The technique of Random Amplified Polymorphic DNA (RAPD) PCR has been used to detect DNA polymorphisms among Toxoplasma gondii strains. Seven arbitrary oligonucleotides (10-mer) were used as primers to amplify total genomic DNAs and significant genetic heterogeneity was detected among 11 T gondii strains with different virulence for mice. The polymorphisms observed allowed relationship dendrograms of T. gondii strains to be constructed by PHYLIP and PAUP analyses. The genetic relationships of the T. gondii strains generated by 2 analyses using completely different assumptions were similar. Both analyses revealed 2 groups of T. gondii strains, one formed by the 6 virulent strains and the other formed by the 5 avirulent strains. This suggests that the genus Toxoplasma may actually contain 2 groups, correlated with their virulence, which have probably evolved independently following their initial separation. Significant polymorphisms were also detected between 2 different laboratory stocks of the T. gondii RH strain.

To understand the relationship between mode of transmission and virulence, we investigated 2 microsporidian parasites in Daphnia magna laboratory populations. Pleistophora intestinalis is only transmitted horizontally, while Tuzetia sp. is transmitted vertically with high efficiency from mothers to parthenogenetic male and female offspring. We were not able to transmit Tuzetia horizontally in the laboratory. Tuzetia reduces host life-time reproductive success and host survival to a much greater extent than does P. intestinalis. Tuzetia-infected hosts were rapidly outcompeted by uninfected hosts. We suspect that Tuzetia infections may persist in natural populations by an as yet undiscovered horizontal transmission. It is possible that an alternate host species may be involved. We present a mathematical model to analyse the conditions for the persistence of a parasite with perfect vertical and an additional degree of horizontal transmission. We show that horizontal and vertical transmission contribute additively to a parasite's ability to invade and persist. Since the fitness contribution of horizontal transmission increases with population size, only very low transmission probabilities per host-to-host contact are necessary for the persistence of parasites occurring in large populations such as those commonly found for Daphnia. The detection of such low rates of horizontal transmission appears unlikely under laboratory conditions since the necessary number of host-to-host (or host-to-spore) contacts is not feasible. We review mechanisms that can maintain vertically transmitted parasites in nature.

Immune responses of 4 inbred lines of chickens, that differ in resistance to Eimeria maxima and E. tenella, were examined. Significant differences were found in in vitro proliferation of peripheral blood lymphocytes to E. maxima sporozoite antigen, the more resistant lines C and 72 having higher responses than the more susceptible line 151. These differences existed pre-infection and were enhanced following both primary and a second infection. The proportions of lymphocyte subsets in the peripheral blood following primary infection also differed between lines, with significantly higher percentages of CD8 + and TCR1 + lymphocytes circulating in the more resistant birds. In contrast, there were few differences between lines in either resistance or in in vitro proliferation of peripheral blood lymphocytes to E. tenella sporozoite antigen either pre-infection or following a primary infection. However, after a second infection when there were significant differences in resistance between lines, as measured by oocyst excretion, there were also significant differences in lymphoproliferation with the more resistant lines 151 and 62 having higher responses than the more susceptible line C. Thus for E. maxima there is a direct relationship between resistance to infection and lymphoproliferation in response to parasite antigen. This implies that differences in cellular immunity may account for differences in resistance between lines, and since these specific responses are enhanced by infection they may also reflect important immune mechanisms. For the rather less immunogenic E. tenella, the correlation between resistance and lymphoproliferation is not so clear. However, where there were significant differences between lines, i.e. after a second infection, the direct relationship between resistance and lymphoproliferation was upheld.

The initiation of feeding of infected Rhipicephalus appendiculatus adults induces the rapid development of Theileria parva sporoblasts within the salivary gland acini leading to the production of numerous sporozoites which are inoculated into the mammalian host initiating infection. In this study the pattern of development, host cell specificity and emission of T. parva sporozoites within the salivary glands of heavily infected, 4-day fed adult R. appendiculatus ticks was examined. Infected acini were randomly distributed throughout the salivary gland. Sporozoite development within each gland was not synchronized and wide variation in the rate of parasite development, which correlated with the secretory activity of the individual acinus, was observed in all glands examined. Previous studies had shown that T. parva developed primarily in Type III ‘e’ cells. However, in heavily infected salivary glands sporogony and the emission of mature sporozoites also occurred in ‘c’ cells of Type II acini. Sporozoite emission from infected cells occurred by a process similar to apocrine secretion. The loss of the apical membrane of the infected cell allowed sporozoites free access to the lumen of the acinus and into the collecting ducts of the salivary gland. Sporozoite discharge was gradual since few parasites were found in the acinus valve or in the collecting ducts. Furthermore, the small size of the acinar valve aperature ensures that only small numbers of sporozoites can be released at any one time from an infected acinus.

