ABSTRACTS

the appendages, suggesting lhal ciliates can more easily colo-nize less active body parts.

28Improved Methods for Preparation of a 25 kDa Ciliary Gly-

coprotein of Tetrahymena thermophila. A. W. WAY and L. A.HUFNAGEL, University of Rhode Island, Kingston, RI.

Concanavalin A (Con A)-binding membrane proteins mayhave an important role in initial recognition and formation of theconjugation junction between mating-competent T. thermophilacells. To further investigate this, methods were developed forincreased yield of a ciliary membrane Con A-binding protein, inpreparation for its further analysis. Variables examined inc1udedmethods to achieve high yields of isolated cilia, affinity columnchromatography techniques, alternative protease inhibitors, andgel spin columns to remove salts and low molecular weight poly-peptides. When Con A affinity chromatography was optimized,a polypeptide of approximately 25 kDa was obtained from de-tergent (Nonidet P-40)-solubilized ciliary membranes of non-starved (mating inactive, non-initiated) cells. Consistent with pre-vious results, this polypeptide was either down regulated or nolonger able to bind Con A in starved (mating competent, initi-ated) cells. Blotting verified that the polypeptide is Con A-bind-ing. We estimate lhal, using these methods, from one Jiter of lateexponential phase cells grown on enriched proteose peptone (~16-20 ml packed cells), approximately 0.1 mg of the 25 kDaglycoprotein can be obtained. These experiments establish pro-cedures for providing sufficient quantities of the 25 kDa proteinfor further studies, inc1uding antibody production, isoelectric fo-cusing, and sequence analysis.

29Effects of Latrunculin B on Tetrahymena Mating. R. V.

ZACKROFF* and L. A. HUFNAGEL**, *Massachusetts Col-lege of Pharmacy and Health Sciences, Boston, MA, **Uni-versity of Rhode Island, Kingston, RI.

Early event s in Tetrahymena mating inc1ude tip transforma-tion, in which the anterior ends of the cells become altered inpreparation for mating, and loose pairing, which may involveboth heterotypic and homotypic interactions at cell surface sitesnot restricted to the transformed tips. Tip transformation andloose pairing appear to precede tight heterotypic pairing at thetransformed tips. To shed further light on the mechanism ofthese early events, we investigated the effects of latrunculin B,an anti-actin drog lhal inhibits Tetrahymena phagocytosis (awell characterized, actin-dependent proces s) at sub-micromolarconcentrations. The rate of stable pair formation was signifi-cantly inhibited by sub-micromolar (4-8 x 10-7 M) concentra-tions of latrunculin B. Micromolar concentrations of latrunculinB strongly inhibited the rate of pairing as well as tip transfor-mation. However, the first observable pairs appeared nearly si-multaneously in drug-treated samples and controls. These re-sults suggest lhal tip transformation is dependent on actin dy-namics, and lhal actin may be involved in other aspects of sta-ble pair formation as well.

Czech Section Society of Proto zoology31st Annual Meeting

Apnl 23-27, 2001Prudka, Czech Republic

(Abstracts 30-40)

7A

30Cutaneous Leishmaniasis in Northern Afghanistan. M.L.

AMIRI*, M.Z. AAMOON** and N. JALILI***, *Institute ofMalaria and Parasitic diseases, Mazar-e-Sharif, Afghanistan,**Army Hospital, Mazar-e-Sharif, Afghanistan, ***Institute ofParasitology, Faculty of Medicine, Comenius University, Bra-tislava, Slovak Republic.

From April 1989-December 1999, 21405 positive cases ofcutaneous leishmaniasis, were registered from the town of Ma-zar-e-Sharif and its surroundings in Northern Afghanistan.From these, 17972 cases (84 %) were detected as Leishmaniamajor (rural form), 3433 cases (16%) as Leishmania tropica(urban form) and 15 cases of Lupoid formo In our results werecorded the high incidence of positive cases (mainly ruralform) during the autumn lhal can be explained by the incuba-tion period of leishmaniasis and the activity of vectars espe-cially in summer months.

31Molecular Polymorphism of Tetratrichomonas gallinarum. I.

