XII International Congress of Parasitology

ICOPAMelbourne (Australia),

August 15-20, 2010

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Index

Front page .......................................................................................................................... I

Complement Inhibition by Scabies Mite Inactivated Protease Paralogs. Fischer, K. ........................................................................................................................... 1

Microvascular aspects of pathogenesis in severe malariaCombes V, Schmutzhard E, Kurtzhals J, Hunt NH, Grau GER ............................................... 7

A new approach to modelling macroparasite transmission based on stratified worm burdenGurarie D., King C.H., Wang X. ............................................................................................. 15

Immune regulation of malaria infection: model calibration and Agent-Based CommunitiesGurarie D., Karl S., St. Pierre T.G., Davis T.M.E .................................................................... 23

Parasite-induced B-cell apoptosis results in loss of specific protective anti-trypanosome antibody responses, and abolishment of vaccine induced protective memory responses.Magez S., Bockstal V, Brombacher F, Black S, Radwanska M ............................................. 31

A robust and improved monkey model of human African trypanosomiasisNgotho M., Kagira J.M., Gaithuma A.K., Kariuki C.K., Akinyi M.Y., Maloba F., Mwaliko V.M., Karanja SM, Maina N.W. ..................................................................................................... 39

Application of nematodes romanomermis iyengari in breeding sites of anopheles pseudopunctipennis in endemics communities of malaria in oaxaca, mexico.Pérez-Pacheco R, Flores-Ambrocio G., Martínez-Tomás S., Bautista Cruz M.A. and Ruíz-Vega J. ................................................................................................................. 47

Seroprevalence of toxoplasma gondii infection in goats From Two Counties In Mississippi, UsaKeith D. Frye and Alex D.W. Acholonu ................................................................................. 51

Blood flukes (Spirorchiidae) as a major cause of marine turtle mortality in QueenslandFlint M., Blair D., Patterson-Kane J., Kyaw-Tanner M. , Mills P.C. ....................................... 57

IV XII International Congress of Parasitology ICOPA

Comparation of the effectiveness of two diagnostic methods of leishmania spp. In cats Bresciani KDS, Serrano ACM, Matos, LVS, Savani ESMM, D’ Auria SRN, Perri, SHV, Bonello F.L, Coelho WMD, Aoki CG, Costa, AJ ......................................................................................... 63

First pass Babesia microti genome annotation Cornillot E., Hadj-Kaddour K., Noël B., Dassouli A., Duclos A., Randazzo S., Barbe V., Wincker P., Schetters T., Gorenflot A. .................................................................................. 67

The Babesia divergens Bd37 erythrocyte binding protein: structural analysis of a protective antigenMurciano B, Bres V, Auguin D, Yang YS, Cornillot E, Schetters TPM, Roumestand C, Gorenflot A, Delbecq S ........................................................................................................ 73

Expression and in-silico screening of plasmodium falciparum Dipeptidyl aminopeptidase I Herbert J. Agatheeswaran N. Liu Z. Roach M. Saleh E. & Menz R. ..................................... 77

Do high levels of vertical transmission explain the high prevalence and ubiquity of Toxoplasma gondii?Hide G., Gerwash O., Abushahma M., Hughes J.M., Thomasson D., Wright E.A., Murphy R.G., Dodd N., Haq S.Z.H, Elmahaishi K.H. and Elmahaishi M.S. ................................................. 83

Seasonal dynamics of tick (Acari: Ixodidae) populations in different areas of Romania and the associated risks of tick-borne diseasesIonita M., Mitrea I.L., Buzatu M.C., Dascalu L. .................................................................... 91

Screening of anti-peptide antibodies in vitro to identify potential cattle tick vaccine antigensLew-Tabor, A., Rodriguez Valle, M., Moolhuijzen, P., Bruyeres A., Bellgard, M ..................... 97

Evaluation of resazurin and propidium iodide-based fluorescence assays for the routine screening of drug susceptibilities of Trichomonas vaginalis.Natto Manal J., Savioli Francesca, Quashie Neils B. and de Koning Harry P. ...................... 103

A highly divergent mitochondrial ATP synthase which confirms the monophyly of the chromalveolatesNisbet, R. E. R., Mitton, E. R., Menz, R. I. ............................................................................ 105

Novel polymorphisms in the Cryptosporidium hominis genomeZhang Y, Fairchild M, Chiu A, Bhuvanendran S, Fung W, Ong C. .......................................... 109

