The value of 2 PCR methods, targeting genomic and kinetoplast minicircle DNA respectively, was investigated for both diagnosis and prevalence studies of canine visceral leishmaniasis (CVL). The first method (R) was 5000-fold less sensitive than the second (method KRV). Both were tested for diagnosis of CVL in 44 sick dogs with confirmed disease using different biological samples. Method R was highly efficient when using invasive samples, but the use of method KRV proved necessary for a 100% sensitive diagnosis using peripheral blood. This method was applied to peripheral blood and skin samples in 263 dogs during a mass survey in the Cévennes focus. PCR was compared to serology and all results were analysed according to clinical status. The ‘CVL-infection’ prevalence was found to be 79.8% by PCR compared with 29.6% by serology: 89.4% of symptomatic and 65.2% of asymptomatic dogs harboured parasites in peripheral blood. This study confirms the high prevalence of asymptomatic carriers of Leishmania. In total, for the diagnosis of CVL in sick dogs, method R is recommended in view of its 100% positive predictive value (compared with 30% for method KRV). A strategy best adapted for prevalence surveys might combine serology and highly sensitive PCR on peripheral blood.

The mode of appearance and assembly of cyst wall filaments on the surface of Giardia duodenalis trophozoites committed to encyst was analysed by scanning and transmission electron microscopy (SEM and TEM) and by fluorescence microscopy (FM). SEM showed a progressive appearance of fibril patches, predominantly on the anterior area of ventral and dorsal surfaces, which then spread and coalesced. By TEM, ruthenium red (RR) displayed staining in encysting cells as rodlike spots of variable diameter (3–25 nm), possibly microfibril tips with polyanionic moieties, that displayed tangential associations and random orientations over the cell membrane. In FM assays, the 1,10-phenanthroline derivative of ruthenium red (RR/oPHE) was a specific ligand for these assembling fibrils and this staining was significantly blocked by N-acetylgalactosamine (GalNac) and galactosamine (GalN). Interestingly, RR staining was lost when the cyst wall was completely assembled and thickened as observed by TEM and FM. Kinetic FM assays, in which a mAb specific for a 26 kDa Giardia cyst wall polypeptide was used concomitantly with RR/oPHE staining, showed a differential pattern for the appearance and reactivity of polypeptide and assembling GalN/GalNac-rich moieties of Giardia cyst wall.

During autumn 2000 an extensive cross-sectional survey of the prevalence of Schistosomiasis japonicum was conducted among about 4000 villagers within 20 villages in the Anning River Valley located in the southwestern Sichuan Province. Two procedures were used to assess infection status, the Kato-Katz thick smear procedure and a miracidia hatch test. Whereas the Kato-Katz procedure provides information on both prevalence and intensity, the hatch test provides only prevalence data, albeit on a much larger volume of stool. In addition, we performed Kato-Katz smears for 15 consecutive samples on a subset of 15 individuals. The proportion of both hatch-test and Kato-Katz positive individuals in the larger cross-sectional survey was 25%. The goal of the study was to estimate both the egg and worm distributions among risk groups using both the hatch and Kato-Katz tests from the cross-sectional data and the repeated Kato-Katz smears from the longitudinal data sets. As a prelude to parameter estimation, individuals were classified into risk groups by natural village and occupation; the proportion of Kato-Katz positive subjects among the risk groups varied from 10% to 60%. We used the statistical model of de Vlas et al. (1992) and Bayesian techniques to derive both estimates of and inference about the worm and egg distribution parameters. The parameter estimates imply (1) similar eggs per gram stool (e.p.g.) per worm pair compared with earlier estimates, (2) a range of worm burdens among the risk groups and (3) estimates of risk heterogeneity within groups is sensitive to prior information on the within-person variability in egg excretion.

Previous studies of mating interactions between schistosome species from different evolutionary lineages in mice showed that mating is not random: heterospecific pairs form but, given the choice, the preference is for homospecific partners. Mating competition and change of mate were also demonstrated. Schistosoma mansoni is a medically important parasite of humans belonging to a different evolutionary group from S. margrebowiei, a parasite of bovines of veterinary importance. Experiments were designed to investigate the mating behaviour of these two species in mice. Analysis of the data showed a preponderance of intraspecific pairs over interspecific, demonstrating a specific mate preference system for both species. The existence of mating competition between these species was indicated. Males of both species were able to actively compete for paired females by pulling them away from their partners, thus effecting a change of mate. Overall, neither species appeared to be competitively dominant to the other, and the data suggest that where this is the case in sequential infections, the most successful species in terms of worm return and ability to form pairs will be the first species to infect the host.

An ELISA was developed for serological detection of Echinococcus granulosus infection in dromedary camels. Antigen B (AgB) partially purified from hydatid cyst fluid of camels or sheep naturally infected with cystic echinococcosis (CE) due to E. granulosus, as well as a recombinant antigen B product (r-AgB) were used in an ELISA to screen panels of serum samples from slaughtered camels naturally infected with CE. Native hydatid cyst fluid antigen preparations were able to detect antibodies in sera from a significant proportion of camels with CE, as confirmed at post-mortem. Seroreactivity however, was variable. ELISA specificity for sera from naturally infected camels versus inspection-negative animals ranged from 90 to 99%. Native antigen B gave the highest sensitivity (97%) in ELISA for camel CE confirmed at slaughter. In contrast, r-AgB gave lower sensitivity for camel (84%) and sheep (28%) CE. The r-AgB-ELISA was, however, highly specific (90 and 95%) respectively for both camel and sheep natural CE infection. These results indicate that an ELISA based on serum antibody detection to AgB could be developed for immunodiagnosis of cystic echinococcosis in camels.

