Workshop 1L: Immunodiagnosis

Post Congress Scientific Report selectively inhibited egg production and much of the gross pathology associated with S. mansoni infection. In culture, the effect on egg production was reversed by the addition of mevalonate. Based on these and other results presented, Van de Waa et al. conclude that the conversion of HMG-CoA to mevalonate is a vital component of S. mansoni egg production. A. C. Fusco et al. in an in vitro study of the role of pH in eicosanoid production and subsequent penetration and transformation of S. mansoni cercariae, reported that treatment with esculetin (an eicosanoid inhibitor) altered the pH optima for both penetration and transformation. This alteration coincided with a change in the distribution of eicosanoids produced supporting the author's hypothesis that leukotrienes and HETEs are important in cercarial penetration while prostaglandins are important in the transformation process. As a potential direction for the identification of anthelmintic activity, D.M. Miller et al. have examined the spontaneous muscle potentials of Acanthocephalans in the presence of a series of biologically active compounds (benzimidazole, oxadiazole, phenylhydrazone and thionocarbanilide). They have concluded that these compounds show different and pronounced effects on membrane ion channels. In an effort to develop compounds active at stages of the parasitic life-cycle other than the adult, P.J. Waller and E. Lacey reported the high in vitro and in vivo larvicidal activity of a series of insecticidal benzoylureas against Trichostrongylus colubriformis. They suggest that such compounds offer a potential adjunct to current anthelmintic treatment, possessing potential for use in sustained release devices. As part of studies into the site of action of benzimidazoles, L. Tang et al. reported the isolation of tubulin from Brugia malayi, B. pahangi and Nippostrongylus brasiliensis using polylysine linked sepharose followed by gel permeation chromatography (by HPLC). In continuation of studies into the benzimidazole-tubulin interaction, E. Lacey reported on the development of a (3H)-mebendazole binding assay to investigate the intrinsic activity of microtubule inhibitors in Haemonchus contortus. In this study, he reports that known inhibitors of mammalian microtubule activity (colchicine, podophyllotoxin, tubulozole-C and others) were 20 to 2000-fold less active against nematode tubulin, highlighting the origins of the selective toxicity of benzimidazoles as anthelmintic agents. WORKSHOP 1J: HOST-PROTECTIVE IMMUNE

RESPONSES D. D. DESPOMMIER*and M. D. RICKARDt * Public Health & Microbiology, Columbia University, New York, U.S.A. t Veterinary Clinical Centre, University of Melbourne, Werribee, Victoria, Australia This workshop contained presentations on a wide variety of related subjects. H. O. Bogh, M. D. Rickard

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and M. W. Lightowlers reported on stage specificity to larval cestode infection and gave convincing evidence that immune mice kill larval Taenia taeniaeformis in their post-oncospheral state of development. The mechanisms of killing or the antigens which elicited the death of the parasite were not identified. Using a related parasite, T. saginata, candidate antigens which evoke host protection were identified by monoclonal antibodies and affinity chromatography. L. J. S. Harrison, G. W. P. Joshua and R. M. E. Parkhouse went on to describe other mAbs which did not confer protection, but which did react with stagespecific antigens on the oncosphere. Crude fractions of Nematospiroides dubius fractionated into molecular weight classes were tested in mice for vaccine efficacy. The largest molecular weight fraction was best at protecting mice. This work was carried out by F. G. Monroy and C. Dobson. The protective immune mechanism(s) against Trichinella spiralis were investigated in which adoptive transfer of welt-characterized sub-populations of lymphocytes were transferred into nonimmune recipients by R. K. Grencis and D. Wakelin. They found that expulsion of adults is facilitated by L3T4 4- ve, L32-ve helper T cells. These cells released interlukin-2, interlukin-3 and immune gamma interferon when placed in the presence of a crude antigen extract. Small numbers of these cells mediated significant immunity in naive mice. Immunity to Anaplasma marginale was correlated with the appearance of antibody against a 38,000-43,000 molecular weight protein. J. H. Adams and R. D. Smith speculated that this antigen, also present in crude lysates of the organism, is responsible for the protection that the crude.lysate can induce in mice. WORKSHOP 1 L: IMMUNOI)IAGNOSIS

GEORGE V. HILLYER * 8£ MARSHALLW. LIGHTOWLERS'~

• Department of Biology, University of Peurto Rico, Puerto Rico t Veterinary Clinical Centre, University of Melbourne, Werribee, Victoria, Australia Significant advances on the immunodiagnosis of cestode, nematode and protozoan infections utilizing several combinations of conventional and modern technologies were presented at the workshop.