Schistosoma mansoni has anti-arthritic effects accompanied with IL-17’s down-modulation dependent on viable eggsOsada Y, Shimizu S, Kumagai T, Yamada S, Kanazawa T ..................................................... 113

Trypanosoma cruzi infection induces nitric oxide production and S-nitrosylation of cellular FLIP in host cellsShimada J., Hatabu T. ......................................................................................................... 117

Development and Comparative Analysis of a 384-well Luciferase-Based Via-bility HTS Assay for Trypanosoma brucei brucei BS427 cellsSykes, M and Avery, V. ........................................................................................................ 121

VMelbourne, Australia, 15-20 August, 2010

High Throughput Screening of the WEHI Cpd Library Against Human African TrypanosomiasisSykes M, Avery V, Parisot J, Street I, Baell J,Chatelain E, Ioset J ........................................ 125

Paragonimus westermani possesses aerobic and anaerobic mitochondria in different tissues, adapting to fluctuating oxygen tensionTakamiya S., Fukuda K., Nakamura T., Aoki T., and Sugiyama H. ........................................ 129

Can humoral host immune responses prevent Giardia infection?: The urinary antibody titers in Indonesian school childrenTokoro M., Syafruddin D., Itoh M., Asih P., Rahman M. ........................................................ 135

Blastocystis sp. zoonotic subtype 6 infection associated with gastrointestinal illness Vassalos C.M., Vassalou E., Spanakos G., Papadopoulou C., Vakalis N. .............................. 139

Prevalence de sarcocystis sp. Dans les carcasses bovines au niveau des abattoirs de rouiba (alger)Harhoura Kh., F. Khouni, Aissi M. ........................................................................................ 143

Heterologous expression in Pichia pastoris of gene BMTI optimizedAndreotti, R., Soares, M., Cunha, R.C., Leite, F.P.L. .............................................................. 149

Intestinal Microsporidiosis in HIV Positive Patients with Chronic DiarroheaBáez-Abréu de Borges E., Arcay L., Salaverría C., Otero E., Bruzual E., Brito A., De La Parte M., Elster C., Faría E., Isernia M., Castro E. ..................................................... 153

Effect of TGF-β and TNF-α treatment in Trypanosoma cruzi-infected cardiom-yocytes: new insights on Chagas’ disease extracellular matrix regulationCalvet C.M., Oliveira Jr, F.O.R., Araujo-Jorge T.C., Pereira M.C.S. ........................................ 159

Identification and characterization of components of small silencing RNA pathways in Trypanosoma cruzi.Garcia-Silva M.R., Tosar J.P, Cabrera-Cabrera F., Bonilla B., Güida M.C. , Parodi A., Rovira C., Robello C., and Cayota A. .................................................................................................... 165

Toward Automation of the Diagnosis of Enteroparasitosis via Computational Image AnalysisGomes J.F., Suzuki C.T.N., Papa J.P., Hoshino-Shimizu S. and Falcão A.X. .......................... 169

In-vitro antimalarial activities of Withania somnifera, Gymnema sylvestre, Ocimum gratisimum and Cajanus cajan.Morenikeji O.A, Pillai C.R, Valecha N and Dash A.P ............................................................. 175

Studies on the local names, knowledge, attitude and practices of loa loa infection in okigwe zone of imo state nigeria.Orji N., Nwoke B., Ukaga C., Obi R. and Anyaegbunam L. ................................................... 181

Preliminary Analysis of in vitro released Excretory/ Secretory (E/S) Cysteine Proteases of the progenetic metacercariae of Clinostomum complanatum.Rizvi A., Fatima T., Shareef P.A.A., Saifullah M.K., Bano B. .................................................. 187

VI XII International Congress of Parasitology ICOPA

A seroepidemiological study of toxoplasmosis in pregnant women in Ardabil, Iran, Using ECLIA techniqueAhady M.T., Chinifroush M., Shokr-abadi M., Amini S., Jafari A.R. ...................................... 193

Polymorphisms of Pvmdr1 gene associated with Chloroquine Resistance Phe-notype among Plasmodium vivax isolates in IndonesiaAsih PBS, Sadikin M, Baird JK, Leake J, Sorontou Y, Sauerwein RW, Vinetz J and Syafruddin D ................................................................................................................ 199

