. Moreover, using cysteine protease inhibitors we developed a protocol to isolate the PVM from PEMS to analyse the protein repertoire of this unique subcellular structure.(Supported by the DFG).

Generation of transgenic Plasrnodium reporter parasites to study sporozoite and liver stage biology Engelmann, S. 1 t, Matuschewski, K. 1 1Hygiene Institut, Abteilung Parasitologie; Universit~t Heidelberg; Heidelberg; Germany

Malaria transmission occurs upon an infectious mosquito bite. Typically less than 100 sporozoites become injected into the skin, where they eventually reach the liver most likely via the bloodstream. Invasion of hepatic cells occurs via transmigration or an active, actin-dependent process followed by formation of a parasitophorous vacuole and subsequent development into an exo-erythrocytic form (EEF). Within the hepatocyte the parasite undergoes major morphological changes and numerous replication cycles, until it enters the blood stream again. Though the liver stage is an essential step in the life cycle of Plasmodium, information has remained scarce. The liver stage is clinically silent, but provides an interesting drug- and immuno-target for intervention strategies. Our work aims at developing a drug screening test for EEFs in Plasmodium berghei. By reverse genetics we develop recombinant parasites, which express the reporter gene lacZ at different time points during EEF development, in order to discriminate early, middle and late stage EEFs. The recombinant parasites will provide a valuable tool to screen for novel drugs and will help to elucidate the parasite's molecular biology of the hepatic stage.

Is the differential gene expression in Trypanosoma cruzi strains group specific? Dost, C. K. 1 t, Saraiva, j.2, Monesi, N. 2, Engels, W. 3, Albuquerque, S. 1 1Allgemeine Genetik; Universit~t TiJbingen; TElbingen; Germany; 2FCFRP; University of Sao Paulo; Ribeirao Preto; Brazil; 3Lehrstuhl for Entwicklungsphysiologie; Universit~t TEJbingen; TObingen; Germany

Trypanosoma cruzi is not a homogeneous population but is rather composed by a pool of strains which circulate in both the domestic and sylvatic cycles involving humans, vectors and animal reservoirs of the parasite. Studies of isolated T. cruzi populations from different origins demonstrated the presence of a large range of strains with distinct characteristics. This intriguingly intraspecific variation has been extensively investigated by characterizing the morphology of blood forms, parasitemic curves, virulence, tissue tropism, pathogenicity and sensitivity to drugs. These phenotypic differences between strains of the same species are determined by

93


differential gene expression. At least two distinct groups of T. cruzi were identified by a number of molecular markers and named group I and II. The differential expression of genes in these groups was analyzed by SSH, suppression subtractive hybridization. Two T. cruzi strains, TA and BoI-SB belonging to group I and II, respectively, were cultured in LIT-Medium and, after 3 passages, RNA from epimastigotes was extracted at the log phase. The cDNA was synthesized from mRNA and SSH Analysis was performed using the cDNA subtraction kit. Two reactions, were performed, the forward reaction with TA strain as tester and BoI-SB as driver, and the reverse reaction. Subtracted cDNA fragments from both reactions were directly cloned and 40 clones were sequenced. Sequence alignments and homology searches identified several cDNA clones that should be differentially expressed in these two 7-. cruzi strains. A couple of clones have been identified which did not reveal homology with any known 7-. cruzi gene but

shared similarity with either Leishmania, Plasmodium or T. Bruce/ genes. According to the 7-. cruzi genome project, so far the gene products of several genes are still unknown and may lead to the characterization of new proteins with interesting functions. Experiments are currently under way in order to analyze whether the genes identified from SSH analysis are also differentially expressed in other 7-. cruzi strains, and whether there is a correlation of the expression pattern with parasitemic curves, pathogenicity, morphology and virulence.

Cloning and characterization of a defensin-encoding cDNA of Triatoma brasiliensis Araujo, C. A} , Waniek, P. J.~, Jansen, A. M. 2, Kollien, A. K. ~, Schaub, G. A } * 1Department of Special Zoology; Ruhr-University; Bochum; Germany 2Department of Protozoology; Instituto Oswaldo Cruz-FIOCRUZ; Rio de Janeiro; Brazil

Insects possess different antimicrobial activities such as lysozyme, cecropins and defensins. The latter belong to the group of cystein-rich antibacterial peptides that are active against Gram-positive bacteria. Defensin is expressed in the midgut, fat body and hemolymph of blood-sucking insects [1]. Since triatomines, the vectors of the etiologic agent of Chagas disease, Trypanosoma cruzi, ingest sterile blood, there seems to be no necessity for intestinal antibacterial compounds. However, triatomines swallow air before moulting, offering air- borne bacteria access to the intestine. In addition, the development of triatomines strongly depends on possessing endosymbiotic bacteria, which they obtain via coprophagy. These bacteria multiply after blood ingestion in the cardia and stomach. The passage of the blood from the stomach to the digesting small intestine causes considerable destruction of symbiont populations, and only about 0.01% of the total population is still present in the rectum [2]. Investigating these interactions of symbionts and triatomines we focussed on the defensin genes of Triatoma brasiliensis. We used degenerate oligonucleotides derived from amino acid sequences of Rhodnius prolixus defensins A, B and C, and RACE-PCR to amplify the 5'- and 3'- end of the defensin encoding cDNA. A complete, 282bp nucleotide sequence of T. brasiliensis was cloned. The deduced amino acid sequence showed 75% identity R. prolixus defensin and an estimated molecular mass of 10 kDa. A nineteen-residue N-terminal signal peptide is followed by a thirty-residue activation peptide with putative cleavage sites at Ser19 and Leu49.

1 Lopez L., Morales G., Ursic R., Wolff M., Lowenberger C. 2003. Insect Biochem. Mol. Biol. 33, 439-447.

2 Eichler S., Schaub G.A. 2002. Exp. Parasitol. 100, 17-21.

Supported by Volkswagen Foundation, Funda~o Oswaldo Cruz-FIOCRUZ and Conselho Nacional de Desenvolvimento Cientffico e Tecnol6gico (CNPq).

Differential gene expression in Cryptosporidium parvum infected intestinal epithelial cells HObner, M. 1, Petry, F. 2, Hommer, V}, Najdrowski, M?, Zelck, U. 1 t 1Institute for Tropical Medicine, Molecular Parasitology Unit; University -FObingen; TElbingen; Germany 2johannes Gutenberg-University; Institute of Medical Microbiology & Hygiene; Mainz; Germany 3InsUtute for Parasitology; University Leipzig; Leipzig; Germany

Cryptosporidium parvum is an obligate intracellular parasite which infects intestinal epithelial cells. To identify transcriptional responses during early time points of infection (2-24h), gene expression of C. parvum-infected and non-infected human colon turnout cells (HCT-8) were analysed by differential display (DD)-RT-PCR. Seventy two differentially expressed transcripts were found, 58 of these were confirmed. Changes in host cell gene expression correlate with a particular parasite-dependent event such as differentiation or replication. Surprisingly, very few genes were induced 2 and 6h post-infection while immediate repression of several host cell transcripts was observed. During penetration and establishment of C. parvum at 2h p.i., differences were detected in genes involved in transport, energy metabolism and in gene regulation. These, in addition to genes involved in cell division, were also mainly down-regulated during the development of trophozoites at 6h p.i. When the reproduction and differentiation to meronts typ I takes place after 12h, gene expression profile was altered: the previously down-regulated clusters did not appear as frequently but membrane associated proteins were more abundant. The profile during penetration of merozoites after 24h, however, resembled that after 2h. We also determined the expression of three oxidative stress key enzymes (iNOS, p22phox and Cu/Zn SOD). While the expression of iNOS was elevated in C. parvum4nfected host cells, no changes were detected in the expression of p22phox or Cu/Zn SOD. As compared with global transcriptional responses evoked by other intracellular pathogens, C. parvum elicits only few changes in host cell transcription during the initiation of infection. The unique localization of the parasite within an intracellular but extracytoplasmic parasitophorous

94

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in W(Jrzburg

vacuole at the apical surface of infected cells may be responsible for the minimal induction of host cell mRNA synthesis.

Cysteine proteinases in the digestive tract of t r iatomines Hendgen-Cotta, U. ~ t, Kollien, A. ~, Schaub, G. ~ 1Department of Special Zoology; Ruhr-University; Bochum; Germany

Triatomines are important vectors of Trypanosoma cruzi, the etiological agent of Chagas disease in Latin America. The digestive tract of these blood-sucking bugs is divided into five functional regions: oesophagus, cardia, midgut, which is subdivided into stomach and small intestine, and rectum. Ingested blood is stored and concentrated in the stomach, and the digestion and absorption occur in the small intestine. In contrast to other insects, triatomines have an acidic midgut lumen and digest blood via cysteine and aspartic proteinases. The activity of two cysteine proteinases, cathepsin B-like and cathepsin L-like proteinases, were investigated at different times after feeding using Z-Phe-Arg-pNA as the substrate and E-64 and CA-074 as a general and specific cysteine proteinase inhibitor, respectively. Using in situ hybridization, cathepsin B mRNA was localized to cells in a distinct region of the stomach and to cells distributed in patches along the small intestine indicated the presence of the corresponding mRNA.

Cell Biology & Biochemistry

Analysis of sequence elements responsible for traff icking of the P.falciparum stevor mult i -gene fami ly Przyborski, j.1 ,, Miller, S. 2, Pfahler, j 1 Brendan, C. 2, Lanzer, M. 1 1Abt. Parasitologie Hygiene Institut; Universit~tsklinikum Heidelberg; Heidelberg; Germany 2Infection and Immunity; WEHI; Melbourne; Australia

Plasmodium falciparum is the causative agent of the most severe form of malaria which is responsible for 1.5- 2.7 million deaths each year. The particular virulence of this species of Plasrnodium is due to the ability of infected erythrocytes to cytoadhere to certain receptors on capillary endothelium, which may result in organ dysfunction or failure. The expression of a parasite derived protein, PfEMP-1 has been linked to this cytoadhesion phenotype, and to the formation of rosettes. PfEMP-1 is expressed on the surface of the infected erythrocyte, and is encoded by the var gene family. Recently, a multi-gene super family has been described consisting of the stevor(Sub Telomeric Variable Open Reading Frame) and rifin genes which, like var , are predominately sub-telomerically located. The products of the rif multi-gene family (RIFINS) have been Iocalised to the surface of infected erythrocytes, those of stevor to membranous structures in the erythrocyte cytoplasm (Maurer's clefts). We have used transfection technology to express STEVOR-GFP fusion proteins and have used these to characterise signal sequences that may be required for trafficking of STEVOR to its eventual destination. We have identified elements that are responsible for trafficking of STEVOR to the parasitophorous vacuole, the erythrocyte cytoplasm, and to the Maurer's Clefts. Dissection of this unusual and complex trafficking pathway may point to novel anti-malarial strategies.

Glyoxalase I of the Malarial Parasite Plasmodium falciparum: Evidence for Subunit Fusion Rahlfs, S} t, Iozef, R. 1, Akoachere, M}, Schirmer, H. 2, Becker, K. ~ ~Interdisciplinary Research Center; Giessen University; Giessen; Germany 2Biochemistry Center; Ruprecht Karls University; Heidelberg; Germany

The cytosolic glyoxalase system comprises two enzymes, glyoxalase I and glyoxalase II, and converts toxic 2- oxoaldehydes into the respective nontoxic 2-hydroxycarboxylic acids, using reduced glutathione as a coenzyme (see 1 for review). The glyoxalase detoxification system is of particular importance to organisms largely depending on glycolytic energy production such as tumor cells and malarial parasites. Here we provide first insight into the malarial glyoxalase system and substantiate the hypothesis of a second gene duplication postulated for large glyoxalases. Recombinant Plasmodium falciparum glyoxalase I (PfGIx I) was characterized as monomeric Zn 2+ -containing enzyme of 44 kDa (2). The KM value of the methylglyoxal-glutathione adduct is 77 + 15 pM, the kcat value being 4000 min -~ at 25°C and pH 7.0. PfGIx I consists of two halves each of which is homologous to the small 2- domain glyoxalase I of man. Both parts of the pfglx I gene were overexpressed; the C-terminal half of PfGIx I was found to be a stable protein and formed an enzymatically active dimer. These results support the hypothesis of domain-swapping and subunit fusion as mechanisms in glyoxalase I evolution.

1) Thornalley, P. J. (1998) Glutathione-dependent detoxification of alpha-oxoaldehydes by the glyoxalase system: involvement in disease mechanisms and antiproliferative activity of glyoxalase I inhibitors. Chem. Biol. Interact. 111-112, 137-151. Review.

95

Scientific Program DGP, 21st Annual Meeting March 17 th - 20 th, 2004 in W0rzburg 2) Iozef, R., Rahlfs, S., Chang, T., Schirmer, R.H., and Becker, K. (2003) Glyoxalase I of the Malarial Parasite Plasmodium falciparum: Evidence for Subunit Fusion. FEBS Lett. in press (available online http://www, febsletters.org/febs/14/show/toc.htt since Oct iz 2003).

X-ray Structure of Glutathione S-Transferase from the Malarial Parasite Plasmodium falciparum: a novel isoform of GST Fritz, K}, Rahlfs, S. 2, Harwaldt, p.2, Becker, A. ~, Schirmer, H?, Kabsch, W. ~, Becker, K. TM

iDepartement for Medical Research; Max-Planck-Institute; Heidelberg; Germany 2justus-Liebig-University; Interdisciplinary Research Center; GieBen; Germany 3Biochemistry Center; Ruprecht-Karls-University; Heidelberg; Germany

Cytosolic glutathione S-transferase isoforms have been grouped into seven species-independent classes (alpha, mu, pi, theta, sigma, kappa and zeta) on the basis of sequence similarity, immunological cross-reactivity, and specificity toward the electrophilic second substrate. These GSTs catalyze the conjugation of glutathione with a wide variety of hydrophobic compounds generally resulting in non-toxic products that can be readily eliminated. In contrast to many other organisms, the malarial parasite Plasmodium falciparum possesses only one GST isoenzyme (PfGST). This GST is highly abundant in the parasite, its activity was found to be increased in chloroquine resistant cells, and it has been shown to act as a ligandin for parasitotoxic hemin (1). Thus, the enzyme represents a promising target for antimalarial drug development.

We now have solved the crystal structure of PfGST at a resolution of 1.9 ~ (2). The protein represents a new GST-isoform that cannot be assigned to any of the described GST classes. Structural alignment of PfGST with members of the alpha-, mu- and pi-classes revealed an rms deviation of at least 1.2 ~. In comparison to other known GSTs, and in particular to the human isoforms, PfGST possesses a shorter C-terminal end resulting in a more solvent accessible binding site for the second substrate. The structure furthermore reveals unique features in the H-site region that could be exploited for the design of specific PfGST inhibitors. Inhibition studies as a basis for further, structure-derived inhibitor development are under way.

