A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 μl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at −20 °C.

Invasive amoebiasis is the result of infection of Entamoeba histolytica. The closely related Entamoeba dispar can colonize the human gut but does not cause invasive disease. In this study, E. dispar was analysed for the presence of the lipophosphoglycan-like (LPG) glycoconjugate known to be present on the cell surface of E. histolytica. E. dispar cells were radio-isotope labelled with [3H]galactose or [3H]inositol. The acidic glycoconjugates were extracted and analysed by hydrophobic chromatography over phenyl–Sepharose and by sodium dodecyl sulphate polyacrylamide gel electrophoresis. No LPG-like molecules could be identified in E. dispar in contrast to E. histolytica, suggesting that these molecules may be absent in the non-pathogenic species.

The first long-term (27 month) survey of single species of Eimeria occurring in a population of Apodemus sylvaticus in the UK showed that Eimeria apionodes, E. hungaryensis and E. uptoni occurred throughout the period whilst E. sp. E. was only found during 4 months. In first-captured animals, overall prevalence of Eimeria spp. was 73%: a figure higher than previously published, but possibly an underestimate. Multiple infections (2 or 3 species) occurred in 34% of first-capture infected animals, but without significant associations. There were no significant differences of prevalence rates between the sexes, nor between adults and juveniles, except for E. uptoni which was more common in juveniles than in adults. E. hungaryensis and E. uptoni probably occurred as near-continuous infections whilst E. apionodes occurred more intermittently but with no long-term total immunity. Monthly prevalence data indicated ongoing trends over the 27 months. Prevalences of E. apionodes indicated an annual cycle, lowest in January–May and with a peak in October–November, correlating positively with both the number of animals and the percentage of juveniles in the host population and also with the environmental relative humidity. There was no consistent pattern for E. uptoni and an annual cycle for E. hungaryensis was not apparent.

The immune response of European sea bass after intracaelomic immunization with Sphaerospora dicentrarchi was studied. Fish were injected with S. dicentrarchi spores (DIC), with spores plus adjuvant (DIC+FCA), with adjuvant alone (FCA) or with PBS. Several parameters of the immune response were measured. Serum lysozyme increased significantly in DIC fish 1 week after immunization (p.i.) and it remained significantly higher in DIC+FCA fish 4 weeks p.i., and in DIC fish 8 weeks p.i. than in PBS-injected fish. The number of nitroblue tetrazolium-positive blood cells was significantly higher in DIC+FCA fish 1, 4 and 8 weeks p.i, but the highest values were detected 1 week p.i. The highest stimulation index was detected in phagocytes from DIC+FCA fish. The number of S. dicentrarchi antibody-secreting cells was significantly higher in DIC+FCA fish than in DIC fish. Serum from DIC and DIC+FCA fish, stained the polar capsules and the valves of S. dicentrarchi spores in immunohistochemistry. Serum antibodies could not be detected using immunoblot assay. All these results show that immunization with S. dicentrarchi resulted in the activation of the non-specific immune response, mainly 7 days p.i. A specific humoral response against the parasite was also demonstrated but it had a low magnitude.

The present review discusses the immune responses to gastrointestinal nematodes in cattle and the different immunological and parasitological parameters used to assess acquired immunity. Measuring acquired immunity to gastrointestinal nematodes in cattle (e.g. for the evaluation of candidate parasite vaccines) is hampered by the limited understanding of bovine immune responses against gastrointestinal parasites. In this paper the available data on protective immunity against gastrointestinal nematodes, and especially Ostertagia ostertagi, in cattle are compared with the current knowledge of protective immune responses against gastrointestinal nematodes in rodent models and small ruminants. In contrast to the immune response in mice, which is controlled by T helper 2 (Th2) lymphocytes and results in mast cell- or goblet cell- mediated expulsion of adult worms, bovine immune responses to O. ostertagi do not show a clear Th2 cytokine profile, nor do they result in rapid expulsion of the parasite. The first manifestation of immunity to O. ostertagi in calves is a reduction of worm fecundity, possibly regulated by the local IgA response. Worm numbers are only reduced after a prolonged period of host–parasite contact, and there are indications that O. ostertagi actively suppresses the host's immune response. Until the mechanisms of protective immunity against O. ostertagi are revealed, the use of immunological parameters to estimate acquired immunity in cattle is based on their correlation with parasitological parameters and on extrapolation from rodent and small ruminant models. Assessing the resistance of calves against a challenge infection by means of parasitological parameters is probably still the most accurate way to measure acquired immunity against gastrointestinal nematodes.