Activation of heme oxygenase-1 by antimalarial drugs in vitroCheeramakara C, Suthisai N, Tanaka I, Ishihara H, Nontprasert A ...................................... 205

Genetic polymorphism of Leishmania major in Isfahan., Iran: Relationships with clinical presentation.Dabirzadeh Mansour, Hejazi Hossien, Mirmohammad-sadeghi Hemmid, Baghaie Mehdi ... 209

Strengthening malaria detection in Rwanda laboratory network by decentralized quality control programFlandin J-F, Mucaca J.B., Ruzindana E., Munyaneza T., Twahirwa M., Gatabazi J.B. and Mukabayire O. .............................................................................................................. 221

Study on Lernaeosis in Arwana Fish (Scleropages jardinii) from Papua, Indo-nesia Shatrie D. N., Kurniasih, Nurcahyo W. ................................................................................. 225

Study on Rhipicephalus (Boophilus) in IndonesiaSahara A., Kurniasih, Prastowo J. ....................................................................................... 229

The New Species of Myxobolus from Gill of Cyprinus carpio in Java, Indo-nesiaPriyono A., Kurniasih, Dewi T.C.A. ....................................................................................... 233

Immunotherapeutic Role of Radio Attenuated Leishmania Parasites in Expe-rimental Murine Visceral LeishmaniasisDatta S., Naskar K., Chakraborty A., Manna M. ................................................................... 239

Building Leadership and Capacity in Health Research in Ghana Quakyi Isabella, Tsui Amy ................................................................................................... 245

Survey of congenital toxoplasmosis by PCRDr Rasti S., Dr Bandepour M., Dr Kazemi B., Dr Behrashi M., Dr Fatahian A., Namakchian M., Mousavi Gh. .............................................................................................. 251

Pantropic retroviral vectors pseudotyped with the glycoprotein of vesicular stomatitis virus (VSVG) mediate gene transfer and transgene expression in Schistosoma japonicum schistosomulesYang S.H., Brindley P.J., Zeng Q.R., Zeng T.B., Li Y.S., Zhou J., Liu Y., Liu B.Y., Cai L.T., Wu C.R., Lu F.G., Tan Z.J., Lan L.M., McManus D.P. ............................................................................. 255

Author Index ....................................................................................................................... 261

2 XII International Congress of Parasitology ICOPA

Importantly, these cases correlate to high rates of observed GAS pyoderma in over 80% of these children.

Based on recent data [11] we hypothesise that the complement-inhibitory properties of excreted scabies mite proteins promote the growth of bacterial pathogens in the microenvironment of the epidermal burrows [12](Fig. 1). We aim to investigate these mechanisms in vitro and in vivo.

Materials and Methods:The nomenclature of the molecules investigated here is based on a phylogenetic tree

illustrating the evolutionary relationships between the SMIPP-Ss [13]. Recombinant SMIPP-Ss I1 and D1 and the active serine protease Sar s 3 were produced in Pichia pastoris by cloning SMIPP-S coding sequences retrieved from the original cDNA clones Yv6023A04 (Genbank accession number AY333081) and YvT004A06 (Genbank accession number AY333085) corresponding to the mature sequences predicted [14]B. Expression of SMIPP-S and Sar s 3 protein in P. pastoris and purification have been described [15; 11; 13]. Complement Proteins were sourced as described [11]. Methodology regarding C3b/C4b degradation assays, hemolytic assays, complement activation assays, direct binding assays, assays to assess the Assembly and decay of

Fig. 1: A female scabies mite burrowing in the upper epidermis. Scabies mite defense (gut and fecal proteins). Host response (immunoglobulins, complement,

coagulation factors, host proteases). Bacteria entering the skin. Picture credit: Andrew A. Erwin.

3Melbourne, Australia, 15-20 August, 2010

the alternative C3-convertase, localization of human C1q and IgG by immunohisto-chemistry has been outlined [11; 16].

