We report the molecular characterization of a middle repetitive DNA sequence, named C6, isolated from the Trypanosoma cruzi genome. C6 appears to be a composite repeated element since 3 subregions may be defined within it on the basis of sequence similarities with other T. cruzi genomic sequences. Sequences homologous to C6 are interspersed in the genome and can be mapped out on most chromosomal bands of different T. cruzi strains. The copy number of the C6 element is about 1000 per haploid genome. Given the species specificity and different genomic distribution of C6 homologous sequences among the T. cruzi strains the C6 element could be a useful probe for diagnosis and typing of parasites. C6 is a polymorphic marker with potential as a tool for physical mapping of the T. cruzi genome.

We compared 19 stocks of Trypanosoma brucei rhodesiense collected in 1991 and 1994 from Tanzania with representative stocks from other foci of Rhodesian sleeping sickness in Zambia, Kenya and Uganda. Stocks were characterized by isoenzyme electrophoresis, restriction fragment length polymorphisms in variant surface glycoprotein genes and random amplification of polymorphic DNA; the banding patterns obtained were coded for numerical analysis. In addition, the Tanzanian stocks were compared by pulsed field gel electrophoresis. Overall the Tanzanian stocks formed a homogeneous group and the predominant genotype isolated in 1991 was still present in the 1994 sample, although at a reduced level. The Tanzanian stocks were distinct from representative stocks from other East African foci. This observation does not support the proposal that there are northern and southern strains of T. b. rhodesiense, but is consistent with the view that T. b. rhodesiense stocks form a mosaic of different genotypes varying from focus to focus in East Africa.

Neospora caninum is an apicomplexan parasite of veterinary importance which invades many different cell types and tissues. N. caninum tachyzoites proliferate intracellularly by endodyogeny. Eventually the massive proliferation of tachyzoites leads to host cell lysis and the newly formed parasites are released and invade neighbouring cells. Tachyzoite cell surface molecules could serve as ligands, mediating host cell adhesion and invasion. Nc-p43 is a recently identified N. caninum tachyzoite surface protein which is functionally involved in the processes leading to host cell invasion in vitro. Affinity-purified antibodies directed against Nc-p43 were used to screen a lambda gt22A-cDNA expression library constructed from N. caninum tachyzoites. The cDNA insert of one immunoreactive clone was subcloned and expressed in E. coli as a poly-histidine fusion protein. The identity of the resulting recombinant antigen termed recNc-p43 was confirmed by immunoblotting, immunofluorescence and electron microscopy using affinity-purified antibodies. The sequence of the cDNA insert encoding recNc-p43 was determined. Analysis of the deduced amino acid sequence revealed that Nc-p43 exhibited similarity to SAG1 (p30) and SAG3 (p43), 2 major surface antigens of Toxoplasma gondii tachyzoites. These similarities were not reflected on the immunochemical level, since no cross-antigenicity between SAG1, SAG3 and Nc-p43 was observed.

We present observational and experimental evidence that cycles of the Lyme disease spirochaete, Borrelia burgdorferi s.l., can be maintained by sheep in the virtual absence of alternative hosts. A 2-year field study in upland moorland habitats of northwest UK established that sheep feed up to 80% of larval, >99% of nymphal and all of the adult female tick (Ixodes ricinus) population. Infection prevalence of B. burgdorferi in questing ticks reaches over 20%, but amplification of infection occurs principally as nymphs (20- to 30-fold), rather than larvae (4- to 7-fold), feed on sheep, and transmission from sheep to ticks occurred only during peak tick abundance in May and September. Experimental transmission studies confirmed that sheep, previously exposed to infected ticks on the moorland site, do not support systemic infections of B. burgdorferi, but they can transmit localized infections from infected to uninfected ticks co-feeding at the same site on the sheep's body.