CEPICKA, K. KUTlSOV A and J. FLEGR, Department of Par-asitology, Charles University, 12844 Prague, Czech Republic.

Tetratrichomonas gallinarum is a widespread parasite ofbirds, mainly Galliformes and Anseriformes. This species usu-ally infects caecum, bul it was found also in beak, salpinx anddifferent visceral organs. Several strains were also isolated fromhuman mouth and lungs. There were originally described sev-eral Tetratrichomonas species from different avian hosts, bulon the basis of morphological and infection studies auly onespecies is presently considered to be valid. We performed thephylogenetic analysis with 19 T. gallinarum strains, inc1udingtwo human strains and 17 strains isolated from 8 different avianspecies. The sequences of 5.8S rRNA with the flanking areasITSl and ITS2 (ITS) of these 19 strains were determined andused for construction of the phylogenetic tree by the Neighbor-joining and Maximum parsimony method. The strains formed5 c1usters A-E; sequences in each c1uster were identical. Humanisolates were placed in c1usters A and B. The sequence ho-mology among c1usters A, Band C (92.9% - 95.4%) was high-er lhali the sequence homology among rour valid Trichomonasspecies (86% - 89%) . However, the sequence homology ofc1usters D or E to c1usters A, Band C, as well as the sequencehomology between c1usters D and E was unexpectedly low(78.2% - 91%) . We also sequenced and analyzed the gene for16S rRNA from one strain of each c1uster. Again, the sequencehomology between c1usters D and E, as well as between c1usterDarE and c1usters A, Band C, was lower lhali the sequencehomology between two Trichomonas species. Therefore wesuggest lhal our strains of Tetratrichomonas gallinarum couldrepresent at least three cryptic species.

32Malic Enzymes of Trichomonas vaginalis: Purification,

Function and Phylogeny. P. DOLEZAL*, J. TACHEZY*, P.PROOST** and I. HRDY*, *Department of Parasitology, Fac-ulty of Science, Charles University, 128 44 Prague 2, CzechRepublic, **Rega Institute for Medical Research, Departmentof Microbiology and Immunology, Leuven, Belgium.

Trichomonas vaginalis possesses two types of malic en-zymes: hydrogenosomal NAD(P)-specific and cytosolic NADP-specific malic enzyme. Hydrogenosomal type is a homotetramerof the 60 kDa subunits. It utilizes NAD+ preferentially to

8A ABSTRACTS

NADP+ and supplies hydrogenosome with pyruvate for furthercatabolic processes. In this study we focused on NADP-specificmatic enzyme from the cytosoL One of the several present iso-forms was purified to homogeneity by liquid chromatography.Molecular weight determination of the native purified isoformsuggested an unusual dimeric arrangement of the 42 kDa sub-units. The enzyme utilizes exclusively NADP+ (Km, 2,8 /-LM)with pH optimum between 7,5-8,5. The main function of thecytosolic matic enzyme is probably production of NADPHwhich is then utilized in the formation of the major glycolyticend product, glyceroL Moreover, together with the consecutiveaction of phosphoenolpyruvate carboxykinase and malate de-hydrogenase the cytosolic matic enzyme forms a pathway thatbypasses pyruvate kinase reaction and transfers reducing equiv-alents from NADH to NADP+. We cloned and sequenced thecomplete gene of NADP-specific malic enzyme. The codingregion is preceded by initiator element while 3' -fianking regioncontains polyadenylation signal, thus corresponding to knownarrangement of transcription unit in T. vaginalis. Amino acidsequence comparisons with homologous proteins revealed eu-bacterial features of cytosolic malic enzyme and indicated to-tally different evolutionary origin of both types of T. vaginalismalic enzymes. Hydrogenosomal type appears to be one of themost divergent eukaryotic matic enzyme. Apart from their ob-vious role in metabolism of T. vaginalis, several studies havedescribed specific extracellular release of the matic enzyme sand suggested their adhesive function. To verify this hypothesis,we analyzed the proteins excreted by T. vaginalis into Doranmedium. In addition to described secretion of both matic en-

zymes we detected other cytosolic (LDH, MDH) and hydro-genosomal (STK) proteins as well. We also examined effect ofT. vaginalis secreted proteases on the activity of MDH, LDHand matic enzymes. In cell-free supernatants obtained after one-hOUf cell incubation both malic enzyme remained active overfour hours while activities of MDH and LDH were undetectable

after 30 minutes. We propase that extracellular localization ofmalic enzymes does not indicate their adhesive function blitrather refiects unspecific exocytosis of cell content in combi-nation with resistance of matic enzymes to proteolytic degra-dation.