Avian trypanosomes are widespread parasites of birds, the transmission of which remains mostly unclear, with various blood-sucking insects mentioned as possible vectors. A search for vectors of trypanosomes of sparrowhawk (Accipiter nisus), buzzard (Buteo buteo), lesser-spotted eagle (Aquila pomarina) and kestrel (Falco tinnunculus) was performed in Czech and Slovak Republics. Black flies (Eusimulium spp.), hippoboscid flies (Ornithomyia avicularia), mosquitoes (Culex pipiens pipiens) and biting midges (Culicoides spp.), trapped while attempting to feed on raptor nestlings, were found to contain trypanosomatids in their intestine. Trypanosomes from the raptors and blood-sucking insects were isolated, and their 18S rRNA sequences were used for species identification and for the inference of intra- and interspecific relationships. Together with the trypanosome isolated from a black fly, the bird trypanosomes formed a well-supported Trypanosoma avium cladeThe GenBank accession numbers of the determined sequences are: AF416559 (Trypanosoma ‘avium’ APO1), AF416560 (Herpetomonas sp. CER1), AF416561 (Trypanosoma sp. CUL1), AF416562 (Trypanosoma sp. OA6), AF416563 (Trypanosoma ‘avium’ SIM3).. The isolates derived from hippoboscid flies and mosquitoes are most likely also avian trypanosomes infecting birds other than the studied raptors. Analysis of the kinetoplast, that has features characteristic for the avian trypanosomes (minicircle size; dimensions of the kinetoplast disc), provided further evidence for the identification of vectors. It is suggested that all trypanosomes isolated from raptors included in this study belong to the T. avium complex and are transmitted by the ornithophilic simuliids such as Eusimulium securiforme.

The tick Boophilus microplus is a 1-host tick that causes important losses to bovine herds, and protective antigens are being investigated in order to develop vaccines that avoid the use of acaricides. Paramyosins are multi-functional invertebrate muscle proteins, whose roles may include host immunomodulation, and seem to be a prominent candidate in a schistosomiasis vaccine. We report here the cloning, expression and characterization of a B. microplus paramyosin (BmPRM). Sequence analysis of the full length coding sequence cDNA shows high identity to other arthropod paramyosin sequences, and the predicted molecular weight, pI and secondary structure are consistent with a typical paramyosin. Western-blot expression analysis indicates the presence of BmPRM in all tissues and developmental stages tested, but not in saliva. The recombinant protein (rBmPRM) was shown to bind both IgG and collagen. Possible implications of these activities with host evasion mechanisms are discussed.

Hyperplasia of Paneth and intermediate cells is a recently described component of the response of the small intestine of mice to infection with the nematode Trichinella spiralis. To investigate whether this hyperplasia is parasite specific or represents a generic intestinal response to infection, mice were infected with T. spiralis, Nippostrongylus brasiliensis, Heligmosomoides polygyrus or Schistosoma mansoni and tissue samples taken at various time-points post-infection to determine Paneth and intermediate cell numbers. All infections induced Paneth and intermediate cell hyperplasia, but the patterns of response varied between the parasite species concerned, reflecting differences in their relationships with the host. Increases in the numbers of these cells appeared to correlate with known patterns of T-helper-2 immune responses.

Nematode infections are subject to density-dependent effects on their establishment, survivorship and fecundity within a host. These effects act to regulate and stabilize the size of nematode populations. Understanding how these density-dependent effects occur is important to guide the development of control strategies against parasitic nematodes and the diseases that they cause. These density-dependent effects have been hypothesized to result from intraspecific competition between parasites for limited resources or from the action of host immune responses. However, no specific evidence exists to distinguish between these two hypotheses. We find that in nematode (Strongyloides ratti) infections, density-dependent effects on parasite establishment, survivorship and fecundity are mediated by the host immune response. These density-dependent effects are only observed late in primary infections and no density-dependent effects are observed in infections in immuno-compromised animals. We find no evidence for intraspecific competition between parasites in experimental infections over a range of doses that encompasses all that is observed in natural infections. We conclude that density-dependent effects due to the immune response will act to regulate S. ratti infections before competition for space or nutrients within the host gut ever occurs.

The effect of cadmium and zinc at concentrations ranging from 0.1 to 10000 μg/l on tail loss in cercariae of Diplostomum spathaceum was investigated at 3 temperatures (12, 20 and 25 °C) and 3 levels of water hardness (distilled water, soft water and hard water). Increasing tail loss over time was found to be linked with a parallel decrease in cercarial survival in controls. Exposure to the heavy metals induced, especially at high concentrations, a change in the relationship between cercarial tail loss and survival, causing either stimulation or inhibition of tail loss dependent on the individual toxic exposure. Under most environmental conditions the rate of tail loss over time was reduced by increasing metal concentrations. Inhibition of tail loss occurred in a limited number of both control and metal-exposed cercariae, with a number of low metal concentrations inducing greater inhibition than in controls. Stimulation of tail loss causing an increased tail loss rate above controls also occurred at certain high metal concentrations. Increasing water hardness and decreasing water temperature caused a reduced tail loss rate over time in both control and metal-exposed cercariae. However, with decreasing temperature a reduced rate of tail loss over time in metal-exposed cercariae compared to controls occurred at some low metal concentrations. When tail loss was compared against cercarial death of the experimental population toxic exposure induced changes in the parallel relationship of these parameters, dependent on individual metal concentration, water temperature and hardness. Differences in the relative effects of cadmium and zinc on cercariae were dependent on the environmental conditions of exposure. Both metals showed limited effects during the period of maximum cercarial infectivity (0–5 h). The mechanisms and importance of metal toxicity to cercarial tail loss are discussed.