Results:A multigene family of proteolytically active and inactive serine proteases (Fig. 2)

[14], homologous to the group 3 major allergens produced in the gut of closely rela-ted HDMs may have specific roles in suppressing the host’s response. SMIPP-Ss and Sar s 3 have been shown to be localised in the mite gut and released with the feces

Fig. 2: A phylogenetic tree illustrating the inferred evolutionary relationships between the SMIPPs was gene-rated in MEGA4 [18] = with maximum likelihood branch lengths inferred using the WAG amino acid matrix in PAML4.1[19]. The tree is rooted at the vertebrate-arthropod divergence. On the right hand side of each clade, mutations of the catalytic triad residues for that clade are shown as horizontal triplets, corresponding with the H63, D107 and S200 positions, respectively (human trypsin numbering). Outgroup sequences are D. pteronyssinus Der p 3 (accession DERP3_DERPT), D. melanogaster gamma trypsin (TRYG_DROME), H. sapiens trypsin 2 (TRY2_HUMAN), and D. rerio trypsin (NP_571783). Figure adapted and simplified from [13].

4 XII International Congress of Parasitology ICOPA

into the upper epidermis (Fig. 3) [15; 11; 16]. They co-localise with serum compo-nents such as host IgG and complement factors (Fig 3). Host IgG has previously been localised to the mite gut [17] and serves as a marker for gut localization.

We have functionally characterized the Scabies Mite Inactivated Protease Paralogues (SMIPP-Ss) as potent inhibitors of the human complement system [11]. SMIPP-Ss show no proteolytic activity but inhibit the haemolytic activity of human serum, as shown in haemolytic assays [11]. Their proteolytically active counterpart Sar s 3 has no effect on complement (unpublished) but plays a pivotal role in digesting filag-grin, a dominant protein forming the functional barrier of the stratum corneum [15]. SMIPP-Ss specifically prevent the activation all 3 complement pathways by binding directly complement factors C1q, MBL and properdin Fig. 4 [11]. The SMIPP-Ss interact mainly with the collagenous stalk of C1q (Fig. 4). To understand the structu-ral basis for SMIPP-S function two crystal structures at 1.85 Å and 2.0 Å resolution

Fig. 3: (A) Immunohistochemical staining demonstrated gut localisation of SMIPP-Ss, Sar s 3 and complement. In consecutive human scabies mite infested skin sections antibodies against a representative Smipp-S, Sar s 3 and co-localised with anti-human IgG (used as a marker to label the gut). (B) The mite gut molecules are also detected in the mite feces within the burrows (bottom row). Figure modified from [15; 11; 16].

5Melbourne, Australia, 15-20 August, 2010

were generated [13]. Occlusion of the S1 subsite by a conserved Tyrosine residue due to the lack of the third disulfide bond, which is present in the active Sar s 3, are predicted to block substrate ingress [13]. The structural changes together with the mutations in the catalytic triad render the SMIPP-Ss proteolytically inactive. We postulate that SMIPP-Ss lost the ability to bind substrates in a classical “canonical” fashion and instead have evolved alternative functions [13]. We are currently inve-stigating the SMIPP-S binding mechanism to complement factors, with the aim to develop complement-inhibiting peptides.

Conclusions:Any successful pathogen must have a strategy to resist complement-mediated kil-

ling. Burrowing scabies mites imbibe epidermal protein and plasma containing various host immune components, in particular the complement cascades. Hence, essential gut proteins must have evolved facilitating simultaneous digestion of the skin and evasion of host defence. SMIPP-Ss may be only one of several strategies that scabies mites have invented to overcome host defences.

This study was supported by Program Grant 496600 and a fellowship to D.J.K. from the Australian National Health and Medical Research Council and by grants from the Swedish Research Council and Swedish Foundation for Strategic Research.

References:[1] M. McDonald, A. Brown, T. Edwards, A. Hope, M. Amu, F. Morey, B. J. Currie, and J.

R. Carapetis, ‘Apparent contrasting rates of pharyngitis and pyoderma in regions where rheumatic heart disease is highly prevalent’, Heart Lung Circ (4) 16 (2007) 254-259.

[2] T. Stewart, R. McDonald, and B. Currie, ‘Acute rheumatic fever: adherence to secondary prophylaxis and follow up of Indigenous patients in the Katherine region of the Northern Territory’, Aust J Rural Health (4) 15 (2007) 234-240.

[3] J. R. Carapetis, M. McDonald, and N. J. Wilson, ‘Acute rheumatic fever’, Lancet (9480) 366 (2005) 155-168.

[4] M. McDonald, B. J. Currie, and J. R. Carapetis, ‘Acute rheumatic fever: a chink in the chain that links the heart to the throat?’, Lancet Infect Dis (4) 4 (2004) 240-245.