In total 445 faecal samples were collected from 259 European badgers (Meles meles) in Wytham Woods, Oxfordshire, UK (462080). Microscopical examination revealed infection with 2 species of coccidia Eimeria melis and Isospora melis. From the initial examination of each animal, point prevalence rates of 0·44 and 0·35 were calculated for Eimeria and Isospora respectively. The intensity of infection was significantly greater for Eimeria than Isopora and the distribution of intensities was highly skewed for both species, with a few individuals shedding the majority of oocysts. Incidence and recovery rates for both coccidia species were calculated from longitudinal data collected at 3-monthly intervals from a subset of the adult badger population, and the predicted prevalence rates based on these were similar to the point prevalence rates. This suggests little, if any, parasite-induced mortality in the adult population. In contrast, there was a marked and significant reduction in the point prevalence and intensity of infection with Eimeria from cub to adult badger suggesting a degree of acquired immunity to Eimeria melis on initial exposure and/or that there is significant Eimeria-associated mortality in the cub population. No such relationship was found for Isospora infection. In those adult badgers with co-infections there was a direct relationship between the intensity of Eimeria and Isospora. The taxonomic status of these parasites suggests a heteroxenous life-cycle for I. melis, and direct transmission of E. melis. However, the greater than expected prevalence of co-infection is consistent with a common source of infection, such as communal latrines.

Schistosomiasis is a health problem in Brazil and the role of rodents in maintaining the schistosome life-cycle requires further clarification. The influence of Schistosoma mansoni on a population of Nectomys squamipes was studied by capture-recapture (1st phase, from June 1991 to November 1995) and removal (2nd phase, from April 1997 to March 1999) studies at Sumidouro, Rio de Janeiro, Brazil. During both phases coproscopic examinations were performed. At the 2nd phase the rodents were perfused and worms were counted. The population dynamics of parasites was studied. During the 1st phase, female reproductive parameters, longevity, recruitment and survivorship rates and migration patterns were studied in relation to schistosome prevalence. Water contamination (source of miracidia), abundance intermediate host and rodent migration were related to prevalence. The N. squamipes population was not obviously influenced by the infection, as shown by the high number of reproductive infected females, high longevity of infected individuals and the absence of a relationship between recruitment or survivorship rates and the intensity of schistosome infection. The data indicate that N. squamipes can increase transmission of S. mansoni in endemic areas and carry it to non-infected areas. Furthermore, this rodent can be used as an indicator of a transmission focus.

Information on the spatial pattern of African animal trypanosomosis forms a prerequisite for rational disease management, but few data exist for any country in the continent. The present study describes a raster or grid-based Geographic Information System for Togo, a country representative of subhumid West Africa, with data layers on tsetse, trypanosomosis, animal production, agriculture and land use. The paper shows how trypanosomosis prevalence and packed cell volume (PCV) map displays may be predicted from correlations between representative field data and environmental and satellite data acquired from the National Oceanographic and Atmospheric Administration (NOAA) and Meteosat platforms. Discriminant analytical methods were used to assess the relationship between the amount of field data used and the accuracy of the predictions obtained. The accuracy of satellite derived predictions decreases from tsetse abundance to trypanosomosis prevalence to PCV value. The predictions improve when eco-climatic and epidemiological predictors are combined. In Togo, and probably elsewhere, the patterns of trypanosomosis prevalence and PCV are much influenced by animal husbandry and other anthropogenic factors. Additional predictor variables, incorporating these influences might therefore further improve the models.

Behavioural, parasitological and immunological data were obtained from 48 children up to 6 years old, resident in a Schistosoma haematobium endemic area in Zimbabwe. The children averaged more than 1 contact with infective water bodies every 3 days and all showed immunological evidence of exposure (an anti-cercarial and/or anti-egg antibody response). IgM was the dominant isotype and appeared in the youngest children, followed by IgA, IgE and IgG3. However, only 38 children showed evidence of infection (an anti-egg response or eggs in urine) and only 14 were excreting eggs. The best estimates from these data are that less than 1 in 100 contacts result in infection and less than 1 in 1000 result in egg output. This suggests that there may be substantial attrition of invading cercaria even in naïve individuals.