A guinea-pig immunoglobulin-G linked agarose column was used to detect the immunoglobulin-G binding proteins (IGBP) in the ixodid ticks, Rhipicephalus appendiculatus, Amblyomma variegatum and Ixodes hexagonus. IGBPs were found in salivary gland extract (SGE) of the unfed ticks of all 3 species. The sizes of the IGBPs, as determined by SDS–polyacrylamide gel electrophoresis, differed between species. In R. appendiculatus 2 IGBP bands were detected in haemolymph collected from unfed females and males, and an additional 78 kDa IGBP was detected in haemolymph samples of male and female ticks that had fed for 6 days. Although there was no difference in the IGBP profile of SGE derived from unfed male and female R. appendiculatus, 2 new IGBPs were detected in the SGE of the male ticks after they had fed for 6 days. The possible function of IGBP in ticks is discussed.

Partial 28S ribosomal RNA (rRNA) gene sequences, including the variable domains D1, D2 and D3, were determined for representative species from the 4 Schistosoma species groups. On an alignment of 1345 bp from S. mansoni, S. haematobium, S. spindale and S. japonicum (with Heterobilharzia americana chosen as an outgroup), both maximum likelihood and maximum parsimony analyses provide a robust molecular phytogeny for the genus; ((((S. haematobium, S. spindale), S. mansoni), S. japonicum), H. americana). When analysed separately, both domain D1 and domain D2 yielded similarly informative data whereas D3 failed to resolve the phylogeny. These results confirm a phylogeny previously suggested by 18S rRNA gene sequences, corroborating the status of S. spindale as a sister taxon to S. haematobium, and demonstrate the utility of 28S rRNA gene sequence data for resolving phylogenies within the Schistosomatidae.

Note: nucleotide sequence data reported in this paper are available in the EMBL database under the accession numbers Z46503/04/05/06/21.

In order to investigate the mode of action of a new class of schistosomicides, the N-alkylaminoalkanethiosulphuric acids, the ‘outer 35S] 2-[(1-methylpropyl)amino]-1-octanethiosulphuric acid was synthesized. Labelling studies of adult Schistosoma mansoni were performed in infected mice and in in vitro incubations. After a single oral dose of the drug to infected mice, 5 metabolites were detected by thin-layer chromatography in organic extracts of male and female adult schistosomes. In vitro studies showed that the same compounds were present in organic extracts obtained from adult male and female worms. One of these metabolites was identified by mass spectroscopy as being the dimeric disulphide derivative of the parent labelled thiosulphuric acid.

Estimates of the time delay between the infective bloodmeal and maturation (incubation or maturation time) for 4 trypanosome stocks (2 Trypanozoon and 2 Trypanosoma congolense) show that maturation time in tsetse is not a parasite species-specific constant. The mean incubation time of a Trypanosoma brucei rhodesiense stock (EATRO 2340 – 18 days) was not significantly different from one T. congolense stock (SIKUDA88 – 15·5 days) but was significantly greater than another (1/148 FLY9 – 12·5 days). There was no significant difference in incubation times between male and female Glossina morsitans morsitans for any of the stocks but in both of the Trypanozoon stocks the proportion of female flies producing mature infections was significantly less than in males. However, estimates of gene frequency, assuming a model in which maturation is controlled by an X-linked recessive allele, gave inconsistent results indicating that maturation cannot be controlled by a single sex-linked gene. Maturation was shown to be a tsetse sex-dependent phenomenon in Trypanozoon but not in T. congolense infections. Incubation time was quite variable even for a single trypanosome stock (e.g. standard deviation of 5 days for one Trypanozoon stock); we discuss how this variability can affect disease transmission, and the interpretation of age-prevalence data.