1) Harwaldt P., Rahlfs, S. & Becker, K. (2002) Biol. Chem. 383, 821-830

2) Fritz-Wolf, K., Becker, A., Rahlfs, S., Harwaldt, P., Schirmer, R.H., Kabsch, W., and Becker, K. (2003) PNAS, in press.

Lipoic acid metabolism in the human malaria parasite Plasmodium falciparum. Wrenger, C.1; M011er, S. 1 1School of Life Sciences, Wellcome Trust Biocentre, University of Dundee, Dundee, UK

Lipoic acid is an essential cofactor of the keto acid dehydrogenase complexes (KADHC). This study shows that the malaria parasite possesses two distinct lipoylation pathways which are found in separate subcellular localisations. Lipoic acid synthesis comprimising lipA (lipoic acid synthase) and lipB (lipoic acid-ACP ligase) is present in the parasite's apicoplast whereas the second pathway compromising IplA (lipoic acid ligase) is located in the parasites's mitochondrion. Both Iocalisations were established by over expression of GFP fusions of the N-terminal sequences of lipA and IplA. RT-PCR, Northern blot and Western blot analyses revealed that all three genes/proteins are expressed in the early and late stages of P. falciparum erythrocytic development, suggesting that that the two pathways act at the same time. All three genes were cloned and truncated forms lacking the targeting sequences were recombinantly expressed in E. coil and their functionality verified by complementation of bacteria lacking the respective genes. Our results show that P. falciparum possess two independent pathways with different locations, responsible for the post-translational modification of KADHCs. Both pathways fundamentally differ from those in the human host. As KADHCs provide metabolites that are required for essential biosynthesis processes such as fatty acid biosynthesis, isoprenoid biosynthesis and heme biosynthesis, the two lipoylation pathways of P. falciparum might be attractive therapeutic targets against malaria.

Clonal variation in calcium homeostasis in Plasmodium falciparum Rohrbach, p.1 t, Friedrich, 0. 2, Lanzer, M. 1 1Hygiene Institute, Dept. of Parasitology; University of Heidelberg; Heidelberg; Germany 2Insitute of Physiology and Pathophysiology; University of Heidelberg; Heidelberg; Germany

Calcium is widely used by eukaryotic cells and is known to serve a wide range of regulatory and signalling functions. Although a number of studies concerning calcium homeostasis in Plasmodium falciparum have been published, still little is known about its physiological relevance. Recent studies have shown that the food vacuole is an intracellular calcium store (Biagini et. al., JBC, 2003). Since the food vacuole is a major digestive organelle of the malaria parasite and a proven chemotherapeutic target, it is important to understand what role Ca 2* may play. We have used a variety of approaches to expand our knowledge of the calcium signalling network of the parasite.

96


In our lab, we have noted that the calcium concentrations within the food vacuole of Plasmodium falciparum differ in various strains. Chloroquine resistant strains, such as Dd2 and K1, show higher Ca 2+ concentrations than chloroquine sensitive strains (eg. HB3 and 3D7). Our current investigations focus on understanding these discrepancies in calcium concentration. Chloroquine resistance may be associated with altered Ca 2÷ homeostasis in CQR strains. Using confocal microscopy, we have been able to show that some dyes widely used are subject to photobleaching. These dyes can give misleading results by not taking into account laser induced photobleaching in various intracellular compartments. Thus, it is crucial to critically evaluate dye properties such as affinity and binding kinetics.

Ref.: GA Biangini, PG Bray, DG Spiller, MRH White, SA Ward (2003) The digestive food vacuole of the malaria parasite is a dynamic intracellular Ca 2* store. J Biol Chem. 278(30):27910-5.

The bifunctional PfAdoMetDCIODC - the key enzymes of polyamine biosynthesis - is crucial for the survival of Plasmodium falciparum MQller, I. B. ~ t, Krnajski, Z} , Langer, C. ~, LQersen, K. ~, Walter, R. D} ~Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany

Polyamines are ubiquitous and essential cellular compounds involved in various processes such as proliferation and differentiation. Two key enzymes, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), deliver the polyamine precursors putresdne and decarboxylated S-adenosylmethionine (dcAdoMet), respectively. Putrescine and dcAdoMet are subsequently utilized by the spermidine synthase to form the polyamine spermidine. Inhibitors of the polyamine biosynthesis as well as polyamine analogues are already used in clinical studies on cancer, and successfully applied as antitrypanosomal. In contrast to the host,

where AdoMetDC and ODC are two separate proteins, Plasmodium falciparum possesses a single bifunctional protein, PfAdoMetDC/ODC. The advantage of the bifunctional organisation is thought to be the control of polyamine synthesis by regulating the abundance and activity of only one protein. PfAdoMetDC/ODC has been biochemically well characterized in previous studies. To evaluate the importance of the bifunctional protein for the survival of the parasite, attempts were made to completely knock out the PfAdoMetDC/ODC gene and to disrupt the ODC domain via double-crossover. In mind that the missing ODC domain as such negatively influences the AdoMetDC domain, an additional construct was made carrying a double mutation within the substrate-binding site of the ODC, thereby leading to an inactive ODC. The major impediment to the disruption of essential genes is the impossibility to isolate nullmutants. To bypass this, the stable transfectants were complemented with putrescine and spermidine. Till now integration was obtained only in cells transfected with the mutated ODC construct. Surprisingly, the double mutation, located near the end of the gene, was in none of the transfectants integrated into the genome, assuming a strongly negative effect on the survival of the parasite, which could not be circumvented by the addition of putrescine.

A mult i -domain adhesion protein family expressed in Plasmodium falciparum gametocytes is essential for sporozoite midgut to salivary gland transit ion Pradel, G. 1 ~, Hayton, K. 2, Bonawitz, A. 1, Mejia, C. 1, Templeton, T. 1 IDepartment of Microbiology and Immunology; Weill Medical College of Comell University; New York; United States 2National Institute of Allergy and Infectious Diseases; National Institutes of Health; Bethesda; United States

An understanding of the evolutionary and functional adaptations of the pathogenic Apicomplexa will provide a framework for the development of diagnostic and therapeutic markers and in dissecting host-parasite interactions and determinants of pathogenesis. To identify new mosquito transmission stage antigens we screened the Plasmodium falciparum genome sequence for genes encoding extracellular multi-domain putative adhesive proteins and identified five proteins, termed PfCCpl through PfCCp5, which have multiple adhesive modules, including a common Limulus Coagulation factor C (LCCL) domain. Orthologs were identified in the Cryptosporidium parvum and Theileria annulata genome sequences, indicating an evolutionary conserved function. Transcript and protein expression analysis show gametocyte-specific expression of PfCCpl through PfCCp3 in P. falciparum parasites, and cellular localization studies revealed that proteins associate with the parasite surface. During gametogenesis, PfCCps are released and localize surrounding complexes of newly emerged micro- and macrogametes, while expression ceases following emergence. To begin to address protein function we disrupted the gene loci for PfCCp2 and PfCCp3 by homologous recombination. Targeted gene disruptant clones are capable of forming oocyst sporozoites but are blocked in the salivary gland transition. PfCCp protein expression cannot be detected in PfCCp3 gene disruptant lines, indicating dependent protein interactions between the three PfCCp proteins. Our data describe a highly conserved apicomplexan protein family expressed in the sexual phase of the Plasmodium life cycle that may represent candidates for study as subunits of a transmission blocking vaccine.

97


A novel member of the GCNS-related N-acetyltransferase superfamily from Leishmania major catalyses the N6-acetylation of S-(aminoethyl)-L-cysteine L0ersen, K} t Clos, j.2, Walter, R. ~ iBiochemie; Bernhard-Nocht-Institut for Tropenmedizin; Hamburg; Germany 2Bernhard-Nocht-Institut for Tropenmedizin; Hamburg; Germany

The GCN5-related N-acetyltransferase (GNAT) superfamily encompasses enzymes that catalyse the transfer of an acetyl-group from acetyl-coenzyme A to a primary amine of acceptor molecules. Numerous diverse GNATs have been reported that derivatise, for example, lysine residues of proteins such as histones and transcription factors or small molecules such as arylalkylamines or polyamines. Acetylation of the latter is catalysed by spermidine/spermine-N-acetyltransferase (SSAT). A gene with considerable homology to known SSAT has been cloned from Leishmania major. The 477 bp ORF encodes a deduced polypeptide of 18.4 kDa. Accordingly, the recombinantly expressed His-tagged protein forms a homodimer with a molecular weight of 44.000. However, neither polyamines nor diamines were effectively acetylated by the enzyme. Instead, the L-lysine analogues S- (aminoethyl)-L-cysteine, followed by O-(aminoethyl)-L-serine, S-(aminoethyl)-homocysteine and 5-hydroxy-L- lysine were found to be the best substrates. Reversed-phase HPLC analyses revealed that N-acetylation takes place exclusively at the side chain amino group of these rare amino acids. Orthologues of the S-(aminoethyl)-L- cysteine N-acetyltransferase have been characterised also from the nematode Caenorhabditis elegans and from mammals. In order to elucidate the biological significance of this novel GNAT, we are currently generating a gene replacement mutant in L. major. The effect of this gene replacement on general viability and virulence will be assessed.

Map kinases in defence cells of the schistosome intermediate host snail Lymnaea stagnalis Edele, F. ~, Oberl~nder, M. 1, Zelck, U} t 1Institute for Tropical Medicine, Molecular Parasitology Unit; University TObingen; TObingen; Germany

Mitogen-activated protein kinases (MAPKs) or extracellular signal regulated protein kinases (ERK) and stress- activated protein kinases (SAPKs/JNKs) comprise an evolutionarily conserved family of serine/threonine protein kinases that mediate intracellular phosphorylation events linking receptor activation to the control of cell proliferation, differentiation, chemotaxis and stress response. Oxidative stress has been implicated in the defense against trematodes by their intermediate host snails. Although it is known that oxidative stress stimulates various MAP kinase families in mammals and plants, the signal events in invertebrates remain largely unknown. In the present study, we determined whether MAPKs are transcriptionally regulated and/or activated by oxidative stress in the hemocytes of the snail Lymnaea stagnalis. We designed degenerate PCR primers based on the conserved amino acid sequences of various MAPKs and detected ERK, JNK and p38 MAP kinase expression by PCR. Sequence homologies indicate conserved MAPK modules for signal transduction and allowed their identification based on the consensus motifs TXY. H202 (100 or 200mM) or Zymosan A were used to induce extracellular oxidative stress and intracellular oxidative burst, respectively. Transcript abundance of MAPKs was neither enhanced by H202 nor by Zymosan-stimulation of the cells. The activation/phosphorylation status of MAPKs was evaluated by immunoblotting. MAPKs were activated by phosphorylation after hemocyte stimulation with H202 or Zymosan. The extent to which each intracellular signalling pathway contributes to the induced stresses and to killing of trematodes will be discussed.

Gonad-preferential activity of protein tyrosine kinase in schistosomes Grevelding, C. G. 1 t, Knobloch, J}, Kapp, K. 2, Philipp, C}, M0nnich, M}, Sch~Bler, P.~, Sroka, S. ~, Lammers, R. 2, Kunz, W. 1 IInstitute for Genetics; Heinrich-Heine-University; 40225 DOssledorf; Germany 2Medizinische Klinik IV; Universit~tsklinikum TObingen, Eberhard-Karls-University; 72076 TObingen; Germany

Female schistosomes depend on signals from the male to induce mitoses in the vitellarium, where vitelline-cells are formed for egg production. If the male is separated from the female, vitelline-cell divisions are stopped, and egg production ceases. Upon remating, mitoses are reinitiated, and egg production is restored. The aim of our study was to find out whether protein tyrosine kinases (PTKs) may be involved in processes that control mitoses and/or differentiation in schistosomes. A novel technique was established that allowed us to measure mitogenic activity in S. mansoni confirming that males control mitogenic processes in females. Herewith we investigated the influence of chemical inhibitors on mitoses in paired schistosomes. The used inhibitors selectively block different classes of cellular tyrosine-kinases (Src or Syk). Evidence is presented that Src- specific inhibitors gender-specifically reduce mitotic activity in females, whereas Syk-specific inhibitors do not affect mitoses in males or females. These results correlate with data obtained from the characterization of cloned PTK genes from S. mansoni. In situ hybridizations and immuno-localizations showed the tissue-specific activity of the cloned Src-like (TK3/ TK5) or Syk-like (TK4) molecules in both genders. TK5 is ubiquitously expressed, whereas TK4 shows activity in parenchyma of both genders~ and in the testes of the male or the ovary of the female, but not in the vitellarium. In contrast, TK3 exhibits a gonad-preferential expression including the vitellarium. From these experiments we conclude that Src-kinases are involved in mitogenic processes during vitelline-cell development. Functional assays and yeast-two-hybrid screenings in heterologous

98


or homologous systems were performed to identify substrates and/or binding partners of TK3. Conserved molecules were identified that are known to be involved in the organization of the cell architecture, pinpointing to a role which could be assigned to TK3.

Structural and functional characterizat ion of TGF-beta signaling systems in Echinococcus mul t i locular is Zavala-Gongora, R. 1, Kroner, A. ~, Wittek, B. 2, Knaus, p.2 Brehm, K. ~ t iInstitut for Hygiene und Mikrobiologie; Universit~t WOrzburg; WOrzburg; Germany 2Lehrstuhl for Physiologische Chemie II; Universit~t WOrzburg; WOrzburg; Germany

Members of the transforming growth factor-beta (TGF-beta)/bone morphonegentic protein (BMP) family of cytokines and their corresponding receptors regulate cellular key processes such as proliferation and differentiation, and could be involved in communication mechanisms between parasitic helminths and their hosts. In this study, we provide evidence for at least two different TGF-beta signaling pathways in E. multilocularis. We have identified three distinct members of the TGF-beta type I receptor family of which two (EmRTK1, EmRTK3) are structurally related to BMP receptors and of which one (EmRTK2) shows significant homology to TGF-beta receptors from mammals and several invertebrates. All three receptors were functionally active and expressed in Echinococcus larval stages during natural infections. In addition, we could identify three members of the Smad-family of intracellular TGF-beta signal transducers of which two (EmSmadA, EmSmadC) are putatively involved in TGF-beta signaling processes while one (EmSmadB) formed part of BMP signaling processes in the parasite. Upon heterologous expression in HEK 293 cells, the Echinococcus Smad factors interacted with mammalian TGF-beta and BMP receptors and were phosphorylated at conserved C-terminal SSXS motifs. Interstingly, although one Echinococcus Smad (EmSmadB) showed a conserved domain structure composed of MH1- and MH2-domains, no MHl-domain was present in the two Echinococcus Smads EmSmadA

and EmSmadC. Like the Echinococcus TGF-beta/BMP receptors, all three Smad factors were expressed in metacestode and protoscolex larval stages during natural infections. Taken together, our data indicate the presence of structurally and functionally conserved TGF-beta and BMP signaling pathways in E. multilocularis and provide a solid basis for future investigations on the role of TGF-beta signaling in cestode development and in mechanisms of host-parasite interaction.