Vaccination against complex metazoan parasites has become a reality with the development and registration of recombinant protein-based vaccines against the cattle tick Boophilus microplus and the sheep cestode Taenia ovis. Progress towards the development of similar vaccines against gastrointestinal nematodes, primarily of ruminants, is outlined within a framework of defining the practical requirements for successful vaccination, antigen selection, recombinant protein production and antigen delivery, be it mucosal delivery or DNA vaccination. Antigen selection strategies include the fractionation of complex, but protective, parasite extracts, the use of antibody probes, evaluation of excretory-secretory components and gut-expressed hidden antigens as well as antigens targeted on the basis of function such as enzyme activity. The difficulties being encountered in recombinant protein production and their solution are discussed as are the requirements for successful antigen delivery. Recent technological developments such as the use of functional genomics to identify new vaccine candidates and DNA vaccination to present the selected antigen to the host immune system are discussed and are anticipated to have a profound effect on vaccine development in the future.

This study applies flexible statistical methods to morphometric measurements obtained via light and scanning electron microscopy (SEM) to discriminate closely related species of Gyrodactylus parasitic on salmonids. For the first analysis, morphometric measurements taken from the opisthaptoral hooks and bars of 5 species of gyrodactylid were derived from images obtained by SEM and used to assess the prediction performance of 4 statistical methods (nearest neighbours; feed-forward neural network; projection pursuit regression and linear discriminant analysis). The performance of 2 methods, nearest neighbours and a feed-forward neural network provided perfect discrimination of G. salaris from 4 other species of Gyrodactylus when using measurements taken from only a single structure, the marginal hook. Data derived from images using light microscopy taken from the full complement of opisthaptoral hooks and bars were also tested and nearest neighbours and linear discriminant analysis gave perfect discrimination of G. salaris from G. derjavini Mikailov, 1975 and G. truttae Gläser, 1974. The nearest neighbours method had the least misclassifications and was therefore assessed further for the analysis of individual hooks. Five morphometric parameters from the marginal hook subset (total length, shaft length, sickle length, sickle proximal width and sickle distal width) gave near perfect discrimination of G. salaris. For perfect discrimination therefore, larger numbers of parameters are required at the light level than at the SEM level.

18S rDNA sequences from 4 isolates of Babesia gibsoni originating from Japan, Malaysia and Sri Lanka were compared with a previously published, 0·5 kb portion of the 18S rDNA from a B. gibsoni isolate from California, USA, and with the corresponding 18S rDNA sequences of other Babesia spp. Distance, parsimony and maximum likelihood analyses showed almost identical genotypes among the small canine Babesia from Asia, but an unexpectedly distant genetic relationship to that from the USA. While the American isolate segregated together with B. equi, the Asian isolates showed a close relationship to B. divergens and B. odocoilei. These results indicate that small Babesia of dogs originating from North America and Asia belong to different, genetically distantly related species.

The snail host signals releasing host-finding responses in miracidia and cercariae of Echinostoma caproni were analysed by fractionation of snail-conditioned water (SCW). Cercariae responded non-specifically to organic and hydrophilic, low molecular weight components of SCW showing their typical turning response. Hydrolysis of peptides in SCW had no effect on cercarial responses. An artificial mixture of amino acids in concentrations determined from SCW as well as glycine alone in a concentration corresponding to the total concentration of amino acids in SCW showed nearly the same efficacy as SCW itself. Miracidia responded to a high molecular weight glycoprotein fraction, which could be isolated from SCW by ion-exchange and size-exclusion chromatography. In contrast to an Egyptian Schistosoma mansoni strain, the echinostome miracidia were not able to differentiate between different snail species. The results show for the first time that miracidia and cercariae of the same species may use different signals to identify the same snail host species. This indicates an independent evolution of host-finding mechanisms in the two parasite stages.