During their growth in vitro, promastigotes of Leishmania amazonensis undergo differentiation from complement-susceptible to complement-resistant forms. Here, we demonstrate that both forms bind comparable amounts of C3 on their surfaces, with the predominant molecule species being the haemolytically active C3b. Likewise, equivalent amounts of C9 are deposited on both forms of promastigotes. However, while C9-bearing complexes are exposed on the cell surface of resistant promastigotes, they are cryptic in the susceptible stage of the parasites. The membrane fraction of complement-resistant promastigote lysates has the ability to inhibit complement-mediated haemolysis, blocking C9, but not C3 deposition to complement-activating complexes. Moreover, the membrane fraction of complement-resistant promastigote lysates can inhibit the late steps of guinea-pig erythrocyte lysis much more efficiently than complement-susceptible ones. Our results indicate that L. amazonensis promastigotes evade complement killing by inhibiting the cytolytic pathway of the complement cascade.

Leishmania infantum immunoelectrophoretic antigen 24 (AG 24), a visceral leishmaniasis associated immunodominant antigen, has been characterized with a monospecific antiserum by combining SDS–PAGE, immunoblotting, metabolic labelling, radio-immunoprecipitation and in vitro poly A+ mRNA translation. AG 24 appeared to correspond to a multi-antigen family of 6–9 members ranging from 20 to 31 kDa and proteinic by nature with no post-translational modifications. A similar banding pattern was recognized by infection sera. AG 24 was not found exposed on the cell surface.

Infection with Schistosoma mansoni was studied in 5 troops of olive baboons (Papio cynocephalus anubis) in Gombe Stream National Park, Tanzania. Three troops were infected with S. mansoni. An aggregated distribution of parasites was observed among hosts. Troop membership was found to be the most significant factor influencing parasite prevalence. Age and reproductive status had no significant effect, but there was a trend for males to acquire higher levels of infection. However, age–prevalence curves showed a high infection in young baboons declining in the older baboons. Behavioural components of exposure – as measured in water-contact pattern – may be related to parasite burden. A ‘peak shift’ between infection in different age-classes in the different troops was observed: troops with higher schistosome prevalences displayed an earlier peak in prevalence of infection. The baboon troop with the most contact with people showed highest prevalence of infection possibly due to longer exposure to the parasite than the other troops and/or higher host density.

The maturation of female Schistosoma mansoni depends on pairing with a male which induces mitotic activities in the reproductive organs of the female worm. Since in other organisms cell proliferation is regulated by well-conserved signal transducing molecules, we looked for such molecules on immunoblots of schistosomes, using antibodies against conserved epitopes of Ras, GAP and MAP kinases. We identified all 3 molecules in schistosomes and found that they are developmentally regulated. Furthermore, there is evidence for their involvement in the male-directed maturation of the female.

Gene expression studies in adult females of Schistosoma mansoni cultured in vitro revealed that the transcription of female-specifically expressed genes is influenced by pairing. In contrast, the activity of genes that are expressed in both genders was not affected by contact with the male. The transcription of genes was monitored in paired, separated and remated females. The transcript level of female-specifically expressed genes decreases within a few days following separation from males. Remating of uncoupled females with males leads to the reinitiation of transcription. These results provide strong evidence for the influence of the male on gene transcription in the female and contribute a molecular basis for the classical histological observation that the maturation of females is male dependent. The data also show that the culture system is suitable to monitor gene expression and, furthermore, they indicate de novo RNA synthesis in vitro.