1. Cestode infections Regarding human cysticercosis, E. Nascimento, J. D. Lopes and C. A. P. Tavares developed three stagespecific monoclonal antibodies (McAbs) to a scolex protein antigen (SPA) of the rnetacestodes. These McAbs were then used to purify antigen from SPA to homogeneity (defined as a single band in PAGE). Applying this antigen in ELISA for diagnosis of Taenia solium cysticercosis 100% of cases were detected as positive. A. Landa, M. Merchant, K. Willms and J. P. Laclette chromatographed T. solium metacestode antigens through Con A sepharose and showed 22 protein bands (coomassie) of which one

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glycoprotein (GPI 180 kD) was found to be common to both T. solium metacestode and adult worm preparations as well as different Taenia spp. metacestodes. C. Larralde, F. Goodsaid and co-workers found that the vesicular fluid of T. solium metacestodes when used in ELISA or IHA had 95% sensitivity when normal human sera from nonendemic areas was used as controls and 100% specificity to detect human cysticercosis. The sensitivity dropped to 80% (ELISA) or 91% (IHA) when the "normal" sera used were from endemic areas. A prominent protein (103 kD) was identified by protein transfer (ErFB) as the most conspicuous detected by the sera of neurocysticercosis patients and clearly warrants further study. This is also found in T. crassiceps making this a potential antigenic source. B. Gottstein and P. M. Schantz found that enzymelinked immunoelectrotransfer blot (EITB) alone could be used for the species-specific imrnunodiagnosis of human cysticercosis. This Z solium metacestode antigen separated by PAGE and reacted in EITB with the sera from patients with cysticercosis consistently showed two 8 and 26 kD bands which were diagnostic for infections. Regarding human Echinococcus multilocularis , a species-specific 54 kD polypeptide with pI of 4.8 was first identified by EITB and IEF (with serum from infected patients) isolated by antibody affinity chromatography and applied to an ELISA which was shown to have a sensitivity of 94% (78 patients) and specificity of 99% with no cross-reactivity with sera from patients with other cestode, nematode, trematode or protozoan infections. D. J. Jenkins and M. D. Rickard investigated antibody responses in helminth-free dogs showing monospecific infection with E. granulosus. Using protoscolex ES antigens, specific antibodies were detected and cross-reactivity studies with other taeniid and nematode antigens demonstrated high specificity of the antibodies for Echinococcus. In addition they investigated ES antigens from larval scoleces of Taenia hydatigena, T. pisiformis and E. granulosus and oncosphere antigens in EITB with sera from dogs with homologous or heterologous cestode infections. Although cross-reactivity was observed between the scolex ES antigens of the Taenia species, no cross-reactivity was observed with ES antigens from E. granulosus protoscoleces. The oncosphere antigens of Taenia spp. also showed a high degree of specificity and 31 and 44 kD antigens were found unique to T. pisiformis and a 22 kD antigen was unique to T. hydatigena. In discussion of the presentations on cestode immunodiagnosis, M.W. Lightowters indicated that the finding of high titre antibodies against taeniid cestode infections in definitive hosts by D. J. Jenkins and M.D. Rickard could have important implications for immune diagnosis of neurocysticercosis where man also acts as definitive host. Should antibodies against adult worms cross react with

metacestode antigens used in cysticercosis immunodiagnosis, this could contribute to "false" positive reactions. K. Willms responded that this was indeed the case and six of ten patients with adult T. soliurn infections were positive in antibody tests with cysticercus antigens.

2. Nematode infections Sera from filariases patients are notorious for their cross-reactivity with filarial spp. antigens. M.M. Kliks, D. C. F. Chan, J. F. Duncan and C. DeiSanti found that sera from patients with nodular and lymphatic filariasis reacted equally well with the sera of patients with Dracunculus medinensis adult female extracts. However filariasis and dracunculiasis sera could be descriminated by EITB and only the latter recognized bands at 60, 57, 19 and 16 kD. Further work to isolate and test the antigenic polypeptides is needed. Regarding human onchocerciasis, R. M. E. Parkhouse and Z. Y. Cabrera found that the detection in ELISA of IgG4 antibody to a low MW antigen gave the highest combined sensitivity and specificity over total IgG alone. In the discussion, a comment was made by D. de Savigny that although the ELISA described by Parkhouse was found to have a sensitivity of 97% with a group of sera from an area with 50% prevalence of onchocerciasis, practical diagnosis in the O.C.P. would need to be demonstrated with sera from low prevalence areas. C. del Aguila, C. Cuellar and J.L. Guillen developed five McAbs with stage specific reactivity with 7bxocara canis second stage larvae and no crossreactivity with Ascaris or Toxascaris. A sandwich ELISA was developed for the detection of circulating immune complexes in humans with visceral larval migrans. Carlow and Philipp developed a stage- and speciesspecific IgM McAb against the infective stage of Brugia malayi found in the mosquito. This was then applied to ELISA to detect larvae in mosquitoes with a high degree of sensitivity and specificity. The authors hope that with this species- and stage-specific probe they can determine immunologically the number of mosquitoes carrying B. malayi third stage larvae. 3. Protozoan infections J. Upcroft and co-workers constructed plasmid cDNA libraries from high molecular weight Giardia intestinalis mRNA and screened clones from the libraries utilizing antibodies raised in rabbits. A clone useful for the detection of infection sera still needs to be identified. E. Linder reported the development of two McAbs for Pneumocystis. One McAb was Pneumocystis specific and detected an 82 kD antigen; the second cross-reacted with Giardia flagella and was shown to be associated to microtubules. In general, most poster and oral presentations used state of the art technologies and combined several

Post Congress Scientific Report strategies to identify and isolate the target immunodiagnostic antigens: PAGE and Western blot (EITB) to identify potential antigenic polypeptides; electroelution of the antigens from gels; development of monoclonal or polyclonal monospecific antibodies followed by the use of these antibodies to isolate the relevant antigens; and finally, the application of these antigens to EL1SA.