33Phylogenetic Position and Relationship of Flagellates of the

Genus Monocercomonas from Different Host Species. V.HAMPL, I. CEPICKA, J. KULDA, J. TACHEZY. and J.FLEGR, Department of Parasitology, Faculty of Science,Charles University, 128 44 Prague, Czech Republic.

The genus Monocercomonas Grassi, 1879 (order Trichomon-adida) comprises more than 20 species mostly parasitic in rep-tiles blit also in other vertebrates and in insects. Important mor-phological feature of this genus is the absence of costa andundulating membrane, characteristic structures of a typicaltrichomonad cello Family Monocercomonadidae Kirby, 1944,established for trichomonads with similarly reduced cell mor-phology, comprises besides parasitic also free-living genera(Pseudotrichomonas, Ditrichomonas, Monotrichomonas). Thisfamily was regarded to be the most primitive group of tricho-monad s from the evolutionary point of view. However, resultsof sequencing of 16S rRNA of some species suggested that theyprobably represent higher branches in the trichomonad tree.Moreover, they indicate that the free-living genera are unrelatedto the genus Monocercomonas. Here we sequenced the gene for16S rRNA of two isolates of Monocercomonas ruminantiumfrom cattle and eight isolates of Monocercomonas sr. from dif-

ferent reptile species. Strains from reptiles formed one cladewith the bootstrap value 100 in the phylogenetic tree of tricho-monads. On the other hand sequences of the two isolates ofMonocercomonas ruminantium were placed with 100% boot-strap support among the free-living genera of trichomonads, i.e.into a branch of species unrelated to other representatives ofthe genu s Monocercomonas. The sequences of these isolatesdiffered only in one nucleotide and were clo sely related (97.1%) to the sequence of Pseudotrichomonas keilini. Our resultsstrongly suggest that taxonomic classification of Monocerco-monas ruminantium should be revised. Furthermore, close re-lationship of this parasitic species with free-living trichomonadsrose interesting questions regarding the evolution of parasitismin this group.

34What is the Cause of the Deteriorated Performance in Subject

with Latent Toxoplasmosis? J. HAVLI

ABSTRACTS

Until now about 45 coccidia species (Apicomplexa: Eimeri-idae, Sarcocystidae) parasiting anuran amphibians are known.The aim of presented research was to study the prevalence ofcoccidian species in ranid frogs in some localities in the CzechRepublic. Hyaloklossia lieberkuehni Labbe, 1894 and Eimeriasr. were found in R. kl. esculenta, whereas the examination ofR. temporaria and R. arvalis revealed E. ranae Dobell, 1908 tobe the only coccidian species parasitising these two ranid hosts.E. ranae was relatively common parasite of R. temporaria,reaching a prevalence of 57.5% in adults. Examination of tad-poles of R. temporaria revealed the presence of E. ranae intheir faeces. Consequent histological examination confirmedstages of merogony and gamogony in the intestine of 13% oftadpoles in popu lation examined. Presence of oocysts in bothtadpoles and adults of R. temporaria indicate the completion ofthe life cycle even before the metamorphosis. Additionally, theinfectivity of adult frog-originated oocysts for tadpoles wasconfirmed by results of cross-transmission experiments. How-ever, there was no evidence of any coccidian parasites to bepresent in freshly metamorphosed specimens of R. temporariain nature. Morphologically identical oocysts were found also inadults of R. arvalis and possible conspecificity of coccidia fromR. arvalis and R. temporaria, as well as the natural life cycleof E. ranae is discussed.