Several monoclonal antibodies (Mabs) were raised against the L1 muscle stage (L1M) of Trichinella spiralis (Ts) and Trichinella pseudospiralis (Tp). Western blot analysis of various antigenic preparations established that Mabs described by different authors recognized 8 antigenic fractions (TSL1–TSL8) in crude extracts of infective larvae. The TSL1 fraction was immunodominant and present on the cuticle of different parasite stages. Mabs against Trichinella T5 (T5) and Ts were selected in order to extend the previous studies to another Trichinella phenotype. Only 35% of the selected Mabs recognized linear epitopes and 71% reacted with soluble or excretory–secretory antigens in a dot blot procedure and ELISA test. The targets of the Mabs were identified by immunoprecipitation with [35S]methionine-labelled L1M worm lysate. Mabs prepared from mice immunized with the whole parasite (T5) recognized a wider panel of antigens in different parasitic organs. Seven antigenic structures were distinguished on the cuticle and several epitopes were identified in the gut, haemolymph and stichocytes. Eleven antigenic groups were established according to their indirect immunofluorescence pattern on cross-sections of the worm. Monoclonal antibodies raised against Ts soluble antigen mainly recognized epitopes in stichocytes and on the cuticle surface. All the selected Mabs recognized T5 and Trichinella britovi (Tb) strengthening the link between these 2 species. Four Mabs were used to differentiate antigenic structures among 6 Trichinella phenotypes and to develop a new tool to follow gene flow within the Trichinella genus.

A single form of cholinesterase was detected in the parasitic nematode Parascaris equorum and purified from a low-salt Triton X-100 extract of whole animals by affinity chromatography on an edrophonium–Sepharose matrix. Based on gel-filtration chromatography, sedimentation analysis and SDS–PAGE, such a cholinesterase is an amphiphilic globular (G2) dimer (125–129 kDa, 6·1 S). It includes some hydrophobic domain other than phosphatidylinositol, which gives auto-aggregation, detergent interaction and also anchors the molecule to the cell membrane. The enzyme, probably functional in cholinergic neurotransmission, is an acetylcholinesterase showing a fairly low substrate specificity with thiocholine esters. Electrostatic interactions seem to play a major role in the catalytic activity. Studies with inhibitors gave complete inhibition with 1 mM eserine, low sensitivity for procainamide and for tetra(monoisopropyl)pyrophosphortetramide as well as higher inhibition with edrophonium chloride and 1,5-bis(4allyldimethylammoniumphenyl)-pentan-3-one dibromide. The enzyme also showed excess-substrate inhibition with acetylthiocholine. No cross-hybridization occurred between the gene(s) encoding acetylcholinesterase in P. equorum and ace-1 from the free-living nematode Caenorhabditis elegans. The expression of a single cholinesterase form in P. equorum, unusual in free-living nematodes, could be due to parasitic life adaptation with resulting reduction of locomotor activity.

Three hymenolepidid tapeworms, Hymenolepis diminuta, H. nana and H. microstoma, are commonly maintained in laboratory rodents and used in many experimental model systems of tapeworm infections. We examined partial sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences to infer phylogenetic relationships of the 3 hymenolepidid species. Parts of the CO1 gene and ITS2 were amplified by PCR and sequenced directly. The CO1 gene sequence obtained was the same in length (391 bp) among all specimens. In the case of ITS2, however, several insertions and deletions were detected (671–741 bp) not only among species but also between an American isolate and a Japanese isolate of H. diminuta. Percentage nucleotide differences between H. diminuta and H. microstoma, or H. diminuta and H. nana were 16·6–18·2% for the CO1 gene and 21·3–22·9% for ITS2. The differences in both sequences between H. microstoma and H. nana were about 14%. Phylogenetic trees inferred from both of the nucleotide sequences showed similar topology, and suggest that H. diminuta may have diverged from the common ancestral line the earliest, and that H. nana is closer to H. microstoma than to H. diminuta.

Regional differences of Hymenolepis diminuta to immune lysis have been investigated by monitoring surface membrane fluidity utilizing fluorescence recovery after photobleaching (FRAP). Although the surface membrane of the newly excysted stage was completely immobile the molecular fluidity of the strobila membrane of 4-day-old parasites was greater than that associated with the scolex/neck region. Significant differences (P>0·001) occurred in the mobility of the strobila membrane of 4-day-old H. diminuta from 100-worm infections compared with 7- and 21-day-old parasites and 4-day-old individuals from 10-worm infections. Exposure to 50% normal rat serum and 1 mg/ml rat C reactive protein decreased or eliminated membrane fluidity. The significance of membrane mobility is discussed with reference to resistance to complement-mediated lysis and destrobilation.