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tive antigen. Antibody columns were used for the purification of protective antigens from Fasciola hepatica and Taenia saginata. In the first case a polyclonal antibody was absorbed with juvenile specific antigen and then used for preparing the column and the corresponding antigen. In the second, a monoclonal antibody reactive with the oncosphere surface was similarly employed. Both preparations gave significant protection in vim.

WORKSHOP 1M: HELMINTH ANTIGENS AND

GENES R. M. E. PARKHOUSE*and M. J. HOWELLt * National Institute of Medical Research, Mill Hill, London, U.K. t Australian National University, Canberra, A. C. T., Australia This workshop focused on four general areas:

(1) The construction and employment of DNA probes for the characterisation of parasite species and variants Probes specific for species of Onchocerca, Echinococcus and Brugia were described. An important point in the case of filarial parasites was the recognition that species-specific DNA probes alone are unlikely to distinguish between microfilariae and infective larvae in the insect vector. An interesting and useful approach to meet situations where homology is extensive was described for Brugia; a repeating DNA sequence was 90% identical between B. malayi and B. pahangi. Fortunately, however, half of the sequence differences were localized to a - 3 0 nucleotide long sequence, and once the appropriate sequences were synthesized it was possible to construct specific diagnostic probes. (2) Expression of parasite antigens in cDNA libraries Successful expression of antigens of Schistosoma, Fasciola, Taenia and Brugia was described in a variety of systems: 2 phage gt 11, Salmonella and mouse C 127 cells. There was general agreement that monoclonal and polyclonal antibodies, including infection sera, to native determinants frequently fail to detect the corresponding products in expression libraries constructed in 2gt 11. On the other hand, antibodies raised against deliberately denatured parasite antigens were more likely to detect fusion proteins expressed in the 2 phage system. A particularly interesting approach, and one likely to receive more attention in the future, was described for Brugia, where an EcoRI digest of genomic DNA was cloned into )~gt 11 to yield an expression library giving a high frequency of antigen expressing clones (1 in 8000). (3) Direct isolation of potentially protective parasite

antigens A surface antigen of Dirofilaria immitis was successfully prepared using conventional biochemical isolation techniques. Yield, although 50% of that predicted, was too low to test its potential as a protec-

(4) Stage-specific expression An interesting antigen of female schistosomes was described. This material was detected in cDNA libraries, was only synthesized by females when coupled to males, and was likely to be an egg component. The use of recombinant nucleic acid technology to explore and define stage-specific parasite components may be expected to increase in the future. WORKSHOP IN: BIOCHEMISTRY OF THE PROTOZOA

G. H. CooMBs* and A. M. GEROt * Department of Zoology, University of Glasgow, Scotland, U.K. t University of New South Wales, Kensington, N.S.W., Australia This workshop contained 17 presentations on a wide variety of parasites and biochemical topics and included a number of exciting and important discoveries. We were reminded that meaningful biochemistry is dependent upon sound parasitological techniques by the report of J. D. Lonsdale-Eccles et al, on purifying African trypanosomes. They showed that bloodstream forms of Trypanosoma brucei brucei purified by the standard ion-exchange chromatography procedures or a density gradient centrifugation method differed in their content of some enzymes, notably proteinases. The former method appeared less satisfactory. The discoveries that trypanothione plays a part in glutathione reductase of salivarian trypanosomes and their metabolism of hydrogen peroxide have been followed up by P.G. Penketh and W. P. K. Kennedy in their studies of other trypanosomatids. They found that both T. cruzi and Leishmania donovani contain more of the cofactor than T.b. brucei and also differ from this parasite in their abilities to metabolize hydrogen peroxide, being able to handle greater concentrations. It was suggested that this could relate to the presence of an intracellular stage in their life cycles. D. J. Grab et al., reported that the enzyme responsible for removing dimyristyl glycerol from the variable surface glycoprotein (VSG), which could play a role in both VSG and membrane recycling, is largely associated with the flagellar pocket and/or endosome-like structures. There were two papers concerning the characterization of T. cruzi stocks from Chile; one by X. Aguilera, W. Apt and M. Miles concerned zymodeme analysis and the other by J. Cerrero et al. involved restriction endonuclease analysis of the mitochondrial