Over a period of 26 months from January 1996 to February 1998, 388 foxes from the city of Zürich, Switzerland, were examined for intestinal infections with Echinococcus multilocularis and other helminths. The prevalence of E. multilocularis in foxes sampled during winter increased significantly from 47% in the urban to 67% in the adjacent recreational area, whereas prevalence rates of other helminths were similar in both areas. Seasonal differences in the prevalence of E. multilocularis were only found in urban subadult male foxes which were significantly less frequently infected in summer than in winter. The distribution of the Echinococcus biomass, as expressed by worm numbers per fox was overdispersed in 133 infected foxes randomly sampled in winter. Ten of these foxes (8%) were infected with more than 10000 specimens and carried 72% of the total biomass of E. multilocularis (398653 worms). Prevalences did not differ significantly in these foxes in regard to age and sex but worm burdens were significantly higher in subadult foxes as compared with adult foxes. In voles (Arvicola terrestris) trapped in a city park of Zürich, E. multilocularis metacestodes were identified by morphological examination and by PCR. The prevalence was 20% among 60 rodents in 1997 and 9% among 75 rodents in 1998. Protoscoleces occurred in 2 of the cases from 1997. The possible risk for human infection is discussed with respect to the established urban E. multilocularis cycle.

Trypanosomes in the dissection-positive proboscis of Glossina pallidipes were identified by PCR using species-specific primers. Of the 3741 flies dissected 643 were proboscis positive. PCR was performed on 406 dissection-positive probosces giving positive identifications in 352 (86·7%) and infection rates of 14·8% for congolense-type infections, 2·8% for vivax- type infections and 1·4% for the unidentified group. Of the 352 PCR identified infections 225 were single, 111 were double, 13 were triple infections and there were 3 quadruple infections. Statistical analysis suggests that mixed infections group into 3 largely separate divisions among the tsetse population (i) Trypanosoma congolense savannah and T. congolense Kenya coast, (ii) T. simiae, T. congolense Tsavo and T. godfreyi and (iii) T. vivax. We conclude that either differing feeding patterns among members of the fly population or the ability of the trypanosomes in each of the infection categories to significantly influence the maturation of trypanosomes in the other categories are the most likely causes of the groupings noted. Chi-squared analysis of dissection and PCR methods of trypanosome identification revealed profound differences (χ = 19·1; D.F. = 1; P > 0·05). If confirmed in other studies these findings have serious implications for our understanding of trypanosome epidemiology in tsetse flies, much of which is founded on data from dissection-based trypanosome identifications.

The compatibility between sympatric and allopatric combinations of Onchocerca volvulus–anthropophilic species of Simulium was studied in the north-eastern focus of human onchocerciasis as well as in a densely populated locality of the Amazonas State in Venezuela. The objectives were to test the conjecture that local adaptation exists between the parasite and its vectors (the Onchocerca–Simulium complex hypothesis), and assess the possibility of the infection spreading from its present distributional range. For the homologous combination, O. volvulus–S. metallicum cytospecies E in Anzoátegui State (north-eastern focus), parasite yield was 45% in contrast to 1% for the heterologous, southern parasite–S. metallicum infection. This was significantly lower than the parasite yield (4–10%) expected after allowing for the effect of density-dependent limitation of infective larval output described in this paper for S. metallicum. The population of S. exiguum s.l. from southern Venezuela allowed no larval development beyond the L1 stage of either northern or southern parasites. Mechanisms for such refractoriness probably operate at the level of the thoracic muscles, not affecting microfilarial uptake or migration out of the bloodmeal. The parasite yield of southern O. volvulus in S. oyapockense s.l. flies biting man at Puerto Ayacucho (Amazonas) was about 1%, in agreement with the figures recorded for highly compatible sympatric combinations such as O. volvulus–S. ochraceum s.l. in Guatemala. No infective larval development of the northern parasite was observed in southern S. oyapockense. These results, together with considerations of typical worm burdens in the human host, presence/absence of armed cibaria in the simuliids, parasite-induced vector mortality, and fly biting rates, suggest a lower potential for onchocerciasis to spread between the northern and southern endemic areas of Venezuela than that between Amazonian hyperendemic locations and settlements outside this focus with high densities of S. oyapockense s.l.