Monoclonal antibody 2A6 binds to the surface of the Dictyocaulus viviparus L3 sheath and identifies a molecule which is highly antigenic in both infected and vaccinated cattle. Immunogold electron microscopy reveals that the antibody binds to a highly stable surface coat overlying the epicuticle of the L3 sheath. The binding to the cuticular surface is stage specific, being restricted to the surface of the L3 sheath, although the antigen can be detected on Western blots of parasite homogenates from the L1 stage. Immunofluorescence on live parasites demonstrates that the antibodies also bind to the L3 sheath surface of many other strongylid nematodes. The antigen detected by 2A6 differs in molecular weight between the species; in D. viviparus the antigen is poorly resolved between 29 and 40 kDa by SDS-PAGE whereas in nematodes from the family Trichostrongyloidea a single band or doublet of approximately 20 kDa is detected, suggesting that a related molecule is present in these species. Although infection and vaccination with D. viviparus elicits a strong immune response to the L3 sheath antigen this does not appear to be the case following infection with other strongylid nematodes.

Adult Trichinella spiralis were maintained in vitro using defined media and the material excreted/secreted (ES) during this time examined for proteolytic enzyme (proteinase) activity using an azocasein assay and gelatin-substrate gels. Several discrete proteinases in the size range 14–100 kDa were observed with optimal activity at pH 7·5. The use of a class-differentiating panel of proteinase inhibitors indicated that serine proteinases were predominant although some inhibition was evident in the presence of cysteine and metalloproteinase inhibitors. Of a panel of potential natural protein substrates tested, ES proteinases only degraded fibrinogen and plasminogen and degradation was, in part, susceptible to the action of serine, cysteine and aspartyl proteinase inhibitors. In addition, antibody harvested from immune but not normal mice inhibited ES proteinase activity, an observation of relevance to the immunobiology of Trichinosis.

A haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

Naturally infected Biomphalaria glabrata snails were collected at two sites near Belo Horizonte, Brazil, and Schistosoma mansoni cercariae isolated from single snails were used to infect individual mice. Genetic comparison of single worm DNA was accomplished by hybridization of Southern blots to a polymorphic repetitive DNA element. Genetic profiles of parasite individuals revealed a diverse array of parasite genotypes in naturally infected intermediate hosts. The observed distribution of schistosome genotypes among intermediate hosts indicates that over half of the infected snails harbour multiple miracidia. Snails were more likely to carry multiple infections than expected by chance. This degree of overdispersion combined with high levels of genetic variability facilitates multi-genotype transmission and helps maintain parasite genetic diversity.

The status of bluegill (Lepomis macrochirus) and green sunfish (Lepomis cyanellus) as homologous hosts for the acanthocephalan Neoechinorhynchus cylindratus was experimentally determined. It was found that the adult parasite did not establish in bluegills, but that these fish could serve as paratenic host. In contrast, complete growth and development to the adult stage occurred in the green sunfish. When green sunfish were intubated with 10 cystacanths/fish, the parasite exhibited a clear preference for the anterior half of the intestine; when 50 cystacanths/fish were intubated, the parasites showed a preference for the posterior half of the intestine. With repeated exposure of cystacanths, the parasites were distributed throughout the intestine. The extent of histopathology induced by N. cylindratus was related to the numbers of parasites present. In light infections (10 cystacanths), the parasite penetrated deeply into the intestinal wall and connective tissue developed around the proboscis. In infections with 50 cystacanths, the proboscis penetration was shallow and little if any connective tissue accumulated. There was also an indication that in crowded areas, the parasites appeared to change their sites of attachment frequently. In both heavy and repeated infections, the parasites evoked significant goblet cell hyperplasia and substantial quantities of mucus covered the intestinal wall. It is suggested that the sticky covering and the presumed presence of antibodies in the mucus combined to create a protective barrier thereby reducing the numbers of parasites that could attach and become established.

The prediction that richness and diversity of helminth communities would be greater in tropical fish was tested by analysing total and intestinal helminth communities in samples of Anguilla reinhardtii taken from 10 localities in tropical and semi-tropical freshwaters in Queensland, Australia. Comparison of findings with those from A. anguilla in the UK revealed that regional species richness was higher in Queensland and that helminth communities harboured a suite of common species, virtually all eel specialists, that were responsible for the observed higher levels of similarity between component communities. All measures of community richness and diversity adopted indicated that the poorest helminth communities in A. reinhardtii were comparable with the richest ones reported from the northern temperate A. anguilla. The richest communities in A. reinhardtii were more diverse than those reported from any other species of fish, whether marine or freshwater, to date, and were comparable with the communities found in some species of aquatic birds. Both tropical conditions and age and endemism of the host, i.e. the time hypothesis, were considered as explanations for this richness but neither could be favoured. A cautious approach to generalizations based on data from temperate regions only is advocated, and the need for more studies on tropical parasite communities is strongly emphasized.