Characterizat ion of a MAP kinase kinase homologue from L. mex icana Kuhn, D. 1 t, Wiese, M. 1 1Parasitology Section; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany

In higher eukaryotes signal transduction pathways like the mitogen-activated protein (MAP) kinase cascades play important roles in differentiation, proliferation, stress response and other regulatory processes. Today a number of genes encoding protein kinases in Leishmania and related kinetoplastids have been described but their function and possible interactions remain largely unknown. One component of MAP kinase pathways are MAP kinase kinases. They specifically phosphorylate and activate MAP kinases, thus indirectly affecting gene regulation which shows their potential as drug targets. Using a partial kinase sequence of the L. major genome project on a genomic DNA library of L, mexicana, we identified a single copy gene coding for a MAP kinase kinase homologue referred to as LmxPK4. The protein is stage-specifically expressed and can be detected exclusively in the promastigote form of the parasite. The null mutant showed a proliferation defect after infection of peritoneal macrophages and delayed development of lesions in mice, which could be partly restored by reintroducing the gene into the deletion background. Several recombinant proteins were expressed in E. coli: the wild-type kinase and an N-terminally truncated mutant which are active in in vitro kinase assays and an inactive KR-mutant. We currently focus on the identification of the substrate of LmxPK4 by using different methods like pull-down experiments with the recombinant kinase- dead mutant or the tandem affinity purification (TAP) tagged protein from transfected L, mexicana. Once identified, a substrate may help us to gain new insights into parasite signal transduction and gene regulation.

A protein kinase involved in f lagel lar length control in Leishmania mex icana Wiese, M. 1 t, Kuhn, D. 1, Scholz, A. ~, Gruenfelder, C. 2 1Parasitology Section; Bernhard-Nocht-Institute for Tropical Medicine; Hamburg; Germany 2Abteilung Membranbiochemie; Max-Planck-Institut fuer Biologie; Tuebingen; Germany

During its life cycle the parasitic protozoon Leishmania mexicana differentiates from a spindle-shaped flagellated form, the promastigote, to the spherical amastigote form carrying a rudimentary flagellum. Besides regulated biochemical changes, this process involves coordinated changes in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue, designated LmxMKK, was identified in a homology screen and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and likely to be regulated by post-translational mechanisms like phosphorylation. A deletion mutant for the single copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in

99


kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology.

Gametogenesis in Plasmodium berghei is controlled by calcium and a calcium-dependent protein kinase Billker, 0.1 t 1Department of Biological Sciences; Imperial College London; London; United Kingdom

The rapid and highly synchronised differentiation of gametocytes in response to a small mosquito molecule, xanthurenic acid (XA), offers an opportunity to study signalling pathways that regulate stage differentiation in malaria parasites. To investigate calcium signalling during gametogenesis we generated a transgenic P. berghei strain, in which expression of a stably integrated fusion gene encoding apoaequorin and green fluorescent protein (GFP) is driven by the EFlo-A promoter of P. berghei. Bioluminescent resonance energy transfer between the chemiluminescent calcium reporter, aequorin, and GFP provides a sensitive measure for cytosolic calcium peaks that is independent of host cell calcium and gametocyte emergence. Using this calcium reporter strain, we show that at a permissive temperature for the induction of gametogenesis, biologically active concentrations of xanthurenic acid trigger a dose-dependent increase in cytosolic calcium within 10 seconds. Calcium release is required for all aspects of gametocyte activation studied, i.e. cell cycle re-entry and axoneme assembly in the male gametocyte and host cell lysis in gametocytes of both sexes. Asexual blood stages obtained from a non-gametocyte producing calcium reporter line failed to respond to XA, suggesting the presence of an XA-responsive signalling pathway specifically in the gametocyte. Targeted disruption of a gene encoding a novel, predominantly male-expressed, calcium-dependent protein kinase, CDPK4, resulted in gametocytes that emerged normally, but in which microgametocytes failed to replicate their genome and exflagellate. We suggest that CDPK4 mediates some, but not all effects of the XA-induced calcium signal and that this kinase is a key regulator of S-phase entry in the male gametocyte upon activation in the mosquito midgut.

The pathogenic amoeba Balamuthia mandrillaris possesses cell-associated phospholipase A, lysophospholipase A, and lipase activities Shadrach, W. S}, Radam, E. 2, Flieger, A. 1, Kiderlen, A. F. 2 t ~Ng5, Pathogenesis of Legionella Infections; Robert Koch-Institut; Berlin; Germany 2Department of Infectious Diseases; Robert Koch-Institut; Berlin; Germany

Balamuthia mandrillaris is a free-living amoeba and opportunistic agent of lethal meningoencephalitis in humans and animals. The amoeba does not grow on agar plates with enteric bacteria but feeds on a variety of eukaryotic cells. Furthermore, B. mandrillaris kills immunodeficient mice when inoculated intranasally. For other free-living amoebae with pathogenic potential, like Acanthamoeba or Naegleria, some virulence factors contributing to the development of disease have been described. One of these determinants is phospholipase A activity, which was found to be enhanced especially in virulent strains of amoebae. To test whether B. mandrillaris also possesses activities for lipid hydrolysis, we here analysed culture supernatants and cell lysates for their ability to destruct different diacylglycerophospholipids, monoacylglycerophospholipids (lysophospholipids) and non-phospholipids. We found that the lipolytic activities of the amoeba are predominantly cell-bound. Enzymatic activity hydrolyzing monoacylglycerol, diacylphosphatidylcholine and to a lesser extent diacylphosphatidylglycerol was primarily detected under acidic reaction conditions with supplementation of calcium ions. The substrate preference of this activity might explain why B. mandrillaris grows on phosphatidylcholine-rich mammalian cells rather than enteric bacteria containing higher amounts of phosphatidylglycerol. Additionally, high lipase activities releasing fatty acids from di- and to a lesser extent from triacylglycerolipids as well as low lysophospholipase A activities hydrolyzing monoacylglycerophospholipids were observed. In conclusion, we have shown that B. mandrillaris possesses a variety of lipolytic enzyme activities associated with the amoebal cell which could contribute to the mammalian cell damage observed both in vivo and in vitro in the course of an infection with the pathogenic amoeba.

Several isoforms of pore-forming peptides of Naegleria fowleri were proteolytical ly released from a precursor protein and display antimicrobial activity Herbst, R. 1 t, Marciano-Cabral, F. 2, Leippe, M. 3 lAG Spezielle Zoologie; Ruhr-Universit~t Bochum; Bochum; Germany 2Department of Microbiology and Immunology; Medical College of Virginia; Richmond; United States 3Zoologisches Institut; Universit~t Kiel; Kiel; Germany

Naegleria fowleri is a free-living amoeboflagellate of soil and freshwater habitats throughout the world. Although the trophozoites are able to fulfil their lifecycle without the intervention of a parasitic stage, they can invade human beings and cause the primary amoebic meningoencephalitis (PAM). This disease is characterized by massive host-tissue destruction indicating that the cytolytic activity of the amoebae is a major factor of their pathogenicity. The amoebic cytolytic capacity has been attributed mainly to pore-forming molecules. We recently described with naegleriapore A2 and B1 proteins that potently display such a pore-forming activity and that kill prokaryotic as well as eukaryotic target ceils (Herbst, R. et al. (2002) J. Biol. Chem. 277, 22353-60). Both naegleriapores were processed from larger muiti-peptide precursor structures; each of them contains

100


several naegleriapore isoformswhich all share the structural motif of six invariant cysteine residues within their highly diverse amino acid sequence. To identify naegleriapore-like peptides without prominent pore-forming activity at the protein level , we screened for small cysteine-rich proteins in crude extracts by fluorescently labelling their cysteine residues. Using this approach, we successfully identified three novel isoforms, all apparently proteolytically released from the precursor molecule of naegleriapore B. The newly recognized naegleriapore isoforms in their highly purified form indeed display remarkably lower pore-forming activity than their relatives naegleriapore A2 and B1 but exert antibacterial activity against Bacillus subtilis and Pseudomonas aeruginosa suggesting that naegleriapores are part of an antimicrobial system of a bacteria- hunting amoeba that accidently can be instrumental in a fatal parasitic disease.

This work was supported by the Deutsche Forschungsgemeinschaft by grant (LE 1075/2-3).

Ident i f icat ion of amoebastat in, a cysteine proteinase inhibitor in trophozoites Entamoeba histolytica Scholze, H. ~ ~, Riekenberg, S}, Witjes, B. 1, Key, G. l, Bakker-Grunwald, T } 1Fachbereich Biologie/Chemie; Universit~t OsnabrOck; OsnabrOck; Germany

Entamoeba histolytica, the causative agent of amoebiasis, is equipped with a range of cysteine proteases, some of which are supposed to be involved in different aspects of pathogenesis. However, little is known about the regulation of their enzymatic activity. Screening of the data base from the Entamoeba genome project (TIGR) for sequences of cysteine proteinase inhibitors led us to an open reading frame with significant similarity to a gene encoding chagasin, a cysteine proteinase inhibitor from Trypanosoma cruzi. In agreement with the hypothesis that the amoebae contain an inhibitor protein, we identified an inhibitor activity towards papain, a model cysteine proteinase, in a homogenate supernatant of Entamoeba trophozoites. The inhibitor assay functioned only after boiling of the extract, which suggest that this inhibitor is a very heat stable protein that may in vivo be complexed with cysteine proteases. Interestingly, the amebic inhibitor encoding gene is localized in close proximity to a gene encoding the amoebic cysteine proteinase, EhCp3, which is present in both pathogenic and non-pathogenic

Entamoeba. We amplified the complete open reading frame by polymerase chain reaction from genomic DNA of E. histolytica and overexpressed it in E. coli. The calculated molecular mass of the recombinant protein including a His~0-tag is Mr 13,600 and agrees well with its migration behaviour in an SDS-PAGE. The protein, which we designate as amoebastatin, is not homologous to the cysteine proteinase inhibitors of the cystatin/stefin-family, but has 30% identity to chagasin of T. cruzi and 31% to a respective putative inhibitor from Leishmania major. Alignment of the amebic inhibitor sequence with these and other hypothetical inhibitor sequences from other parasites as well as from the prokaryote Pseudomonas aeruginosa revealed the conserved sequence motif, LXG/SNP'FI'GY/FXW. Biochemical characterization and immunohistochemical localization of this inhibitor are in progress.

Ident i f icat ion and functional characterization of a -N-acetylhexosaminidase of Entamoeba histolytica Riekenberg, S. 1 ~, Flockenhaus, B. i, Bakker-Grunwald, T} , Scholze, H. i ~Fachbereich Biologie/Chemie; Universit~t OsnabrOck; OsnabrOck; Germany

Entamoeba histolytica is the causative agent of amoebiasis, an infectious disease highly prevalent in developing countries. Apart from causing dysentery, trophozoites of E. histolytica are capable of invading inner organs of the host forming tumour-like abscesses. During penetration of the intestinal tissue trophozoites are confronted with the mucin layer of the epithelium and other glycosylated barriers whose overcoming requires the hydrolysis of glycosidic bonds.

As a candidate for this activity, we have purified a 6-N-acetylhexosaminidase from trophozoites of E. histolytica, which sedimented at 12S, consistent with an apparent Mr of N400,000 as established by gel exclusion chromatography. SDS-PAGE of the purified complex yielded a single protein band at an apparent Mr of 62,000, suggesting that the native enzyme is hexameric. Based on gene sequences identified in the E. histolytica genomic data base having the same derived N-terminus as our purified enzyme complex, we amplified and cloned two genes coding for two predicted, highly similar hexosaminidase chains, which we designated as Ehhexo and EhhexL3. Northern blotting indicated that the two genes were expressed to a similar extent. Accordingly, we assume that the hexosaminidase complex contains both polypeptide chains. By cell fractionation, the hexosaminidase was found to be a major component of cytoplasmic granules; these contain tissue-destructive factors and are released after collagen-induced transfer to the cell surface. Immunocytochemistry with antiserum against the o-chain revealed strong fluorescence both in surface patches, which we interpret as released granules, and in vesicles throughout the cell. In an intestinal monolayer model, inhibition of hexosaminidase activity by N-acetylglucosamine prevented the decrease in monolayer resistance induced by trophozoites. Based on these findings we propose that the hexosaminidase contributes to trophozoite invasiveness.

101


Resistance against a cysteine proteinase inhibitor in Entamoeba histolyt ica correlates with secretion of unprocessed proteinases and reduced pathogenicity Nowak, N}, Tannich, E. 1, Bruchhaus, I } t iMolecular Parasitology; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany

Cultivation of Entamoeba histolytica trophozoites for few days in the presence of the cysteine proteinase inhibitor (CPI) E-64 kills almost all amoebae. But approximately 0.1% of the amoebae develop spontaneously a resistance to E-64 enabling growth in the presence of this inhibitor. E-64-resistant (E-64r) amoebae retain this resistance, even after cultivation for up to two months in the absence of E-64. The resistance to E-64 is specific as no cross-resistance to other CPIs was detected. In contrast to wild-type (WT) amoebae, resistant amoebae are able to secret the unprocessed proform of the CPs in the presence of E-64. This suggests that the secretory pathway of E-64r amoebae is upregulated in the presence of the inhibitor. Furthermore, E-64r amoebae cultivated in the presence or absence of E-64 showed a significantly reduced pathogenicity. All other phenotypical changes observed for E-64r amoebae were also seen for non-resistant WT amoebae when short- term cultivated in the presence of E-64. Both amoebic populations possess no detectable cysteine proteinase (CP) activity and the CPs are present as inactive unprocessed proforms when the amoebae were cultivated with E-64. Furthermore, fibrillar structures of unknown origin were observed within these cells. Despite the induction of an E-64 resistance in vitro, it can be postulated that CPs represent promising drug targets, because of the missing cross-resistance to other CPIs as well as the missing pathogenicity of E. histolytica after induction of an E-64 resistance. This work was supported by the DFG (BR 1744).

