The Leishmania donovani complex is considered to be composed of 3 species; L. donovani, L. infantum and L. chagasi, although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene (mspC) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum, which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum/L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

In order to investigate the phenomenon of age-related immunity to visceral leishmaniasis, a 1 year prospective survey was carried out on 5671 people in a Leishmania infantum focus in north-west Iran. The average incidence rate of infection since 1985 was 2·8%/year with all ages equally at risk. One in 13 infections in children led to visceral leishmaniasis (VL), and this ratio decreased significantly with age. Seroprevalence also dropped rapidly with age, suggesting that the same process may affect both clinical outcome and the humoral immune responses. Cell-mediated immunity was associated with a reduction in the seroconversion rate and an increase in the serorecovery rate. Even amongst people with no detectable cell-mediated immunity to Leishmania, the seroconversion rate decreased and the serorecovery rate increased with age. All current VL patients had a negative leishmanin skin test response. Hence, adults may develop protection against L. infantum through 2 processes, 1 dependent and 1 independent of acquired cell-mediated immunity.

The in vivo expression and distribution of the dense granule protein GRA7 was examined in both the exoenteric (tachyzoite and bradyzoite) and enteric (coccidian) forms of Toxoplasma gondii by immunocytochemistry. There was strong staining of GRA7 in granules within all the infectious stages (tachyzoite, bradyzoite, merozoite and sporozoite). During tachyzoite development, GRA7 was secreted and was associated with the parasitophorous vacuole. In contrast, although there was staining of granules within the bradyzoites of more mature cysts, there appeared to be little staining of the tissue cyst wall or host cell. The apparent stage-specific variation in secretion of GRA7 between tachyzoites and bradyzoites was confirmed by double labelling using stage-specific markers (SAG1 and BAG1). In the enteric forms in the cat gut there was strong labelling of the PV containing early asexual and sexual stages and staining of a few granules in the apical cytoplasm of the merozoite. The positive enteric staining pattern differentiates GRA7 from the other GRA proteins (GRA1–6) which were absent in the merozoites and enteric stages. The staining pattern of GRA7 with strong staining during tachyzoite and enteric development and reduced staining in the tissue cysts is similar to that seen for NTPases. The function of GRA7 is unknown but it is unique among the dense granule proteins in being expressed in all the infectious forms of T. gondii which would point to a basic role in the vacuolar adaptations required for active parasite development.

DNA probes were developed for the detection and identification of 2 microsporidian parasites of marine fishes, Microgemma caulleryi (infecting the liver of the greater sand-eel, Hyperoplus lanceolatus) and Tetramicra brevifilum (infecting muscle, intestine and liver of the turbot, Scophthalmus maximus, a commercially important species). The probe-development procedure used is fast and straightforward, and readily applicable to the development of probes for other microsporidian species. First, genomic DNA of microsporidian spores was isolated and digested with the restriction enzyme Hind III. The fragments obtained were ligated into the vector pBluescript SK(+) and cloned in Escherichia coli. Appropriate inserts were identified and then amplified by PCR, using primers specific for regions adjacent to the Hind III restriction site in the vector sequence (and thus avoiding the need to develop primers specific for the inserts themselves). The copies were labelled with digoxigenin, for subsequent use as probes, during PCR itself. The specificity of candidate probes was tested in dot–blot hybridization assays, with the target DNA being (a) genomic DNA of the microsporidian from which the probe had been obtained, or of another species, (b) the corresponding genomic DNA in the phagemid, or (c) DNA from the corresponding host tissue. These assays identified a ca 1180 bp probe for M. caulleryi, denominated C38, and a ca 1363 bp probe for T. brevifilum, denominated F9. Similar assays designed to assess sensitivity indicated that F9 showed detectable binding to as little as 500 ng of T. brevifilum genomic DNA, and C38 to as little as 125 ng of M. caulleryi DNA; these results were obtained with detection of DIG by enzyme immunoassay (i.e. using a phosphatase-coupled anti-DIG antibody), and could no doubt be improved if a radioactive labelling and detection system were used. The probes developed in this study will greatly facilitate detection and identification of M. caulleryi and T. brevifilum in fish tissues, and may prove useful for identifying possible intermediate hosts used by these species.

The microsporidian genus Nosema is characterized by development in direct control with host cell cytoplasm, diplokaryotic nuclei throughout development and disporous sporogony. The genus Vairimorpha exhibits the same features plus an octoporous sporogony producing uninucleate spores in a sporophorous vesicle. A microsporidium from diamondback moth, Plutella xylostella, falls between Nosema and Vairimorpha in that it initiates but fails to complete the octosporous sequence in this host. The name Vairimorpha imperfecta n.sp. is proposed. Merogony is mainly by formation of buds from multinucleate meronts, the buds remaining attached in chains. Diplokaryotic spores measure 4·3×2·0 μm (fresh) and have 15·5 coils of the polar tube in 1 rank. The octosporous sporogony is aborted owing to irregular formation of nuclear spindles, incomplete cytoplasmic fission and bizarre deposition of electron-dense episporontal secretions. Phylogenetic analyses of the sequences of the small subunit rRNA genes of V. imperfecta and of several Nosema and Vairimorpha spp. place V. imperfecta in a clade with Nosema spp. from Lepidoptera rather than in the clade containing the more typical species of Vairimorpha. It is suggested that the ancestors of the Vairimorpha/Nosema complex of species exhibited both disporous and octosporous sporogonies, as does the type species of Vairimorpha, Vairimorpha necatrix. It would follow that true Nosema spp. have lost the ability to express an octosporous sequence and that V. imperfecta is in the process of losing it. It is proposed that the genera Nosema and Vairimorpha be placed in the same family Nosematidae Labbé 1899, rather than in separate families and orders as at present.