36Use of Random Amplified Polymorphic DNA (RAPD) Anal-

ysis for the Identification of Giardia intestinalis Subtypes. J.SEDINOVA*, J. FLEGR*, P. L. EY** and J. KULDA*, *De-partment of Parasitology, Faculty of Science, Charles Univer-sity in Prague, Vinicna 7, 128 44 Prague 2, Czech Republic,**Department of Molecular Biosciences, Adelaide University,Adelaide SA 5005, Australia.

The random amplified polymorphic DNA (RAPD) methodwas used to investigate genetic polymorphisms among 25 iso-lates of Giardia intestinalis and to assess the utility of RAPDfor subtype detection and genealogical analysis. Using data ob-tained for 6 human and 19 animal-derived isolates in polymer-ase chain reactions with 13 different primers, phylogenetic treeswere constructed and bootstrap values computed by the pro-gram FreeTree. Three major clades were distinguished, corre-sponding to previously defined genetic assemblages A, BandE. The purported specificity of assemblage E genotypes for ar-tiodactyl hosts was supported. Assemblages A and B showedwide host spectra, including human and animal hosts. The iden-tity of RAPD pattems obtained from 14 clones derived fromone G. intestinalis isolate (PI5) indicated that the isolate wasgenetically homogenous. No correlation was found between thegenotype of analysed isolates and the presence or absence ofthe double-stranded RNA Giardiavirus. The results indicate thatRAPD banding data provide reliable genetic information thatcan be used for both 'fingerprinting' and genealogical purposes.

37Leishmania tropica: Field Study in the Endemic Focus in

Turkey and Laboratory Transmission from Asymptomatic Hostsand Non-lesion Sites. M. SVOBODOV A and P. VOLF, De-partment of Parasitology, Charles University, Vinicna 7, 12843Prague 2, Czech Republic.

Sandflies were studied in Urfa, Turkey-a focus of Ltropica.Light and rodent traps were placed in the endemic quarters.Phlebotomus sergenti caught was twice as mailY in number asP.papatasi. These 2 species constitute 99% of sandflies. The

9A

male:female ratio was 1.4 for P.sergenti and 0.9 for P.papatasi.Both species were most abundant in stables followed by cellars,and less so in rooms, yards and on roofs. P.papatasi was rel-atively more numerous in stables, while P.sergenti in rooms.Sources of bloodmeals were determined by ELlSA using anti-IgG. The bloodmeals originate from several vertebrates includ-ing rodents. House mice (Mus domesticus) and black rats (Rat-tus rattus) were abundant and caught in sandfly-infested houses.Black rats were susceptible to infection with Ltropica. The par-asites produced no skin lesion, blit persisted at the inoculationsites of ear and footpad. P.sergenti fed on the tat ears acquiredinfection. BALB/c mice developed lesions after inoculationwith Ltropica, and the infection eventually visceralized. Feed-ing on non-lesion sites resulted in 20% infected P.sergenti.Thus, P.sergenti can acquire infection on inoculation site of anasymptomatic host and on non-lesion site of a symptomatichost, which is important in the epidemiology of the disease.

38A New Paraxonemal Protein in Giardia intestinalis. P. TES-

AROV A and E. NOHYNKOV A, Department of Tropical Med-icine, 1st Faculty of Medicine, Charles University, Studnickova7, 128 00 Prague, Czech Republic.