Molecular analysis of in vivo antigenic variation of Giardia lambl ia clone GS/M-83-H7. M011er, N. 1 t, Bienz, M. ~, yon AIImen, N} 1University of Berne; Institute of Parasitology; Berne; Switzerland

Giardia lamblia is an intestinal protozoan parasite of humans and various animals. Manifestation of the disease varies from asymptomatic carriage to severe diarrhea and malabsorption. Infection occurs upon peroral ingestion of cysts which, following excystation in the small intestine, release two trophozoites each. In the last few years, many studies were focused on the investigation of the parasites' major surface antigen, VSP, which is known to be responsible for the antigenic variability of the parasite. By using G. lamblia clone GS/M-83-H7 (expressing VSP H7) trophozoites and a neonatal mouse model for experimental infections, we quantitatively assessed on the transcriptional level the process of antigenic variation of the parasite. In this study, variant- specific regions identified on different GS/M-83-H7 vsp sequences served as targets for quantitative reverse transcription (RT)-polymerase chain reaction (PCR) to monitor alterations in vsp gene transcription during infection. Respective results demonstrated that antigenic switching of both the duodenal trophozoite and the caecal cyst populations was associated with a massive reduction in vspH7 mRNA production. Conversely, antigenic switching did not cause a significant increase in transcription of any of the subvariant vsp genes analysed. Most importantly, we also explored variant-type formation and vsp mRNA synthesis after infection of mice with cysts that had been collected during the abovementioned infection experiment. In contrast to the infection with trophozoites, infection with cysts was associated with an antigenic reset of the parasite in that the antigenically diversified inoculum (cysts containing non-VSP H7-type trophozoites) "converted" into a non- diversified population of intestinal trophozoites which essentially consisted of the original VSP H7-type. This antigenic reset occurs during the process of in vivo excystation and does not reflect a selective process, favouring expansion of an eventual minor population of VSP H7-types residing within the antigenically- diversified cyst inoculum. Based on these findings, the VSP H7-type has to be regarded as a predominant variant of G. lamblia clone GS/M-83-H7 which (re-) emerges during the early stage of an infection and may contribute to an optimal establishment of the parasite within the intestine of the experimental murine host. Future investigations will address the question whether the strategy of such an antigenic reset is compatible with both (i) a possible participation of surface antigen alterations in an adaptive process facilitating transmission of G, lamblia from one to another host species and (ii) a putative occurrence of repeated G. lamblia infections in the same individuum.

Characterization and subcellular localization of annexin E1 in trophozoites of Giardia lambl ia Vahrmann, A. 1 t, Szkodowska, A. 1, Mueller, M. C. 1, Bakker-Grunwald, T. 1, Scholze, H. ~ iFachbereich Biologie/Chemie; Universit~t OsnabrOck; Osnabr~ck; Germany

Annexins represent a multigene family of eukaryotic proteins with various, but in detail mostly unknown functions. We have identified a novel annexin, ANXE1 (old nomenclature: annexin XXI) in a cell extract of trophozoites of Giardia lamblia. This annexin, in contrast to its homologous a-giardins (ANXE2 and ANXE3), has a true type I I Ca2+-binding motif (endonexin-fold, GXGTD{38}D) in the second annexin repeat and a further rudimentary one in the fourth repeat. Furthermore, in the C-terminal region of the ANXE1 sequence some motifs typically occurring in the a-giardins are conspicuous, such as an ITG/AM- and a KXXYK-motif, and a tryptophan residue followed by a charged residue next to the C-terminus. All these motifs, whose physiological significance is still unknown, are not conserved in the annexins of higher eukaryotes. According to molecular modelling of the sequence, whose secondary structure prediction reveals a mostly a-helical folding, these

102


residues are positioned next to the concave side of the molecule, which in other annexins is orientated to the cytoplasmic face of the molecule. Western blot analyses using polyclonal antibodies raised against recombinant ANXE1, show that this annexin, as the o-giardins, is part of the cytoskeletal fraction of trophozoites of G. lamblia. This fact was confirmed by immunocytochemical analyses which show that ANXE1, in contrast to the a- giardins (ANXE2 and E3) which are associated with the ventral disk, is a constitutive part of the eight giardial flagella as well as of the median body of the trophozoites. Electron microscopy of the purified recombinant annexin E1 suggests that the protein is able to self assemble into fibrils. Together, we hypothesize that ANXE1 may function as a Ca2+-regulated structural element linking the phospholipid bilayer to the underlying axoneme.

Ident i f icat ion and characterization of heme-interact ing proteins in the malaria parasite, Plasmodium falciparum Nickel, C. 1 t, Campanale, N. 2, Tilley, L. 2, Becker, K} 1Interdisciplinary Research Center; Giessen University; Giessen; Germany 2Department of Biochemistry; La Trobe University; Melbourne; Australia

The malarial parasite P. falciparum has to deal with reactive oxygen species derived from toxic free ferriprotoporphyrin IX (FP) which is a by-product of hemoglobin degradation. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited and the build-up of redox-active free FP is thought to be a key mechanism in parasite killing. Apart from its direct toxicity, free FP might, however, also interact with parasitic enzymes thereby modulating their activity. This has been shown to be the case for Pf glutathione S- transferase before 1.

In the study presented here we investigated the effects of free FP on different (antioxidant) enzymes of the parasite 2. Interestingly, Pf glutathione reductase (GR) was less susceptible to FP than its human counterpart. Addition of 25 ~JM FP to the NADPH reduced enzyme resulted in an immediate activity loss of 20% (PfGR) in comparison with 60% for hGR. In contrast, Pf thioredoxin reductase (PfTrxR) was strongly inhibited by FP. Also Pf glutaredoxin was shown to be moderately sensitive to FP. The activity of Pf glyceraldehyde 3-phosphate dehydrogenase was inhibited by FP with a Ki value of only 0.2 mM, while RBC GAPDH was much less sensitive. The data are consistent with the suggestion that low micromolar concentrations of FP may have a regulatory function in malarial parasites. The exquisite sensitivity of PfGAPDH and PfTrxR to FP suggest a metabolic constellation in which glycolysis s and DNA synthesis (requiring reduced Trx or Grx) are suppressed whereas the flux through the pentose phosphate pathway as well as the function of GR are favored. This scenario would ensure sufficient production of reducing equivalents to counteract the oxidative stress induced by FP.

1. Harwaldt et al., 2002, Biol Chem 383:821-30

2. Campanale et al., 2003, JBC 278:27354-61

3. Famin & Ginsburg 2003, Parasite 10:39-50

Two small secretory molecules affect development of Plasmodium sporozoites in hepatocytes M011er, A.-K. 1 t Camargo, N. 2, Matuschewski, K. 1, Kappe, S. 2 IHygiene Institut, Abteilung Parasitologie; Universit~t Heidelberg; Heidelberg; Germany 2Seattle Biomedical Research Institute; Seattle Biomedical Research Institute; Seattle; United States

Plasmodium salivary gland sporozoites are transmitted via the bite of an infected Anopheles mosquito, which releases the sporozoite stage into the skin. Sporozoites enter the bloodstream and, upon reaching the liver hepatocytes, develop into exo-erythrocytic forms (EEF). Salivary gland sporozoites and EEFs are suitable targets for immuno- and drug prophylaxis because they precede the development of the pathogenic blood stages. Liver stages can be considered the terra incognita of the Plasmodium life cycle since very little is known about gene expression in liver stages and how they interact with host hepatocytes. Plasmodium sporozoites follow a developmental program on their way from midgut oocysts to the transmission stage. We previously identified a set of transcripts that are specifically upregulated in infective salivary gland sporozoites (UIS genes). Two UIS genes, UIS3 and UIS4, are abundantly expressed in mature sporozoites and encode members of a family of small secretory molecules. One of these proteins is transferred to the parasitophorous vacuole membrane (PVM) of early liver stages and is continuously present in the PVM throughout liver stage development. Expression of the protein can not be observed in blood stages. Our ongoing work explores the function of UIS3 and UIS4 through reverse genetics, thus enabling us to assess the relevance of these genes in the entire Plasmodium life cycle.

103


Survival of host hepatocytes depends on the developmental stage of Plasmodium berghei parasites Heussler, V. ~ t, Fr6hlke, U. ~ lAG Malaria; Bernhard-Nocht-Institut; Hamburg; Germany

Plasmodium sporozoites infect hepatocytes and develop within several days to large schizonts. Finally thousands of merozoites are produced and released in the blood stream where they invade red blood cells. Little is known on how the host hepatocyte reacts to the enormous growing parasite and how the merozoites are liberated. In previous work we have demonstrated that P. berghei-infected hepatocytes do not show significant changes in the expression, activation or the distribution of pro-and anti-apoptotic markers as cytochrome c, caspase 3 and bcl family proteins during the first 2 days of infection. 3 days p.i., however, P. berghei-infected cells undergo apoptosis documented by dramatic morphological and biochemical changes. Parasitised host cells round off and float into the supernatant and host cell nuclei become condensed and fragmented. The infected cell divides in different vesicles resembling apoptotic bodies. Additionally, activated caspase 3 was found in host cells containing merozoites and the cell membrane asymmetry was disrupted shown by positive Annexin V-FITC staining. After several hours in culture P. berghei merozoites are released from apoptotic host cells in an ordered manner and not by disruption of the host cell. Together, these observations confirm that the parasite protect the host cell from apoptosis until merogony. Upon the development of merozoites, however, apoptosis is induced, most likely to facilitate the release of parasites from the host cell. We are now interested to investigate the molecular events responsible for these dramatic changes in the host cell.

Ident i f icat ion and characterisation of Theileria annulata proteins as to their potential to interact with the proteins of transformed host cells Schneider, I. 1 t, Hailer, D. ~, Seltzer, U. 1, Ahmed, j.1 ~Immunologie und Zellbiologie; Forschungszentrum Borstel; Borstel; Germany

-i-heileria parasites possess the unique property of being able to infect and transform the host mononuclear cells. It is postulated that parasite surface or secretory proteins target signal transduction pathways or directly interfere with the cell cycle machinery to initiate and maintain a transformed state of the host cell. To identify such potentially interacting proteins, a strategy was implemented to detect parasite genes containing leader sequences using PCR techniques. Primers were derived from consensus sequences of known T. annulata 5"-signal sequences. Screening of cDNA derived from T. annulata-infected bovine leucocytes resulted in the identification of two unknown parasite-specific genes containing the corresponding membrane leader peptides. Further studies with cDNA derived from different Theileria annulata stages (sporozoite, schizont and piroplasm) revealed that one of the genes (TaSE) is expressed only in the schizont-stage whereas the other one (TAD) is transcribed in all stages of the parasites life cycle. TaSE is a single copy gene with 6 introns resulting in an ORF consisting of 309 amino acids with a molecular weight of about 37 kDa. TaD, on the other hand, is composed of 147 amino acids with a molecular weight of 20 kDa and does not contain introns. Anti-sera raised against the proteins showed specific reactivity only to cell lysate of infected cells (Western blot) with a protein band corresponding to the respective predicted molecular weight. Analysis by confocal microscopy revealed a potential schizont membrane localization of TaD whereas the TaSE-protein could be found within the parasite as well as in the host cell cytoplasm. Potential binding partners, especially of TaSE, remain to be determined, whereby confocal data reveal a close interaction of TaSE with host cell cytoskeleton structures.

The sporozoan parasite Theileria annulata influences p53-mediated apoptotic events during infection of mononuclear cells Hailer, D. 1 ~, Schneider, I. 2, Ahmed, j.2, Seltzer, U. 1 1Research Center Borstel; Immunology and Cellbiology; Borstel; Germany 2Immunology and Cellbiology; Research Center Borstel; Borstel; Germany

The infection of bovine leukocytes by the intracellular protozoan parasite Theileria annulata results in the transformation and uncontrolled proliferation of the host cells. This transformation is dependent on the continuous presence of Theileria schizonts, since after parasite removal with the naphtochinone derivate buparvaquone. The cells subsequently undergo apoptosis. We were particularly interested in parasite controlled mechanisms that prevent transformed host cells from undergoing apoptosis and thus focused our investigations on the role of the transcription factor p53 since this protein is known to induce apoptosis after different stimuli. Using immunofluorescence and confocal microscopy techniques, we found large portions of the p53 protein to be localized in the cytoplasm of Theileria annulata infected cells, which was in contrast to findings in other transformed cell lines like MDBK, Cos-7, and Jurkat, where p53 was predominantly localized in the nucleus of the cells. During incubations with buparvaquone, p53 is translocated in a time dependent manner to the nucleus of the host cells. In subsequent experiments, genes were investigated which are expressed directly under the transcriptional control of p53 (the pro-apoptotic proteins Bax and Apaf-1, and the anti-apoptotic protein Bcl-2). Both mRNA (real time PCR) and protein (Western blot) expression levels of Bax and Apaf-1 were significantly up-regulated, whereas a slight down-regulation on the mRNA and protein levels were determined for Bcl-2 upon buparvaquone treatment. Lastly, incubations with buparvaquone and the p53 inhibitor Pifithrin-Q

104


or the Bax inhibitor BIP (Bax Inhibiting Peptide), respectively, led to a significant reduction of apoptosis as compared to controls.

In vitro induction of Neospora caninum bradyzoites in Vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites Vonlaufen, N, ~, Naguleswaran, A} , MOiler, N. ~, Hemphill, A. ~ t 1Institute of Parasitology; University of Bern; Bern; Switzerland

We report on an optimized method for the in vitro culture of tissue cyst-forming Neospora caninum bradyzoites in Veto cells, and separation of viable parasites from host cells. Treatment of tachyzoite-infected Vero cell cultures with 17 MM sodium nitroprusside for 8 days severely down-scaled parasite proliferation, lead to reduced expression of tachyzoite surface antigens, and induced the expression of the bradyzoite markers NcBAG1 and the cyst wall antigen recognized by the monoclonal antibody mabCC2. TEM demonstrated that intracellular parasites were located within parasitophorous vacuoles that were surrounded by a cyst wall-like structure. Upon induction of stage conversion, the dense granule antigens NcGRA1, NcGRA2 and NcGRA7 got incorporated into the cyst wall. Adhesion/invasion assays employing purified tachyzoites and bradyzoites showed that tachyzoites adhered to, and invaded, Vero cells with higher efficiency compared to bradyzoites. Quantification of adhesion and invasion experiments showed that, in contrast to T. gondii tachyzoites, N. caninum tachyzoites adhered to host cell surface chondroitinsulfate glycosaminoglycans, but chondroitinsulfate was not involved in the actual invasion process. Similar assays also showed that aspartyle protease inhibitors affected invasion of N. caninum tachyzoites but not of T. gondii. However, removal of terminal sialic acid

residues from either the host cell- or the bradyzoite-surface, increased invasion of Vero cells by N. caninum bradyzoites, but not tachyzoites. These studies showed that N. caninum and T. gondii tachyzoites differ with regard to their adhesive and invasive properties, and also demonstrate intrinsic differences between N. caninum tachyzoites and bradyzoites with regard to host cell adhesion and invasion.