The tegumental membrane complex of Schistosoma mansoni is the site of interaction between the parasite and the host. The tegument is involved in uptake of many nutrients, but also plays a crucial role in the evasion of the actions of the host immune system. Essential for the success of this evasion is maintaining the integrity of the tegumental membranes. The rate of turnover of phospholipids was investigated by pulse-labelling worms cultured in vitro, followed by additional incubation in the presence of unlabelled substrates. Tegumental membranes were isolated, characterized using antibodies against specific tegumental proteins, and analysed. It was demonstrated that the most prominent fatty acid found in tegumental phospholipids, palmitate, incorporated rapidly into the phospholipid fraction during a 30 min pulse labelling. In a subsequent 20 h chase with unlabelled substrates, the incorporated radioactivity was lost again from the tegumental membrane complex. This high turnover of palmitate was found to be limited to phosphatidylcholine (PC) only. The turnover was due to deacylation/reacylation, and not to the sloughing of membranes as is the case in schistosomula. It is speculated that this rapid turnover of PC in the tegument of adult schistosomes plays a new and important role in the immune evasion by the parasite.

The genetic population structures of the freshwater snail Bulinus globosus and its trematode parasite Schistosoma haematobium from 8 river sites in the Zimbabwean highveld were compared using randomly amplified DNA(RAPD) markers. There was significant variability between snail populations collected at different sites, but schistosome populations only showed differentiation at a wider geographical scale (between 2 non-connected river systems). For snails, genetic distance was better correlated with proximity along rivers than absolute geographical separation. In contrast, schistosome genetic distance was better correlated with absolute geographical separation than proximity along rivers. These results are consistent with different dispersal mechanisms for snails and schistosomes and the implications for host–parasite coevolution are discussed.

Two clones, totalling 8068 bp and spanning over half of the coding region of the mitochondrial genome of Schistosoma mansoni, have been sequenced. Complete sequences are presented of the large and small ribosomal RNA subunits, CO2, ND3, ND4, ND6 and ATPase 6 genes. Incomplete sequences were found for the CO1, ND2 and CytB genes. At least 10 tRNAs were also detected and alternative structures for some of these discussed. The gene order of S. mansoni is unique and differs from that of Fasciola hepatica, the only other trematode for which any information is available.

Localization of the type 2 Alu sequence (B2), a highly repetitive DNA sequence in the mouse genome, was examined by in situ polymerase chain reaction (in situ PCR) in schistosomes. The signals to the B2 sequence were detected in the cytoplasm of the tegumental membrane and in the nuclei of the mesenchymal, testicular, ovarian and vitelline cells of 8- week Schistosoma japonicum. In contrast, it was difficult to detect any signals of this sequence in 8-week S. mansoni, whereas in 24-week male S. mansoni the signals were observed in the cytoplasm of the tegumental tubercles and in the nuclei of the mesenchymal and testicular cells. On the other hand, in 24-week female S. mansoni the signals were found in the nuclei of the mesenchymal, ovarian and vitelline cells but not found in the tegument. On the contrary, no hybridization band of the B2 sequence was detected in the amplified DNA of 3-week schistosomula of either species. These observations proved that the host DNA sequences existed in restricted schistosome cells and were accumulated in the schistosome body during their development.

A total of 121 European eels (Anguilla anguilla) from 2 sampling sites on the River Rhine were investigated in respect of their parasite communities. Special attention was given to the swim bladders, intestines, gills and fins of the fish. Twelve different parasite species were found to live in and on the eels. Data from each sampling site were kept separate. Parasites found in descending order of prevalence were: Anguillicola crassus, Trypanosoma granulosum, Myxobolus sp., Paratenuisentis ambiguus, Pseudodactylogyrus sp., Bothriocephalus claviceps, Myxidium giardi, Pomphorhynchus laevis, Trichodina sp., Raphidascaris acus, Acanthocephalus lucii and Acanthocephalus anguillae. Significantly different prevalences were reported for L3 larvae of A. crassus, adult P. ambiguus, B. claviceps and Myxobolus sp. at the 2 sampling sites. The highest number of parasite species was recorded from the intestine, which contained up to 6 different helminths. The coexistence of the acanthocephalans P. laevis and P. ambiguus, which showed clear patterns of distribution within the intestine of the respective hosts, was reported for the first time. Up to 3 different helminth species were found in the intestine of individual fish. Among those, acanthocephalans were the most prevalent worms with the eel-specific parasite P. ambiguus as the dominant species not only of the intestinal but also of the total component communities. Both infra and component communities exhibited low diversity and were dominated by this single species. The evenness reached only approximately 50% or less and it remained unclear why the helminth communities of the eels from the River Rhine with its huge catchment area exhibit such a low parasite diversity and high dominance.