Schistosomes, which live as parasitic adults in the blood vessels of mammals, birds and crocodiles, are unique among trematodes because they have separate sexes. Several studies have shown that the sex ratio is often biased towards males. Sexual dimorphism can be very important in several genera (Schistosoma, Heterobilharzia, Schistosomatium), as larger males grip smaller females in a gynecophoric canal. But not all schistomatid species have developed a gynecophoric canal. It is also striking that the number of testes varies widely, from a small number of testes reported (2–7) to a very high number (>500). We tested the hypothesis of trade-off investment by males: muscle (as estimated by the length of the gynecophoric canal) in order to sequester one or a few females, or testes in order to inseminate numerous females. We used recent comparative methods, which need phylogenetic information of the analysed species. We employed a morphological cladistic analysis of the Schistosomatidae and a construction of a phylogenetic supertree of Schistosoma based on available molecular information. Our phylogenetic analysis supports previous hypotheses on the origin and diversification of the schistosomes. The family seems to have originated as parasites of crocodiles (or possibly other related poikilotherms) and to have secondarily evolved within the Aves and then mammals. Our phylogenetic analyses show that (1) the possession of a gynecophoric canal is a plesiomorphic character, and several reductions of this character have occurred during the diversification of the Schistosomatidae; (2) a small number of testes is a plesiomorphic stage, and an increase of testes number has occurred several times. Finally, our comparative analysis suggests that there is a trade-off between investment in muscle or in number of testes (other variables being controlled for). Male competition for access to females seems to follow two ways: investment in testes in order to fertilize a high number of females or investment in muscle in order to hold and sequester one (Schistosoma) or a few females (Heterobilharzia). We show the existence of the trade-off between muscles and testes in connection with a male biased sex ratio. The development of dioecy cannot explain the trade-off between muscles and testes, which instead appears to be a result of sexual selection possibly driven by the male biased sex ratio.

The 18S nuclear small subunit ribosomal RNA gene of piroplasms from wildlife and human cases of babesiosis in the western USA were isolated by PCR and sequenced. Phylogenetic analyses of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that piroplasm isolates from the human cases were indistinguishable from some of the isolates from the western wildlife species, most notably the isolates from mule deer (Odocoileus hemionus). These results suggest that large ungulates may serve as reservoirs for human piroplasm infection. The western piroplasm isolates from humans and wildlife formed a distinct clade, separate from other piroplasms found worldwide.

This study was performed with Litomosoides sigmodontis, the only filarial species which can develop from the infective larvae to the patent phase in immunocompetent laboratory BALB/c mice. Parasitological features and immune responses were analysed up to 3 months before and after challenge inoculation, by comparing 4 groups of mice: vaccinated challenged, challenged only, vaccinated only, and naive mice. Male larvae were very susceptible to irradiation and only female irradiated larvae survived in vivo. Protection, assessed by a lower recovery rate, was confirmed and was established within the first 2 days of challenge. This early reduction of the recovery rate in vaccinated challenged mice was determined by their immune status prior to the challenge inoculation. This was characterized by high specific IgM and IgG subclass (IgG1, IgG2a and IgG3) levels, high specific IL-5 secretion from spleen cells in vitro and a high density of eosinophils in the subcutaneous connective tissue. Six h after the challenge inoculation, most tissue eosinophils were degranulated in vaccinated challenged mice. Thus, in the protocol of vaccination described, protection appeared mainly to result from the stimulation of a Th2 type response and eosinophils seemed to be the main effectors for the increased killing of infective larvae in vaccinated challenged mice. Two months after challenge inoculation, the percentage of microfilaraemic mice was lower in vaccinated challenged mice as a consequence of this overall reduction in the worm load. In both vaccinated challenged and challenged only groups, the in vitro splenocyte proliferative capacity was reduced in microfilaraemic mice.

Infections with Haemonchus contortus are a major constraint on ruminant health world-wide. Young lambs are very sensitive to Haemonchus infection. Older lambs and sheep acquire immunity after a continuous or seasonal exposure to the parasite. The mechanisms underlying immunity are still not completely understood. Antibodies, in particular local IgA and IgE, certainly play a role. The role of IgG is less clear. Lymphocyte proliferation responses seem to correlate to immunity. Sheep that have high antigen-induced lymphocyte responses have a low susceptibility to infection. Furthermore, several studies have demonstrated that immunity against H. contortus is associated with mastocytosis and hypersensitivity reactions. More recently, increasing attention is being paid to the role of cytokines (interleukins and γ- interferon) in the activation of specific defence mechanisms. Reverse transcriptase–polymerase chain reaction (RT–PCR) assays to study cytokine mRNA expression have become available. The inability of young lambs to mount a significant Th2 response, which is normally characterized by high IgE levels, mastocytosis and eosinophilia, may account for the phenomenon of unresponsiveness in these animals.