Characteristics of Leishmania major-harbouring vacuoles in murine dendritic cells Fuss, V. ~ t, Steigerwald, J} , Moll, H. ~ l Inst i tut for Molekulare Infektionsbiologie; Universit~t WOrzburg; WOrzburg; Germany

Protozoa of the genus Leishmania exist as obligatory intracellular parasites in mammals. They invade macrophages and dendritic cells (DC) where they reside in membrane-bound compartments, called parasitophorous vacuoles (PV). The spezialized functions of both host cells are associated with differences in their interaction with Leishmania. However, most of the present knowledge of the characteristics of PV harbouring Leishmania is derived from studies of infected macrophages. Since emerging data demonstrate that DC contribute an essential part to host resistance against infections, there is a need to understand the properties and biogenesis of the PV in Leishmania-infected DC. Therefore, we used a murine DC line (FSDC) as well as bone marrow-derived DC (BMDC) for infection with L. major and analysis of various endosomal or lysosomal proteins by fluorescence labelling and confocal microscopy. Macrophages were used as controls. L. maJor-containing PV in all cell types had the characteristic of a late endosomal/lysosomal compartment, as hallmarked by the expression of CD68, LAMP1 and LAMP2. However, the biogenesis of PV in DC differed from that in macrophages: The early endosomal marker CD71 disappeared very early after phagosome formation in FSDC and BMDC, indicating a more rapid maturation of the phagosome into a late endosomal compartment in DC as compared with macrophages. Furthermore, hardly any fusion events of the PV with lysosomes could be observed in BMDC, whereas the PV in macrophages and FSDC, which represent an early stage of DC differentiation, had access to lysosomal contents. This block of fusion in BMDC was specific for live promastigotes, as vacuoles containing amastigotes or heat-killed promastigotes fused extensively with lysosomes. In conclusion, the findings show that L. major-containing PV in DC have distinct features that are influenced by the stage of DC differentiation and may be relevant to the specialized host cell functions of DC.

Inhibition of caspase activity by Toxoplasma gondii in a cell-free system indicates novel mechanisms of interference with host cell apoptosis Keller, p.1, Goebel, S. 1, Fischer, S. F. 2, H~icker, G. 2, Gross, U. 1, L0der, C. G. K. ~ t IDepartment of Bacteriology; University of G#ttingen; G#ttingen; Germany 2Institute for Medical Microbiology; Technical University of MOnchen; MOnchen; Germany

Programmed cell death, i.e. apoptosis is an important defence mechanism of the host to counteract the development and replication of intracellular microorganisms. However, several pathogens, including the obligat intracellular apicomplexan parasite Toxoplasma gondii have been shown to interfere with apoptosis induced by a variety of proapoptotic stimuli. In the present study, we investigated the effect of T. gondii on the in vitro- induced activation of the effector caspase 3 after addition of cytochrome c (cyt c) and dATP to cytosolic extracts of human-derived Jurkat T cells. The results show that viable parasites dose-dependently inhibited cyt c/dATP- mediated caspase activation. This indicates for the first time, that T. gondii is able to directly interfere with the apoptotic pathway at the level of apoptosome assembly or caspase 3-activation. The anti-apoptotic activity of T. gondii required the presence of viable, but not necessarily replicating parasites. Furthermore, inhibition of caspase 3-activity was not reversed after treatment of 7-. gondii with cycloheximide suggesting that de novo

105


protein synthesis is not required to exert this effect. A PBS-soluble parasite lysate mimicked the inhibitory activity of viable 7-, gondii on cyt c/dATP-mediated caspase 3-activation. Importantly, excretory-secretory antigens (ESAs) from 7-. gondii, which were prepared by incubation of extracellular parasites with serum and/or ethanol also inhibited the apoptotic signalling pathway. Thus, release of T. gondii proteins from one of the parasites' excretory/secretory organelles possibly mediates interference with the cyt c/dATP-induced activation of the caspase cascade. In conclusion, interference of 7-. gondii with the apoptosis signalling cascade in a cell- free system provides a valuable tool to study the interaction of the parasite with apoptosis of its host cell.

The effect of Trogli tazone in Trypanosoma brucei Denninger, V. 1, Figarella, K}, Duszenko, M. ~* ZPhysiologisch-Chemisches Institut; Universit~t TElbingen; TObingen; Germany

I t has been shown that short stumpy parasites produce PGD2 from arachidonic acid (Kubata et al. 2000). The effect of PGD2 on bloodstream form trypanosomes was recently studied and shown to induce apoptosis (manuscript submitted). Looking for a putative receptor, we considered PPARy, because this protein is a transcription factor in many eukaryotic cells, and 15-deoxy-PGJ2, a metabolite of PGD2, is one of its natural ligands. For experimental research we chose a synthetic agonist called troglitazone. This compound belongs to the family of thiazolidindiones and is already approved as an antidiabetic drug in USA. Troglitazone inhibited the growth of T. brucei bloodstream form but did not induce cell death. Using FACS-analysis of TMRE stained cells, a marker for mitochondria membrane potential, we found only a minor hyperpolarisation instead of hypopolarisation, which would be a marker for apoptosis. Morphology was also studied by fluorescence microscopy.

Differentiation from slender to stumpy bloodstream form is a prerequisite for formation of procyclic insect forms after a bloodmeal. Since light microscopy indicated stumpy morphology induced by troglitazone treatment, we used a transformation protocol to check for this possibility. Thus we treated slender bloodstream forms of strain MITat 1.2 for 16h with or without troglitazone. Cells of all these cultures were transferred to transformation media containing citrate and cis-aconitate. Appearence of procyclic forms was monitored by cell growth, Western blotting to detect procyclic proteins, and morphology. Cells were transferred to procyclic media after 48h. While control cells grew up to lx105 cells ml-1 and died quickly thereafter, troglitazone trated cells reached lx107 cells ml-1 showing clear procyclic morphology. The observed data are consistent with a troglitazone induced differentiation from slender to stumpy forms.

Aquaglyceroporins of Trypanosoma brucei Uzcategui, N. z, Palmada, M. 2, Szallies, A} , Lang, F. 2, Duszenko, M, ~ t ~Physiologisch-chemiches Institut; Universit~t TC/bingen; TE/bingen; Germany 2physiologisches Institut; Universit~t TElbingen; TE/bingen; Germany

Trypanosoma brucei, the causing agent of African sleeping sickness, rely exclusively on glycolysis to obtain energy. Under anaerobic conditions, glucose is quantitatively converted to equimolar amounts of glycerol and pyruvate as the final products of glycolysis. It has been shown that the intracellular increase of both metabolites are toxic for this parasite, which must thus be removed in order to avoid cell death. Previously, we have demonstrated by biochemical tools that glycerol transport in Trypanosoma brucei occurs by specific membrane proteins (Wille et al 1998). Now, we have cloned three putatives aquaglyceroporin genes (TbMIP1, TbMIP2, TbMIP3). Aquaglyceroporins belong to the Major Intrinsic Protein (MIP) family and are channels permeated by small non-ionic molecules in the size range between water to glycerol. TbMIP1 and TbMIP3 contain two highly conserved NPA-box motifs, which form the pore. TbMIP2, on the other hand, contains a NPS motif instead, which is found only in some channels, and even the leucine residue that follows this motif is unique, as all other channels investigated so far, show an arginine residue in this place. In order to perform a functional characterization, we have heterologously expressed these proteins in yeast and in Xenopus oocytes respectively. Yeast expressing TbMIP1, 2 or 3, was able to grow in isotonic and hypertonic conditions. However, they could not accumulate glycerol and the most of the compound, produced intracellularly, was released to the medium. Xenopus oocytes, expressing TbMIP1, 2 or 3, were able to take up glycerol, indicating that these proteins are involved in efflux and influx of glycerol in 7-. brucei. In addition, using a swelling oocytes assay, we determined that others polyols and water may also permeate the MIP channels of 7-. brucei. Currently, we study their localization within the cell, and investigate their roles in metabolism and osmoregulation.

Establishment of knock-down approaches in adult Drosophila melanogaster Gerber, S. 1 *, H0bner, K. 1, Gunkel, N. 1, Seizer, P. M?, Wolf, C} 1Target Discovery; Intervet Innovation GmbH; Schwabenheim; Germany 2Intervet Innovation GmbH; BioChemInformatics; Schwabenheim; Germany

Parasitic arthropods, such as ticks and fleas, cause great damage to companion and domestic animals. We are interested in identifying novel compounds directed against specific gene products of these parasites. The model organism Drosophila melanogaster was chosen for an in-depth molecular analysis. Two techniques were established and evaluated to examine the knock-down of gene products and the effect of compound action on the most interesting adult stage. We were particularly interested in using RNA interference (RNAi) as a tool to

106


test the functional role of genes in adult flies. To bypass the complexity of generating transgenic flies and to exclude early effects caused by embryonic injection, we evaluated a system to inject dsRNA into adult flies. In a first attempt we generated flies which were transgenic for the luciferase gene (firefly) and tried to repress the luminescence by injection of luciferase dsRNA. In addition to these measurements of protein activity, we also analysed the effects on the level of mRNA by RT-PCR. In a second step we focused on the endogenous gene Phosphofructokinase. In this approach we injected dsRNA of gene fragments and measured potential effects by RT-PCR. For further experiments we cloned an RNAi construct in order to investigate RNAi in vivo. In addition we injected known insecticidal compounds into Drosophila melanogaster. In comparison to oral applications, the injection method led to increased effects, regardless of the mode of action of the tested compounds. This was tested by the injection and oral application of different compound concentrations. These experiments demonstrated that adult Drosophila melanogaster are suitable for using as model organisms for insecticidal compound action.

Conformation and function of a lipid binding protein AgFABP from the parasitic nematode Ascaridia gall i Jordanova, R. ~*, Radoslavov, G. ~, Fischer, p.2, Liebau, E. 3, Walter, R. D?, Bankov, I. 1, Boteva, R. 4 ~Biochemistry; Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences; Sofia; Bulgaria 2Helminthology; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 3Biochemistry; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 4Institute of Molecular Biology; Bulgarian Academy of Sciences; Sofia; Bulgaria

AgFABP, a 12 kDa lipid binding protein (LBP) from Ascaridia galli, is a member of the Nematode Polyprotein Allergen / Antigen (NPA) family. The spectroscopic analysis of the protein allowed characterization of its conformation and ligand binding activities. The CD spectra of AgFABP showed a highly ordered, predominantly alpha helical secondary structure, which did not significantly change upon ligand binding. The fluorescence properties of AgFABP were due to its highly conserved single Trp residue (Trp17) and three Tyr residues. Analysis of the efficiency of the energy transfer between these chromophores showed a high degree of the Tyr-Trp dipole-dipole coupling. AgFABP has a single hydrophobic binding site and a high affinity for different ligands. The conjugation of the protein with fatty acids or retinol was accompanied by enhancement of the Trp emission, which allowed calculation of the affinity constants of the binary complexes. By FRET the distance of 1.41 nm between the single Trp of AgFABP and the fluorescent fatty acid analogue DAUDA was estimated. A chemical modification of the Cys residues of AgFABP (Cys66 and Cys122) with the fluorescent marker, dansylated iodacetamide IAEDANS, followed by MALDI TOF analysis showed that only Cys66 was labelled. The observed similar affinities for fatty acids of the modified and non-modified AgFABP suggest that Cys66 is not a part of the protein binding pocket but probably is located close to it. AgFABP is one of the most abundant proteins in A. galli and by immunohistology and immunogold electron microscopy it was found to be extracellulary distributed in most tissues of the parasite, suggesting that AgFABP could play an important role in the transport of fatty acids and retinoids. According to the restricted lipid metabolism we propose that AgFABP is essential for the survival of the parasite.

Caenorhabdit is elegans as a model system for the investigation of stress responsive proteins of parasitic nematodes Burmeister, C. ~, Leiers, B. 2, Walter, R.D. 1, Liebau, E. 1 * ~Biochemistry; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany 2Institute for Genetics; University of D~sseldorf; DOsseldorf; Germany

It is not possible to elucidate the function of stress responsive proteins in the filarial nematode Onchocerca volvulus, since it cannot be maintained in the laboratory and genetic manipulations cannot be performed. This inaccessibility is a common problem in working with parasitic nematodes; the possibility of transgenic overexpression, promoter-reporter constructs and RNA interference (RNAi) predestinates Caenorhabditis elegans as model system to investigate stress responsive enzymes in nematodes and draw conclusions about the biological function of the enzyme in the parasite. The glutathione S-transferases (GSTs) are a family of phase IT enzymes that utilize glutathione in reactions contributing to the transformation of a wide range of exogenous and endogenous compounds, including carcinogens, therapeutic drugs, and products of oxidative stress. The Omega-class GST - C29E4.7 - is a candidate for the protection against oxidative stress in C. elegans. Following affinity-chromatography, the enzymatic activity of the recombinantly expressed enzyme was determined using several model substrates. The ,halo assay" provided a test of resistance to long-term exposure to oxidative stress conditions. Here it was demonstrated that the survival of bacteria, overexpressing C29E4.7, significantly increased under several stress conditions. The specific silencing of the C29E4.7 by RNAi created worms with an increased sensitivity to the stressor Juglone. Furthermore, under ,knock down" conditions, a reduced lifespan was observed. Promoter-GFP (green flourescent protein)-constructs Iocalised C29E4.7 expression exclusively in the intestine of all post- embryonic stages. To determine the mechanism and factors regulating the expression of this stress responsive GST, promoter deletion and mutation analysis are on the way.

107


Unraveling the role of the Leishmania 100 kD Heat Shock Protein Klaholz, L. 1 t, Wiesgigl, M}, CIos, J.~ 1Parasitology Section; Bernhard-Nocht-Institute for Tropical Medicine; Hamburg; Germany

During vector to host transmission, parasites of the genus Leishmania encounter heat stress and an acidic milieu in the phagolysosome of the hosts macrophage. Triggered by these two stimuli the parasites undergo a stage differentiation towards the amstigote stage.

Induced by the increase in temperature the expression of the heat shock proteins is enhanced. The expression of HSP70 and HSP90 is increased from a background of strong constitutive expression in the promastigote stage. By contrast, HSP100 is specifically expressed in the mammalian stage of the parasite. Replacement of the HSP100 gene renders the parasites avirulent in mice. The delta hsplO0 mutants also show irregular morphology after infection of macrophages in vitro. This indicates that HSP100 is crucial for the parasites' intracellular survival. The cellular pathways that require HSP100, however, remain unidentified as yet. Therefore, we took two approaches to unravel the role of HSP100. 1) In order to identify gene loci that, upon overexpression, compensate for the loss of HSP100, we constructed a cosmid DNA library from L. major delta hsplO0 mutants and transfected this library into the same mutant strain. These mutants were inoculated into mice, and restoration of virulence to the delta hsplO0 mutants was observed. The recombinant parasites were reisolated from infected tissue, and the cosmids they harboured were analyzed to identify genes the amplification of which rescue the delta hsp100 phenotype. 2) We started a proteomics approach to analyze differences in the protein patterns of wildtype and delta hsplO0 L. donovani. The differences we observe concern mainly other HSPs such as HSP70 and HSP90, which seem to associate with protein aggregates in the wildtype, but not in the delta hsplO0 strain. This indicates a crucial role for HSP100 in the recruitment of other heat shock proteins to denatured proteins.