[5] A. C. Steer, A. W. Jenney, J. Kado, M. R. Batzloff, S. La Vincente, L. Waqatakirewa, E.

Fig. 4: Direct binding of SMIPP-Ss to various complement proteins. Microtiter plates were coated with complement proteins or BSA as a negative control and incubated with 200 µg/ml of SMIPP D1 or I1. Bound protein was detected using specific polyclonal antibodies against D1 and I1. Figure sourced from Bergström et al. [11] with permission. Copyright 2009. The American Association of Immunologists, Inc.

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K. Mulholland, and J. R. Carapetis, ‘High burden of impetigo and scabies in a tropical country’, PLoS Negl Trop Dis (6) 3 (2009) e467.

[6] J. Heukelbach and H. Feldmeier, ‘Scabies’, Lancet (9524) 367 (2006) 1767-1774.[7] J. R. Carapetis, A. Brown, and W. Walsh, ‘National Heart Foundation of Australia (RF/

RHD guideline development working group) and the Cardiac Society of Australia and New Zealand.Diagnosis and management of acute rheumatic fever and rheumatic heart disease in Australia - an evidence-based review.’, (2006).

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[9] D. Taplin, S. L. Porcelain, T. L. Meinking, R. L. Athey, J. A. Chen, P. M. Castillero, and R. Sanchez, ‘Community control of scabies: a model based on use of permethrin cream’, Lancet (8748) 337 (1991) 1016-1018.

[10] D. B. Clucas, K. S. Carville, C. Connors, B. J. Currie, J. R. Carapetis, and R. M. An-drews, ‘Disease burden and health-care clinic attendances for young children in remote aboriginal communities of northern Australia’, Bull World Health Organ (4) 86 (2008) 275-281.

[11] F. C. Bergstrom, S. Reynolds, M. Johnstone, R. N. Pike, A. M. Buckle, D. J. Kemp, K. Fischer, and A. M. Blom, ‘Scabies mite inactivated serine protease paralogs inhibit the human complement system’, J Immunol (12) 182 (2009) 7809-7817.

[12] K. Fischer and D. J. Kemp, ‘Scabies and bacterial skin infections at a molecular level.’, Microbiology Australia (5) 30 (2009) 177-180.

[13] K. Fischer, C. G. Langendorf, J. A. Irving, S. Reynolds, C. Willis, S. Beckham, R. H. Law, S. Yang, T. A. Bashtannyk-Puhalovich, S. McGowan, J. C. Whisstock, R. N. Pike, D. J. Kemp, and A. M. Buckle, ‘Structural mechanisms of inactivation in scabies mite serine protease paralogues’, J Mol Biol (4) 390 (2009) 635-645.

[14] D. C. Holt, K. Fischer, G. E. Allen, D. Wilson, P. Wilson, R. Slade, B. J. Currie, S. F. Walton, and D. J. Kemp, ‘Mechanisms for a novel immune evasion strategy in the scabies mite sarcoptes scabiei: a multigene family of inactivated serine proteases’, J Invest Dermatol (6) 121 (2003) 1419-1424.

[15] S. A. Beckham, S. E. Boyd, S. Reynolds, C. Willis, M. Johnstone, A. Mika, P. Simerska, L. C. Wijeyewickrema, A. I. Smith, D. J. Kemp, R. N. Pike, and K. Fischer, ‘Characte-risation of a serine protease homologous to house dust mite group 3 allergens from the scabies mite sarcoptes scabiei’, J Biol Chem (2009).

[16] C. Willis, K. Fischer, S. F. Walton, B. J. Currie, and D. J. Kemp, ‘Scabies mite inacti-vated serine protease paralogues are present both internally in the mite gut and externally in feces’, Am J Trop Med Hyg (4) 75 (2006) 683-687.

[17] C. M. Rapp, M. S. Morgan, and L. G. Arlian, ‘Presence of host immunoglobulin in the gut of Sarcoptes scabiei (Acari: Sarcoptidae)’, J Med Entomol (3) 43 (2006) 539-542.

[18] K. Tamura, J. Dudley, M. Nei, and S. Kumar, ‘MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0’, Mol Biol Evol (8) 24 (2007) 1596-1599.

[19] Z. Yang, ‘PAML 4: phylogenetic analysis by maximum likelihood’, Mol Biol Evol (8) 24 (2007) 1586-1591.