Paraxonemal structures of different morphological patremparallel flagellar axonemes in cells of several unrelated groupsof protists, including retortamonads, diplomonads, parabasalids,kinetoplastids etc. Of these, a paraflagellar rod (PFR) of try-panosomatids is well characterized while little is known aboutothers. The PFR consists of two major closely related proteins,PFRl and PFR 2, which together with some minor proteinscompose a highly ordered paracrystalline structure alongsidethe flagellar axoneme. We used two monoclonal antibodies,L13D6 and L8C4, specific for the major paraflagellar proteinsof Trypanosoma brucei (PFR-A and PFR-C) to test whetherrelated proteins are present in paraxonemal structures found inGiardia intestinalis, G. muris and Tritrichomonas foetus. InGiardia, different structures accompany ventral flagellar axo-nemes and intracytoplasmic portions of axonemes of anterolat-eral and posterolateral flagella, in Tjoetus, an undulating mem-brane associated recurrent flagellum contains a rod-like para-crystalline structure. In indirect immunofluorescence, the anti-body L13D6 which recognizes both T.brucei PFR-A and PFR-Cdecorated specifically intracytoplasmic portions of both axo-nemes of anterolateral flagellar pair in G.intestinalis. Double-label staining with monoclonal antibody against alpha tubulinshowed that the L13D6 antibody label was localized anteriorlyto the axonemes in close proximity to axonemal microtubules.During cell division, the paraxonemallabel disappeared in pro-phase in accord with flagellar rearrangement and reappeared intelophase when daughter anterolateral flagella assumed the typ-ical interphase arrangement. Surprisingly, no label was detectedin any phase of G.muris or Tjoetus cell cycles. The antibodyL8C4, specific for the PFR-A only, did not recognize any epi-tope in any of the flagellates tested.

39Cyclophilins of Trichomonas vaginalis. V. VARGA *, M.

DUCHENE**, J. TACHEZY*, I. HRDY*, J. KULDA* and T.THALHAMMER **, *Department of Parasitology, Faculty ofScience, Charles University in Prague, Vinicna 7, 128 44Prague 2, Czech Republic, **Department of Pathophysiology,University of Vienna, Waehringerguertel 18-20, A-1O90 Vien-na, Austria.

lOA ABSTRACTS

Cyclosporin A (CsA), initially developed as an immunosup-pressive drug, possesses activity against various protozoan par-asites in a species and/ar stage specific manner. CsA binds tothe high affinity intracellular receptors, cyclophilins (Cyps).The cyclophilins display the peptidyl-prolyl cis/trans isomeraseactivity and accelerate the rate-limiting step in the folding oftarget proteins. If CsA binds to Cyp, the CsA/Cyp complexinhibits calcineurin-mediated dephosphorylation of transcrip-tion factors, which in their phosphorylated form are unable totranslocate in the nucleus. The present study was undertaken tocharacterize Cyps in Trichomonas vaginalis and to test the sus-ceptibility of the parasite to CsA and related compounds. Usinga CsA-affinity column an 18kDa protein was isolated from acytosolic fraction of T. vagina/is. The protein was unglycosy-lated and peptide sequencing showed its high homology to Cypsof other parasites. The protein possessed peptidyl-prolyl cis/trans isomerase activity, inhibitable by CsA (IC50 - lOnM) ,indicating that this protein belongs to cyclophilins. Further-more, the isoelectric focusing revealed two isoforms with anisoelectric point around 8.6, as observed for other cytosolicCyps. Exposure of T. vaginalis to CsA for 24 and 48 hrsshowed lhal CsA is toxic to the parasite at the minimal lethalconcentration (MLC) of 60 /LM. Interestingly, another CsA-de-rivative, the SDZ PSC833, which does not bind to the humancytosolic Cyp, was more effective lhali CsA (MLC about 7/LM). It indicates, lhal Cyp binding and inhibition of peptidyl-prolyl cis/trans isomerase activity might not be the on ly mech-anism of the CsA action against T. vaginalis. Further studiesare required to elucidate the physiological function of this novelT. vaginalis cyclophilin and to evaluate its potencial as a targetfor therapy.

40Development of Avian Trypanosomes in Mosquitoes. J. VO-

TYPKA *.**, M. SVOBODOV A * and P. VOLF*, *Departmentof Parasitology, Faculty of Science, Charles University, 128 44Prague, Czech Republic, **Institute of Parasitology, CzechAcademy of Sciences, 370 05 Ceské Budjovice; Czech Repub-lic.