Changes in substrate preferences, glucose uptake and enzymatic activities in a glibenclamide- resistant Leishmania mexicana strain, Uzcategui, N. 1, Figarella, K}, Camacho, N. 2, Ponte-Sucre, A. 3 t ~Instituto de Medicina Experimental und Physio/ogisch-chemisches Institut; Universidad Central de Venezuela und Universit~t TObingen; TObingen; Germany 2Instituto de Medicina Experimental; Universidad Central de Venezuela; Caracas; Germany 3Universidad Central de Venezuela und Universit~t WOrzburg; Instituto de Medicina Experimental und Institut for Molekulare Infektionsbiologie; WOrzburg; Germany

ATP-binding-cassette (ABC)-type drug transporters identified in microorganisms including Leishmania are divided into the gene-encoded multidrug resistance P-glycoprotein and the drug resistance-associated protein plasma membrane transporters. Their over expression occur as a result of chemotherapy and questions have been raised on whether and how ABC-type drug transporters can be targeted to improve patient treatment. Drug resistance conforms a plethora of responses with a complexity higher than previously expected that may involve biochemical and functional parasite mechanisms. To further our understanding on the relevance of these additional mechanisms in Leishmania, we have explored in an in vitro selected Leishmania strain [NR(Gr)] resistant to GLIBENCLAMIDEO, an ABC-transporter blocker, if chemo-resistance induces changes in Leishmania substrate preferences, including glucose uptake through the plasma membrane. Our results suggest that resistance to GLIB altered the substrate preferences of Leishmania parasites, decreased the expression of glucose transporters and increased significantly the activity of glycolytic enzymes as hexokinase and phosphoglucose isomerase as well as glutamate deshidrogenase, which supplies substrates to fuel the Krebs cycle. These results indicate that in Leishmania, a glycolytic metabolite imbalance and a preferential utilization of oxidative phosphorylation ,could be a metabolic consequence of drug resistance. These results stress the importance of identifying not only the mechanisms directly involved in drug resistance but also the physiological changes that occur in Leishmania as a consequence of, or concomitantly with, the development of this phenotype.

Trypanosoma brucei transferrin receptor mRNA contains iron-responsive elements in the 5"-UTR Ngazoa, E. S. 1, Kabiri, M. 2, Becket, K}, Steverding, D. 3 t 1Interdisciplinary Research Center; University of Giessen; Giessen; Germany 2Department of Parasitology; University of Heidelberg; Heidelberg; Germany 3School of Biological Sciences; University of Bristol; Bristol; United Kingdom

Bloodstream forms of Trypanosoma brucei require iron for growth. All iron is delivered by transferrin of the host that is taken up via receptor-mediated endocytosis. The transferrin receptor (TfR) of T. brucei is a heterodimeric protein complex encoded by ESAG6 and ESAG7. These genes are located upstream of the variant surface glycoprotein (VSG) gene in a polycistronic telomeric expression site. Although the trypanosomal TfR bears no structural similarity with the mammalian TfR, the expression of both receptors is regulated by the availability of iron. Here we show, that the iron-dependent expression of the trypanosomal TfR is probably controlled by elements in the 5 "-untranslated region (UTR) of ESAG6 and ESAG7. Using folding algorithms for RNA secondary structure prediction, similar stem-loop structures were identified in the 5'-UTR of

108


both ESAG6 and ESAG7 mRNA. To prove that these stem-loop structures function as an iron responsive element involved in the iron-dependent regulation of the trypanosomal TfR, the ESAG6 of the active expression site was replaced with the neomycin gene to give AESAG6::NEO mutants. A second cell line was generated in which also the 5'-UTR of the ESAG6 was partially deleted (nucleotides -16 to -35 of the identified stem loop) to give Z~(-16/-29)5"utrZ~ESAG6::NEO. After selection of stable transfectants, the effect of iron depletion on the expression of neomycin in both mutant cell lines was investigated. Whereas the amount of neomycin in L~ESAG6::NEO cells was regulated by iron availability, the expression of neomycin in A(-16/- 29)5"utrZ~ESAG6::NEO cells was unaffected by iron depletion. These results strongly support that the identified stem-loop structure in the 5'-UTR of ESAG6 mRNA functions indeed as an iron responsive element.

Expression and transport of Toxoplasma gondii protein-complexes in Giardia duodenalis Gaechter, V. 1 t, Deplazes, p.2, Hehel, A. 2 ~Parasitologie; ZOrich; ZOrich; Switzerland; 2; Parasitologie; ZOrich; Switzerland

T. gondii is a protozoan parasite that promiscuously infects a wide variety of hosts and tissues. As a well- adapted intracellular parasite it produces an asymptomatic infection in most susceptible hosts but it can cause severe pathology if acquired during pregnancy or in immunocompromized individuals. Despite many years of research and development, there is still no working subunit vaccine available against Toxoplasma infection. Recently a novel expression system was developed in our lab which enables us to produce correctly folded and therefore immunoreactive membrane-anchored T. gondii SAG-antigens in the axenically growing protozoan parasite Giardia duodenalis. In the present project we applied this method to the microneme protein- complexes, which play an essential role in parasite motility and host-cell invasion. Normally microneme proteins are transported as complexes to the plasma membrane whereby one protein (e.g. TgMIC6) serves as an escorter for the others. First results with ectopic expression of a plasma membrane targeted TgMIC6 variant in Giardia, indicated that it can be expressed efficiently, but is retained in the ER. The failure of the TgMIC6 escorter protein to be exported in transgenic Giardia could be explained with the absence of two soluble proteins TgMIC1 and TgMIC4, which form a stable complex with TgMIC6 in Toxoplasma. This is consistent with data of Reiss et al. 2001 and Soldati et al. 2001 showing that deletion of TgMIC1 has deleterious effects on assembly of the complex and its proper targeting. Based on these results we are expanding the capacity of the Giardia expression system to accommodate all members of this protein-complex, with the aim to obtain a biologically functional and properly targeted TgMIC1/4/6 complex.

Biochemical characterization of LmxMPK1, a mitogen-act ivated protein (MAP) kinase homologue essential for survival of Leishmania mexicana amastigotes - at tempts to identify protein kinase su bstrates Melzer, I. M. ~ t, Schuldt, K. ~, Wiese, M. ~ iParasitology section; Bernhard-Nocht-Institute for Tropical Medicine; Hamburg; Germany

A MAP kinase homologue from Leishmania mexicana, LmxMPK1, was found to be essential for the survival of the parasite in the infected host. However, the deletion mutant of LmxMPK1 was still able to infect macrophages in vitro followed by differentiation from promastigote to amastigote morphology, but failed to proliferate afterwards, thus, losing the capability to cause the disease in Balb/c mice. We overexpressed a His-tagged form of LmxMPK1 using the Baculovirus system in Sf9 cells and purified the enzyme by means of affinity chromatography with different metal ions. Activity assays with radiolabeled ATP were performed to determine the specific properties of the protein kinase. We determined pH- and temperature-optima, ionic composition of the reaction buffer and analysed the stability of the kinase under different storage conditions. To search for possible substrates, the recombinant enzyme was used to screen a h-phage expression library of Leishmania mexicana cDNA in a solid-phase phosphorylation assay with radiolabeled ATP. Here, we show the results of the characterization as well as the procedure and results of the screen for substrates of LmxMPK1.

Glutathione synthesis in parasitic nematodes: Caenorhabditis elegans as a model system Ajonina, C. 1, Sommer, A. 1, LQersen, K. 1, Liebau, E. 1, Walter, R. D. 1 t 1Biochemistry; Bernhard Nocht Institute for Tropical Medicine; Hamburg; Germany

The low-molecular mass thiol, glutathione (GSH), plays an important role in the maintenance of the intracellular thiol redox state and in detoxification processes. Apart from the GSH redox cycle, the control mechanism for the maintenance of intracellular GSH levels is the de novo synthesis of GSH. Synthesis involves two consecutive enzymatic reactions. Ligation of glutamate and cystein, the rate limiting step of the de novo synthesis, is catalysed by gamma-glutamylcystein synthetase; the addition of glycine is catalysed by GSH synthetase (GS). GSH depletion has been discussed as a chemotherapeutic strategy for parasitic nematodes and the GS is proposed as a potential drug target. This is the first study investigating a GS from nematodes. By BLAST search we identified the GS from Caenorhabditis elegans (Ce-GS). The deduced amino acid sequence shares only a moderate degree of identity with other known GSs, but the residues responsible for substrate and co-factor binding are all conserved. Ce-

109


GS was cloned and expressed recombinantly in Escherichia coll. The purified enzyme exhibits a moderate activity. The protein is active as a dimer, with a subunit molecular mass of 55 kDa. Transgenic C. elegans strains were generated by microinjection and GFP (green flourescent protein)-constructs Iocalised expression of Ce-GS in the intestine and the excretory system of all post-embryonic stages. The specific silencing of Ce-GS by RNA interference created worms with an increased sensitivity to oxidative stress. The minimal promoter region was determined by deletion constructs, mutational analysis of the promoter region are on the way. Work is in progress to isolate the GS from Onchocerca volvulus.

Comparat ive examina t ion of the expression of t issue cyst markers in Hammondia sp. isolated from dogs and foxes Meyer, j1 , Riebe, R. 2, Conraths, F. J.1, Bohne, W?, Rohn, K. 4, Peters, M. ~, Schares, G. 1 t 1Institute of Epidemiology; Federal Research Centre for Virus Diseases of Animals; Wusterhausen; Germany 2Institute of Infectology; Federal Research Centre for Virus Diseases of Animals; Greifswald-Insel Riems; Germany 3Department of Bacteriology; University of GOttingen; G~tUngen; Germany 4Institute of Pathology; Tier~rztliche Hochschule Hannover; Hannover; Germany 5Pathologie; Staatliches Veterin~runtersuchungsamt Amsberg; Arnsberg; Germany

A cell culture system was recently established to study the development of tissue cysts in Hammondia sp. (Schares et al. 2003). This system was used to compare the expression of tissue cyst markers in two Hammondia sp. isolates, Hammondia heydorni (Giessen-1999) and Hammondia sp. (FOX-2000/1). The spontaneous development of tissue cysts in cell culture was assessed by using polyclonal antibodies against Toxoplasma gondii bradyzoite antigen 1 (Anti-BAG1), rat monoclonal antibodies against a cyst wall protein (MAbCC2) and a lectin (Dolichos biflorus agglutinin (DBA)). In both parasites BAG1 is expressed 2-3 days earlier than the other tissue cyst markers. Single parasitophorous vacuoles containing both, BAGl-positive and negative parasites suggest that bradyzoite development is asynchronous in a cloned intravacuolar population. In both Hammondia sp. isolates, FOX-2000/1 and Giessen-1999, MAbCC2 positive vacuoles (i.e. tissue cysts) were first seen 6 or 9 days after the infection of the cell culture. High proportions of MAbCC2 positive vacuoles (>=70% of all vacuoles containing >1 parasites) were already observed 10-11 days (FOX-2000/1) or 14 days (Giessen-1999) post infection. After one to two months post infection more than 95% of the vacuoles were MAbCC2 positive. The development of DBA-binding carbohydrates followed a similar pattern. The results show that both parasites spontaneously develop high proportions of tissue cysts in cell culture. Our finding that tissue cyst markers established for T. gondii cross-react suggests that cell culture-derived Hammondia tissue cysts could help to further study the composition and the maturation of tissue cysts in protozoan parasites of importance for human and veterinary medicine, i.e.T, gondii and Neospora caninum. Reference: Schares et al. 2003, A Hammondia-like parasite from the European fox (Vulpes vulpes) forms biologically viable tissue cysts in cell culture, Int. J. Parasitol. 33, 229-234.

Structure-based mutat iona l analyses of the glyoxalase I f rom Plasmodium falciparum Haase, S. 1, K0hnl, J}, Walter, R. ~, Torda, A. 2, Liebau, E. 2 ~ ~Biochemistry; Bernhard Nocht Institute for Tropical Medicine; 20359; Germany 2Zentrum for Bioinformatik; University of Hamburg; Hamburg; Germany

The glyoxalase (GIo) system, composed of GIo I and GIo II, catalyses the conversion of 2-oxoaldehydes into the respective 2-hydroxyacids. The predominant function of the system lies in the glutathione-dependent conversion of cytotoxic and mutagenic methylglyoxal, produced as a glycolysis by-product, to D-lactate. To meet their high energy needs, Plasmodium falciparum have a high rate of anaerobic glycolysis, a high flux of glucotriose and proposedly a concomitant high rate of formation of methylglyoxal. I t is proposed that by inhibition of the GIo system, the concentration of methylglyoxal would rise to toxic levels, making the system an attractive target for the development of novel antimalarial drugs. The glo I gene of P. falciparum is localized on chromosome 11. The GIo I from P. falciparum (Pf-GIo I) has a different protein structure to its host's counterpart. In mammals, the GIo I is a 43 kDa dimer composed of two identical subunits, while in P. falciparum it is a monomeric protein derived from fusion of these monomers. Monomeric GIo I have been identified in yeast and some plants, where several variations have evolved through the process of gene duplication and 3D domain swapping. The Pf-GIo I has been identified and characterised. Recombinant expression of an active Pf-GIo I in Escherichia coil was accomplished. Modelling of the tertiary structure suggests, that the enzyme has two active sites. By site directed mutagenesis, each active site was inactivated to investigate its contribution to the overall enzymatic activity.

110


Role of PUF-proteins in gene expression in Trypanosoma brucei Luu, V.-D. 1, Clayton, C. ~, Guilbride, L. ~ t 1ZMBH; Universit~t Heidelberg; Heidelberg; Germany

Control of gene expression in trypanosomes is mainly post-transcriptional and heavily dependent on mechanisms affecting transcript stability. Most known cis-regulatory elements determining mRNA stability are located in the 3'-untranslated regions (3'-UTRs). Trans-acting factors that bind these, modulate transcript stability and largely mediate the response of mRNA molecules to the intracellular environment. Members of the PUF family are 3'-UTR regulatory proteins that generally repress expression of multiple target transcripts by control of mRNA-degradation or interference with translation. In addition to the previously documented TbPUFl-protein (Cross et al., 2002) database searching identified three new putative PUF genes in the T. brucei genome: Tbpuf2, Tbpuf3, and Tbpuf4. The effect of overexpression and RNAFknockdown of these proteins on gene expression is analysed by microarray, northern blot analyses and growth phenotype. Microarray results indicate that at least three transcripts are upregulated by overexpression of TbPUF1. Similar experiments investigating the roles of TbPUF2, TbPUF3, and TbPUF4, are in progress. We will further identify putative PUF-interacting partners using the Tandem Affinity Purification (TAP) method. Together these data should supply insight into part of the local and global 3'UTR-protein communication network that primarily controls gene expression in trypanosomes.

Functional characterization of TLP-1, a novel member of the TRAP-family in Plasmodium Hei6, K. ~ t, M011er, A.-K. ~, Matuschewski, K. 1 iHygiene Institut, Abteilung Parasitologie; Universit~t Heidelberg; Heidelberg; Germany

Plasmodium host cell entry is an active actin dependent process that is mechanically linked to a specialized form of locomotion, termed gliding motility. All invasive stages require specific proteins on their surface to recognize and infect their target cell. In the case of sporozoites, thrombospondin-related anonymous protein (TRAP) fulfils this function. Proteins of the TRAP-family are typeI transmembrane proteins and defined by homologous regions in their extracellular part, the A-domain and thrombospondin repeat (TSR) and a conserved cytoplasmic domain that confers binding to the actin myosin motor machinery. Sequence analysis revealed a transcript that shows high similarities to TRAP concerning the extracellular domain. Additionally, TRAP-like protein-1 (TLP-1) contains conserved residues in the cytoplasmic tail of TRAP- family members. Gene expression analysis revealed transcription of TLP-1 in red blood cell stages and sporozoites. In order to identify the function of TLP-1 in the life cycle of Plasmodium we use the advantage of reverse genetics. Specifically, we test if TLP-1 performs vital functions during erythrocytic schizogony and if TLP-1 is a functional paralogue of TRAP by tail swop experiments.

Characterisation of the gonad-preferentially expressed diaphanous homolog from Schistosoma mansoni M0nnich, M. 1 ~, Knobloch, J.1, EI-Bahay, A} , Kapp, K. 2, Lammers, R. 2, Grevelding, C. G. ~ 1Institute of Genetics; Heinrich-Heine-University; DElsseldorf; Germany 2Medizinische Klinik IV, Universit~tsklinikum TElbingen; Eberhard-Karls-University; TElbingen; Germany

For sexual maturation, female schistosomes need pairing-contact with a male. Some studies indicated that males control the transcription of genes, which are involved in the differentiation of the reproductive organs of the female. In addition, evidence was obtained that signal transduction processes are involved in the male- female interaction. With the Src-like tyrosine kinase SmTK3, a signalling molecule was isolated from S. mansoni, which, according to Iocalisation studies, may have a function in the gonads. A schistosome yeast-two- hybrid library was screened to identify binding partners of SmTK3. Their identification could provide first hints towards a potential function of TK3, because signalling molecules are conserved. Using the SH3 domain of TK3 as a bait to identify potential downstream binding partners, a member of the FH (formin homology) protein family was identified. FH proteins are involved in actin-mediated processes controlling cell and tissue architecture, cell polarity, cell-cell interaction and cytokinesis. The FH homolog from S. mansoni belongs to the subfamily of diaphanous related formins (DRFs). The Drosophila DRF-homolog was shown to be involved in spermatogenesis, oogenesis and embryogenesis. Since only part of the sequence of the diaphanous homolog of S. mansoni was isolated by the screen, full-length cDNA cloning was achieved by genomic walking. In situ hybridisations confirmed the expression of the diaphanous gene in vitelline cells, oocytes and in the spermatocytes. Here, TK3 is also expressed. In mouse, it was demonstrated that DRF proteins couple Rho and Src during signalling. Since a Rho homologue was identified in schistosomes, it seems likely that a signal transduction pathway including TK3, SmDRF and Rho exists in S. mansoni. To check this hypothesis, protein interactions between Rho and SmDRF were tested by yeast-two-hybrid analyses. To further characterise SmDRF, Northern-, Southern Blots and RT-PCR analyses have been performed.

111

Scientific Program DGP, 21st Annual Meeting March 17 th - -20 th, 2004 in WQrzburg

Identi f icat ion of binding partners of the protein tyrosine kinases TK3 and TK4 of Schistosoma mansoni Philipp, C} t, Knobloch, J.~, Sroka, S}, Grevelding, C. G. ~ ~Institute for Genetics; Heinrich-Heine-University; DBsseldorf; Germany

Schistosomes are the only trematodes which have developed separate sexes. Male and female schistosomes live as couples in the blood streams of their hosts, and the sexual development of the female essentially depends on the pairing contact with the male. Although the male stimulus for female maturation is as yet unknown, it was hypothesised that it may be a chemical factor inducing mitogenic and/or differentiation processes in the female. Because such biological processes are controlled by signal transduction molecules, we started to identify and characterise signalling proteins. Among these, the protein tyrosine kinases (PTKs) are known to be key players in cell proliferation and differentiation. Using a PCR approach with degenerate primers, different PTK genes were isolated. Two of them, the Src-like TK3 and the Syk-like TK4 were analysed in detail. TK4 was shown to be expressed in the testes of males the ovary of females, but not in the vitellarium. TK3 also revealed gonad-preferential expression, but including the vitellarium. To gather experimental indications for a function of these molecules, yeast-two-hybrid screenings were performed to identify binding partners in heterologous and homologous systems. Towards this end, the SH2-domains of both PTKs were subcloned as bait constructs to discover the upstream binding partners of TK3 and TK4. SH2-domains bind to phosphorylated tyrosines to receive a signal following receptor activation. Yeasts, however, do not have kinases specific for tyrosines. To overcome this experimental limitation, the tyrosine domain of TK3 was additionally cloned into the bait vector to ensure the phosphorylation of tyrosines for a successful screen. Experiments such as Western Blots and RT-PCRs were done to confirm the expression of both domains prior to the screening. Results will be presented that demonstrate the feasibility of this approach, and the successful identification of potential binding partners of these PTKs.

I n vivo ratiometric pH measurements of Plasmodium fa lc iparum Kuhn, y.1, Rohrbach, P.1, Lanzer, M. ~ t 1ABT. Parasitologie Hygiene-Institut; Universit~t Heidelberg; 69120 Heidelberg; Germany

As a diprotic weak base chloroquine should accumulate in acidic compartments such as the food vacuole. Of the many proposed models of chloroquine resistance one suggests that changes in intracellular pH may lead to drug resistance. However intracellular pH-measurements and calculated pH values have remained controversly. We have succeeded in producing transfectant parasites, of both CQR (Dd2) and CQS (HB3) P. falciparum strains, that express a pH sensitive GFP either targeted to the cytosol or the food vacuole. Targeting of the GFP to the cytosol required no signal sequence whereas targeting to the food vacuole was achieved by using the N- terminal signal sequence'of a putative plasmepsin. Ratiometric pH measurements using the expressed and targeted GFP were performed. It appears that the cytosolic pH exhibits no difference between the CQR strain Dd2 and the CQS strain HB3.

Development of an axenic in vitro cultivation system for Echinococcus mul t i locular is larvae Spiliotis, M. 1, Tappe, D. 1, Sesterhenn, L. 1, Brehm, K. 1 t i Inst i tut f~r Hygiene und Mikrobiologie; Universit~t W(~rzburg; W•rzburg; Germany

Suitable in vitro cultivation systems for parasitic helminths are a necessary pre-requisite to study the molecular basis of host-helminth interactions. Here we report on the development of an in vitro system for long-term cultivation of E. multilocularis larvae under axenic conditions. In the absence of feeder cells from the host, long- term survival of the parasite strictly depended on low oxygen conditions and the presence of reducing agents in the medium. This indicated that E. multilocularis larvae are highly sensitive to reactive oxygen radicals which are formed during conventional cell culture. Host serum supported survival of the parasite but growth of metacestode vesicles and differentiation towards the protoscolex stage only occurred in the presence of culture medium that was preconditioned by hepatoma cells or several other immortal cell lines, thus providing unequivocal evidence that E. multilocularis growth and development strictly depends on soluble growth factors which are released by actively proliferating host cells and which are not already present in serum. Using the axnic culture system, the effect of several host factors such as insulin, epidermal growth factor and bone morphogenetic proteins on the parasite have been investigated. Taken together, the axenic cultivation system reported herein constitutes an important refinement of already existing in vitro culture methods for E. multilocularis larvae and will greatly facilitate the identification of host-derived growth factors for the parasite.

112

Scientific Program DGP, 21st Annual Meeting March 17 th -- 20 th, 2004 in WOrzburg

Identification and characterization of an insulin receptor homologue from Echinococcus multi locularis, Konrad, C. z, Kroner, A } , Tappe, D}, Spiliotis, M. z, Zavala-Gongora, R. z, Brehm, K. 1 t lInstitut fElr Hygiene und Mikrobiologie; Universit~t W[~rzburg; W(Jrzburg; Germany

Receptor tyrosine kinases play a key role in the communication of cells with their environment and could be important mediators of the effects of host cytokines on endoparasitic helminths. Here we describe the identification and characterization of an E. multilocularis receptor kinase, EMIR, with significant homology to members of the insulin receptor family of vertebrates and insects. EmIR displayed an overall domain structure identical to other members of the protein family and shared considerable homologies with the human insulin receptor in the ligand binding (30 % identical aa) and the tyrosine kinase domains (47 %). Analysis of the chromosomal locus encoding EMIR, emir, showed that the insulin receptor genes from insects, mammals and Echinococcus derive from a common ancestor gene. By immune-histochemistry and RT-PCR studies, EmIR was shown to be expressed in the larval stages protoscolex and metacestode during natural infections. In yeast two-hybrid studies, the ligand binding domains of the human insulin receptor and of EmIR showed comparable affinity to human insulin which indicates that EmIR could serve as a parasite derived factor for the detection of host insulin during an infection. This is the first report on a member of the insulin receptor class of a parasitic helminth and provides a solid basis for further studies on the role of insulin signaling in Echinococcus development and on insulin-based communication between parasite and host during alveolar echinococcosis.

Functional characterization of ERM (ezr in/radixin/moesin) signaling mechanisms in the Echinococcus multi locularis larval stage. Hubert, K. 1, Zavala-Gongora, R. 1, Bergmann, M}, Frosch, M. 1, Brehm, K. 1 * lInstitut for Hygiene und Mikrobiologie; Universit~t WElrzburg; WE/rzburg; Germany

In vertebrates, members of the ezrin/radixin/moesin (ERM) protein family act as linkers between the cytoplasmic membrane and the cytoskeleton which involves binding of their N-terminal domains (N-ERMAD) to several membrane bound receptors, and interaction of the C-terminal region (C-ERMAD) to F-actin. By means of these interactions, ERM proteins are involved in several cellular key processes such as cell-cell adhesion, membrane trafficking, microvilli formation and transmembrane signaling. To initiate studies on ERM signaling in invertebrates, we have investigated the functional properties of the E. multilocularis protein EIp (EM10; EM II /3), an immuno-dominant antigen which shares clear sequence homologies with vertebrate ERM proteins. By yeast two-hybrid and immunoprecipitation studies, we demonstrate that the mechanisms of EIp activation are similar to those observed for its vertebrate counterparts. By screening of a yeast two-hybrid expression library, we could identify binding partners for the EIp N-ERMAD and C-ERMAD domains. One of these, PDZ-1, which specifically interacts with the EIp N-ERMAD region, belongs to a family of PDZ-domain containing scaffolding factors and could serve as a bridging protein between EIp and several surface receptors. As a C-ERMAD specific interaction factor of EIp we could identify tropomyosin, a well known regulator of cytoskeleton dynamics. Interestingly, this interaction between an ERM protein and tropomyosin seems to be specific for the parasite (or for invertebrate systems) since we could not detect interactions between tropomyosins and ERM proteins of vertebrates. Hence, our data indicate both conserved and distinct features of ERM signaling mechanisms in E. multilocularis. On the basis of these data, it will in future studies be feasible to identify Echinococcus surface receptors for EIp activation (via PDZ-1) and to closer investigate the biochemical basis of EIp/cytoskeleton interactions involving tropomyosin.

113


Autorenverzeichnis

Ab EI-Rady A. 019VD Achu-Kwi D. 062PV Adam R. 075VI Adrian M. 008PS Affognon H. 0 5 9 W Ahmed J. 157VB

156VB Ahmed J,S. 053VV Ajonina C. 171PB Akoachere M. 134VB Alaeddine F, 092VC Albuquerque S. 083PI

128PG Al~ns R. 111PC AIgner M. 076VI AI-Jawabreh A. 044VE

001VS Ammann P. 079VI ANDREA G. 101VC Arada ib I . 041VE Araujo C.A.C. 129PG Ardana I.B. 063PV Arkush K.D. 055VV Asp~ck H. 042VE

003VS AuerH. 042VE Azzouz N. 110PC

Bachmair A. 120VG Bakheit M.A. 053VV Bakker-Grunwald T. 152VB

149VB 150VB

Bankov I. 164PB Barry M. 0 5 9 W Barutzki D. 0 5 2 W B~rwald A. 052VV

043VE B~sslerT. 067VI Bauer C. 063PV Bauer H. 108VC

099VC Baumeister S. 125VG Baz A. 074VI Becker A. 135VB Becker K. 168PB

103VC 134VB 135VB 153VB

Bente M. 116VG 117VG

Bergmann M. 181PB Betzel C. 104VC Beyer C. 102VC Bienz M. 151VBA Billker O. 146VB Blot C. 108VC Blackhall W. 060PV Blei6 W. 121VG Bocoum Z. 059VV Bohne W. 172PB Bonawitz A. 139VB Boteva R. 164PB Brehm K. 180PB

143VB 181PB 179PB

Brendan C. Brindley P.J.

Bruchhaus I.

Bruhn H.

Burmeister C,

Buschbaum S, B0ttner M.

Camacho N. Camargo N, Campanale N. Choudhury K. Clausen P.H.

Clayton C. Clayton C.E. CIos J.

Conrad P.A, Conraths F.J.

Copeland C.S. Coulibaly B. Cramer J,

Damriyasa I.M. Das Gupta R. Daugschies A. Davioud-Charvet E.

Dematteis S. Denninger V. Deplazes P.

Deterding A. Diall O. Diallo B. Diarra B. Dietz K. Dimitr i jevic B. Dinkel A.

Doering H. Doligalska M. Dorbic T. Dost C.K.

Dr~ger K.