Parasitic flagellates of the genera Trypanosoma and Leish-mania have a two-host life cycle and altemate between verte-brate and insect hosts. Transmission of the parasite juto theinsect vector is due to uptake of flagellates with peripheralblood. However, transmission to the vertebrate host may occurin several ways, for example, by inoculation with insect salivaor contamination with insect feces. The location of parasites inthe insect digestive tract or in the salivary glands is critical tothe mechanism of transmission to the vertebrate host. Aviantrypanosomes (strain CULI, which originates from naturallyinfected Cu/ex pipiens pipiens) were used to infect laboratoryreared female mosquitoes (Cu/ex pipiens quinquefasciatus). In-fected mosquitoes (16 days post infection) were dissected andheavy infections of trypanosomes were found in the divisionbetween the foregut and the thoracic midgut. The other partsof the digestive tract including salivary glands were parasite-free. Semithin and thin cross-sections of the fore part of thealimentary tract containing trypanosomes were analyzed bylight and electron microscopy, respectively. The parasites ob-served were in close contact with chitin in the cuticular liningof the stomodeal valve auly; the region of epithelium coveredby microvilli was not colonized. The adhesion of the parasiteto this cuticle-lined region occurs by the formation of zonalhemidesmosome-like plaques at the extremities of the expandedflagella of epimastigote. The high number of trypanosomes in

the anterior part of the thoracic midgut creates a plug, whichcould result in the regurgitation of flagellates juto the vertebratehost with a backflow of ingested blood during sucking. Addi-tionally, some damage to the chitin layer or impairment of thestomodeal valve motility could help such a transmission pro-cess. The development of avian trypanosomes in mosquitoes issimilar to lhal of the leishmanias in sand flies and thereforecould represent a new model for the investigation of host-par-asite relationships.

Italian Section Society of Protozoologists22nd Annual Meeting

September 20-21, 2001La Spezia, Italy

(Abstracts 41-54)

41Environmental Effects on Cholinergic Molecule Activity in

Protists. A. AMAROLl*, C. FALUGI**, L. SANNA *, V.EVANGELlSTI***, A. VIARENGO*** and M.U. DELMON-TE CORRADO*, *Dipartimento per 10 Studio del Territorio edelle sue Risorse, **Dipartimento di Biologia Sperimentale,Ambienta1e, Applicata, Universita di Genova, 16132 Genova,***Dipartimento di Scienze e Tecnologie Avanzate, Universitadel Piemonte Orientale, 15100 Alessandria, Italy.

It is well known lhal neurotoxic drugs, such as organophos-phate (OP) and carbamate (CB) compounds, are currently em-ployed in agricultural sites, because of their insecticide activity.The first target of these compounds is the cholinergic neuro-transmitter system, by inhibiting cholinesterase activity. There-fore, this enzyme activity is usually exploited as a biomarkerof neurotoxic drugs. Recently, we found acetylcholinesterase(AChE) activity in Paramecium primaurelia and its role wasinferred in modulating the pre-conjugant cell-to-cell interac-tions. The results of our investigations extended to Dictyoste-lium discoideum by histochemical, electrophoretic, and spectro-photometric methods showed the presence of a cholinesterase(ChE) activity able to cleave both the acetyl-beta-methyltioch-oline jodide and propionyltiocholine jodide substrates, sensitiveto neurotoxic drugs, and possibly involved in regulating cell-to-cell interactions during aggregation. In this study, we char-acterized the D. discoideum ChE activity by evaluating spec-troscopically the effects of specific ChE inhibitors, such as iso-OMPA, BW, and eserine, as well as by a spectrophotometricanalysis performed under varying conditions of chemico-phys-ical parameters, i.e. pH and temperature. Furthermore, wechecked the chance to exploit P. primaurelia as a bioassay forthe pre-chemical screening of fresh water environments in re-lation to the occurrence of neurotoxic drugs. The presence ofpropionylcholinesterase (PrChE) activity was histochemicallyrevealed and the spectrophotometric analysis showed signifi-cantly higher AChE activity lhali PrChE activity. Exposure bothof AChE and PrChE to 10-6 M Diazinon or 10-6 M Carbaryl,the active principles of OPs or CBs, respectively, resulted insignificant inhibition of AChE activity.

42Evaluation of a Rapid Immunochromatographic Test for the

Serodiagnosis of Visceral Leishmaniasis. o. BRANDONISIO,L. FUMAROLA, D. LEOGRANDE and P. MAGGI*, Diparti-mento di Clinica Medica, Immunologia e Malattie Infettive, Se-