Dr6gem011er M. Duerr H.P. Dungey F.A. Duszenko M,

133VB 121VG 120VG 117VG 116VG 151VB 097VC 065VI 088PI

124VG 165PB 104VC 094VC 062PV

167PB 154VB 153VB 113PC 058VV 059VV 019VD 174PB 114PC 140VB 113PC 166PB 055VV 040VE 046PE 172PB 052VV 043VE 120VG 103VC 102VC

063PV 109VC 061PV 103VC 108VC 099VC 074VI 161PB 169PB 047PE 100VC 059VV 0 5 9 W 0 5 9 W

050PEA 015VD 049PE 013PS 039VE 041VE 050PE 085PI

121VG 128PG 083PI

043VE 046PE 094VC

050PEA 100VC 162PB

Edele F. Eichholz J. Eichner M. Eiras A.E.

EI-Bahay A. EI-Bahay A.O, EImahdi I.E. EI-Matbouli M. Engelmann S. Engels W.

Epe C. Erb K.J. Estzerbauer E. Eubel J.

Fasen W.

Figarella K.

Fischer P. Fischer S.F. Fischer S. Fleischer B. Flieger A.

FIockenhaus B. Franzen C.

Friedrich O.

Fritz K, Fr~hlke U. Frosch M. Fuss V.

Gaechter V. Gall Y, Gaworski I. Geier M.

Geiger S. Gelhaus C.

Gerber S. Goebel S. Goretzki J. Gottstein B,

Grabe K. Grace D. Graefe S. Greiser-Wilke I. Grevelding C.G.

Gross U,

Gr6tzinger J. Gruenfelder C. Guilbride L. Gunkel N.

161PB

141VB 096VC

050PEA 021VD 018VD 176PB

121VGA 013PS 056VV 127VG 083PI

128PG 005VS 078VI 056VV 103VC

043VE 046PE 161PB 167PB 164PB 160VB 035PD 071VI 147VB 027VD 150VB 069VI 035PD 012PS 137VB 107VC 135VB 155VB 181PB 159VB

169PB 058VV 071VI

021VD 018VD 062PV 126VG 115PC 163PB 160VB 040VE 093VC 079VI 074VI

025VD 0 5 9 W 071VI

057VV 142VB

121VGA 177PB 176PB 160VB 076VI 088PI

145VB 174PB 163PB

114


Haas W. 025VD 056VV 023VD

Haase S. 173PB Habedank B. 016VD Haberl B. 023VD H~cker G. 160VB Hailer D, 156VB

157VB HAMELS I. 101VC Hammerschmidt K. 024VD Harder S. 117VG

116VG HarrisJ.R 008PS Hartel K.S. 050PE H~rter G. 073VI Hartmann P. 069VI

012PS Hartmann S. 075VI

089PI 080VI 082PI 085PI

123VG Harwaldt P. 135VB Hayton K. 139VB Hecht O. 088PI HehelA. 169PB HeineIS. 039VE Hei6 K. 175PB Hemphi l lA. 092VC

074VI 158VB 093VC

Hendgen-Cotta U. 014VD 132PG

Hepp S. 061PV Herbst R. 148VB Hermann A.J. 089PI Hermosilla C. 070VI

081PI 086PI

054VV Herrmann K.U.-. 084PI He~eIJ. 023VD He~rampf B. 061PV HeselhausJ. 057VV He6 R.G. 043VE Heussler V. 155VB Heyers O. 120VG

121VG Hoerauf A. 050PE Ho~cker I.L. 120VG Hoffmann T. 020VD Hoffmann W.H 084PI Holland G. 027VD Hommer V. 131PG

096VC H6ppnerJ. 122VG HORAUF A. 101VC HubeR K. 181PB H0bner K. 163PB H0bner M.P 084PI H0bner M. 131PG H0tt S. 020VD

Zozef R. 134VB Irsch T. 098VC

Jacobs T. 071VI J~keIT. 068VI Janje B. 067VI

Janko C. Janse C. Jansen A.M. Joachim A. Jordanova R.

Kabiri M. Kabsch W. Kafatos F.C.

KAISER A. Kalbacher H. Kalbe M.

Kalinna B. Kalinna B.H.

Kallert D. Kampen H.

Kapp K.

Kappe S. Kayser O. Kedves K. Keller P. Kern P.

Key G. Khomutov A. Kiderlen A.F.

Klaholz L. Klawonn W. Kliemt D. Klinkert M.Q.

KI6ss D. KIotz C.

Knaus P. Knobloch J.

Kollien A.

Kollien A.H. Kollien A.K. K~nig A, Konrad C. Krakau M. Kranich S. Krasky A. Krause E.

Krause T. Krauth-Siegel R.L

Kretschmer U. Krnajski Z. Kroner A.

Kuhls K. Kuhn D,

Kuhn Y. K0hnl J. Kunz W. Kurtz J.

039VE 109VCA

129PG 051VV 164PB

168PB 135VB 022VD 066VI 101VC 084PI

026VD 034PD 017VD 121VG 120VG 123VG 056VV 032PD 033PD 142VB 176PB 154VB 112PC 023VD 160VB 073VI 038VE 149VB 109VC 112PC 028VD 147VB 027VD 166PB 046PE 068VI

121VGA 105VC 043VE 095VC 091VC 143VB 176PB 177PB 142VB 014VD 132PG 016VD 129PG 039VE 180PB 037PD 080VI

119VG 116VG 117VG 109VC 099VC 114PC 098VC 077VI 138VB 180PB 143VB 009PS 144VB 145VB 178PB 173PB 142VB 024VD

Kusel J.R.

Labohm R.

Lammers R,

Lang C. Lang F. Langer C. Langner K.F. Lanzer M.

Laube U.

Laufer S. Lay K. Lee B. Leibold W. Leiers B. Leippe M.

Leutenegger C.M. Liebau e.

Liedtke S. Lieke T. Lindenthal C. Lingelbach K, Loy C. Lucius R.

L0der C.G

L0ersen K.

Luu V.D. Luz J,

Mackenstedt U.

Maetzel D. Mai B.

121VGA

043VE 046PE 176PB 142VB 076VI 162PB 138VB 057VV 107VC 106VC 137VB 178PB 133VB 028VD 027VD 112PC 084PI

062PV 099VC 057VV 165PB 148VB 115PC 124VG 126VG 088PI 064VI 065VI 055VV 171PB 173PB 104VC 122VG 164PB 165PB

121VGA 071VI 105VC 125VG 023VD 017VD 123VG 095VC 089PI 085PI

121VG 082PI 080VI 077VI 075VI

091VC 160VB 076VI 140VB 109VC 138VB 171PB 174PB 068VI

061PV 068VI 047PE 090PI 049PE 111PC 013PS 041VE 039VE 033PD 072VI

115

Scientific Program DGP, 21st Annual Meeting March 17 th -- 20 th, 2004 in WOrzburg Maier W.A. 033PD

032PD Manfras B.J. 073VI Marciano-Cabral F. 148VB Marh6fer R. 095VC Matthes H.F.-. 017VD Matuschewski K. 154VB

109VCA 127VG 175PB

Matzk P. 028VD McDermott J. 059VV McLean J. 106VC Meints G.A. 097VC Meiser C, 015VD Mejia C. 139VB Melzer I.M. 170PB Merli M. 049PE

090PI Meyer J. 172PB Meyer V. 025VD Michel K. 066VI Michel R. 003VS Miller M.A. 055VV Miller S, 133VB Moll H. 087PI

159VB 072VI

Monesi N. 128PG Mueller H.M. 066VI Mueller M. 075VI Mueller M.C. 152VB Mueller N. 079VI M011er A. 012PS

069VI M011er A.K. 154VB

175PB MOiler H,M. 022VD M011er I.B. 138VB M011er M. 089PI

082PI M011er N. 151VBA

158VB MOiler S. 109VC MOiler S. 136VB MOnderle M. 031VD M0nnich M. 142VB

176PB 121VGA

M0nstermann S. 059VV Naguleswaran A. 158VB Najdrowski M, 131PG

0 5 1 W

Nessler S. 107VC Ngazoa E.S. 168PB Nickel C. 153VB Njoroge E.M. 013PS Nowak N. 151VB Nozaki T, 097VC Nyalwidhe J. 125VG

Oberl~nder M. 141VB OIdfield E. 097VC Omer R.A. 041VE Ozel M. 027VD

Palmada M. 162PB Pape M. 060PV Patino P.J. 078VI Pelte N. 066VI Perbandt M. 104VC Peters M, 172PB

Petry F,

Pfahler J. Philipp C.

Pillai S. Planelles G. Plappert S. Pleydell D. Pogonka T.

Ponte-Sucre A, P6tzsch C. Pradel G. Prelezov P.N. Presber W. Pr61s F. Propping S, Przyborski J.

Radam E.

Radoslavov G. Rahlfs S.

Randolph T. Rausch S.

Rauser M. Reck C. Reich B. Reichhart J.M. Reiner G. Reiter-Owona I. Reitze F. Renner C. Renz A. Reule M. Ribeiro F, Riebe R. Riekenberg S.

Rinder H. Robledo S.M. Roeder T. Rogl A. Rohn K. Rohrbach P.

Rohwer A. Romig T.

Rose A.

Rossi A, Rossignol J,F. Rzepecka J,

Salzberger B.

Sanchez C.

Santos de Macedo C. Saraiva J.

011PS 008PS 131PG 096VC 133VB 177PB 142VB

121VGA 123VG 107VC 111PC 047PE 095VC 091VC 077VI 167PB 040VE 139VB 063PV 007PS 016VD 006VS 133VB

028VD 112PC 147VB 164PB 134VB 135VB 0 5 9 W 085PI 082PI

043VE 068VI

017VD 066VI 061PV 050PE 010PS 047PE 062PV 047PE

121VGA 172PB 149VB 150VB 006VS 078VI 064VI

020VD 172PB 178PB 137VB 119VG 038VE 039VE 049PE 013PS 047PE 041VE 018VD 021VD

121VGA 093VC 085PI

012PS 069VI 107VC 106VC 110PC 128PG

Sayed Ibrahim Aly A. Schares G.

Schaub G.

Schaub G,A.

Scheid P.L. Schelzke K. Schirmeister T.

Schirmer H.

Schirmer R.H

Schlecker T. Schlote S. Schneider I.

Schneuwly S. Schnieder T,

Schnur L.F. Schoenian G. Sch61merich J. Scholz A. Scholze H.

Scholzen T. Sch6nfelder K, Sch6nian G.

Schreiber T. Schr6der R, Schroeder J. Schuldt K. Schulz-Key H. Schumacher S. Sch061er P. Schwarz A. Schwarz R.T. Schwarz S. Schwenkenbecher J. Seltzer U.

Seizer P. Seizer P.M.

Sereda M.J. Sesterhenn L. Shadrach W.S.

Sidibe I. Skupch K,C. Soboslay P.T Sommer A. Sonnenburg B.

Spiliotis M.

Spit t ler H. Sroka S.

083PI 109VCA

052VV 172PB 046PE 043VE 015VD 132PG 014VD 020VD 129PG 016VD 048PE 077VI

126VG 115PC 135VB 134VB 103VC 108VC 114PC 057VV 157VB 156VB 035PD

123VGA 060PV 005VS 094VC 001VS 044VE 035PD 145VB 149VB 152VB 150VB 053VV 090PI

001VS 007PS 009PS 039VE 043VE 119VG 170PB 084PI 084PI

142VB 032PD 110PC 040VE 007PS 157VB 053VV 156VB 095VC 102VC 119VG 163PB 080VI 179PB 027VD 147VB 059VV 026VD 084PI 171PB 082PI 089PI 180PB 179PB 050PE 142VB

116

Scientific Program DGP, 21st Annual Meeting March 17 t" - 20 th, 2004 in W0rzburg

177PB Stadler P.F. 120VG Stambusch R. 046PE Staubach C. 043VE Steigerwald J. 159VB Steigerwald M. 087PI Stein W. 106VC SteinbOchel M. 022VD Stett ler M. 093VC Steuber S. 019VD Steverding D. 168PB

100VC Stirati R. 080VI Stock P. 014VD Stoverock M. 005VS Strathmann K.P. 011PS Strelkova M.V. 001VS Strube C. 123VGA SOhwold A. 054VV Sures B. 031VD

030VD 029VD

Sutor A. 040VE Szallies A. 162PB Szkodowska A. 152VB

Tackmann K. 040VE Tannich E. 151VB Tappe D. 180PB

179PB Taraschewski H. 031VD

010PS Tata P.S. 028VD

112PC Taubert A. 070VI

086PI 081PI

054VV Templeton T. 139VB Tenter A.M. 055VV

004VS Thielen F. 031VD

030VD Thieltges D.W. 036PD

037PD Thoma B.R. 048PE Thoma D. 047PE

039VE Thompson K.A. 100VC Tilley L. 153VB Torda A. 173PB Tourneux F.P. 047PE Traub K. 090PI Trees S. 111PC Truj i l lo-Vargas C.M. 078VI

Uzcategui N. 162PB 167PB

Vahrmann A. 152VB Velez I,D. 078VI Vicik R. 115PC

126VG Vogl S. 002VS Volkmer-Engert R. 080VI Volz J. 022VD von Allmen N. 151VBA von Blumr6der D. 046PE von Samson- Himmelst jerna G. 005VS

060PV 004VS 094VC

von Witzendorff C. Voncken F. Vonlaufen N. Vorwerk N.

Wackwitz C. Waibel H. Walduck A.K. Walker M.

Walochnik J. Walter R.D. Walter R. Walter R Walter R.D.

W~lz M. Waniek P. Waniek P.J.

Waters A.P. Wegner K.M Weible A.K. Weich N. Wiedemann M. Wiese M.

Wiesgigl M. Winkelmann J.

Wippersteg V. Witjes B. Wittek B. Wittig B. Wolf C. Wrenger, C, Wulff C. Wurm R. WOrth S.

Zahler-Rinder M.

Zahner H.

Zahnert H. Zavala-Gongora R.

Zelck U.

Zentgraf U. Zeyhle E. Zil ler M. Z immermann A. Z immermann S.

123VGA 017VD 114PC 158VB 004VS

051VV 059VV 121VG 074VI

093VC 003VS 173PB 140VB 165PB 138VB 164PB 109VC 122VG 104VC 171PB 013PS 014VD 129PG 016VD

109VCA 034PD 047PE 105VC 006VS 145VB 144VB 170PB 166PB 050PE 065VI

124VG 121VGA

149VB 143VB 121VG 163PB 136VB 025VD 043VE 045VE

002VS 045VE 061PV 070VI 081PI

054VV 086PI 181PB 180PB 143VB 067VI 141VB 131PG 083PI 013PS 043VE 013PS 030VD

117

Date post:	04-Jan-2017
Category:	Documents
Upload:	doantu
View:	219 times
Download:	0 times

Scientific program DGP, 21st Annual Meeting March 17th – 20th, 2004 in Würzburg

Documents