An automated flow cytometric (FCM) detection method has been developed and validated with a simple diagnostic procedure in parasitized erythrocytes of Plasmodium berghei-infected rats using the nucleic acid-binding fluorescent dye YOYO-1. High levels of reticulocytes were detected during the course of the infection, ranging from 1·2–51·2%, but any RNA potentially confounding the assay could be removed by digestion with RNAse. The cell counts of uninfected, infected, and nucleated cells occurred with high precision. The cells were divided into different populations according to their physical or chemical properties but various factors within the assay such as cell fixation, RNA digestion, and compensation required optimization. In this study, FCM greatly simplified and accelerated parasite detection, with a mean precision of 4·4%, specificity of 98·9% and accuracy of 101·3%. The detection and quantitation limits in the assay were 0·024% and 0·074% parasitaemia, respectively. Overall, the parasite counts by FCM measurement correlated highly (r2=0·954–0·988) with the parasitaemia measured by light microscopical analysis when animals treated with suppressive, clearance, and curative doses of novel antimalarial drugs were examined. The lower levels of parasitaemia (30%) detected by microscopy compared to FCM may be related to a number of schizonts externally attached to the erythrocyte membranes that normally would not be included in microscopy counting. Lower sampling error and reliable identification of rodent erythrocyte parasites based on the principles of FCM have replaced the traditional blood smear in our laboratory.

The ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits and tiger puffer (Takifugu ruburipes). Serum from rabbits and fish immunized with theronts had agglutination/immobilization activity against theronts in vitro; fish serum antibody levels (measured by enzyme-linked immunosorbent assays: ELISA) correlated with this activity. Anti-theront antibody levels in fish were significantly higher in the immunized group as compared with control fish at 2 weeks after booster immunization (injection of bovine serum albumin; Student's t-test, P<0·01). Biochemical analyses indicated that a Triton X-114-soluble 32 kDa theront integral membrane protein may be the agglutination/immobilization antigen. Indirect immunofluorescence staining of theronts suggested that this 32 kDa antigen was expressed on the surface of cilia. The full-length 32 kDa antigen cDNA contained 1147 basepairs, encoding a 328-amino acid protein including hydrophobic N- and C-termini. As with Tetrahymena and Paramecium spp., TAA and TAG appear to be used as glutamine codons in the 32 kDa antigen gene.

Leishmania produce several types of mucin-like glycoproteins called proteophosphoglycans (PPGs) some of which are secreted while others are found on the surface of promastigotes and amastigotes. These proteins are thought to be important in the transmission, invasion and subsequent intracellular survival of parasites. The structure and function of PPGs are species and stage-specific in the case of L. major and L. mexicana, but no such information has hitherto been available for L. donovani. This study presents, for the first time, an initial characterization (localization) of PPG in sodium stibogluconate (SSG)-resistant and sensitive clinical isolates of L. donovani from Bihar (India) by confocal microscopy, flow cytometry and Western blotting using antibodies to L. major PPG. Confocal microscopy analysis revealed that both promastigotes and amastigotes possess PPG on their cell membrane and flagellar pocket membrane but its expression was variable in different isolates. The quantitative analysis by FACS and Western blotting showed that the expression and intensity of PPG bands was higher in SSG-resistant isolates. This study suggests the possibilities of involvement of PPG in drug-resistant mechanisms and of using PPG abundance as a marker for identifying drug-resistant clinical isolates in Indian kala azar.

We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.

The 70 kDa heat-shock protein (Hsp70) sequences are considered one of the most conserved proteins in all domains of life from Archaea to eukaryotes. Hammondia heydorni, H. hammondi, Toxoplasma gondii, Neospora hughesi and N. caninum (Hammondia-like organisms) are closely related tissue cyst-forming coccidians that belong to the subfamily Toxoplasmatinae. The phylogenetic reconstruction using cytoplasmic Hsp70 coding genes of Hammondia-like organisms revealed the genetic sequences of T. gondii, Neospora spp. and H. heydorni to possess similar levels of evolutionary distance. In addition, at least 2 distinct genetic groups could be recognized among the H. heydorni isolates. Such results are in agreement with those obtained with internal transcribed spacer-1 rDNA (ITS-1) sequences. In order to compare the nucleotide diversity among different taxonomic levels within Apicomplexa, Hsp70 coding sequences of the following apicomplexan organisms were included in this study: Cryptosporidium, Theileria, Babesia, Plasmodium and Cyclospora. Such analysis revealed the Hammondia-like organism to be the lowest divergent group when compared to other groups within the phylum Apicomplexa. In conclusion, the Hsp70 coding sequences proved to be a valuable genetic marker for phylogenetic reconstruction and may constitute a good candidate to be used with other genes for species phylogeny within this group of organisms.

The ‘Muguga cocktail’ live vaccine, delivered by an infection and treatment protocol, has been widely deployed in Eastern, Central and Southern Africa to protect cattle against East Coast fever, caused by Theileria parva. The vaccine contains 3 component stocks (Muguga, Serengeti-transformed and Kiambu 5). In a previous study, parasites from vaccinated and unvaccinated animals were genotyped with a panel of micro- and minisatellite markers (Oura et al.2004a) and it was shown that only the Kiambu 5 stock establishes a long-term carrier state but there was no evidence for the transmission of this stock. Also parasite genotypes different from the 3 component vaccine stocks were identified in vaccinated animals. We now report a follow-up study on the same farm, some 4 years after the initial vaccination, aimed at establishing the source of the novel parasite genotypes identified in vaccinated cattle, determining the longevity of the carrier state established by the Kiambu 5 vaccine stock and re-examining whether vaccine transmission can occur over a longer time-scale. To do this, samples were taken from vaccinated and unvaccinated cattle and the parasites were genotyped with a series of micro- and minisatellite markers. The data indicate that the vaccine stabilates contain at least 6 parasite genotypes, the Kiambu 5 stock can be detected in many but not all vaccinated cattle for up to 4 years and can be transmitted to unvaccinated cattle which share grazing and that some of the vaccinated animals become infected with local genotypes without causing overt disease.

The mechanism of action of praziquantel (PZQ), the drug of choice against schistosomiasis, is still unclear. Since exposure of schistosomes to the drug is associated with calcium influx and muscular contraction, calcium channels have been suggested as the target, although direct combination of PZQ with their subunits was never demonstrated. We report a hitherto unknown effect of PZQ, namely the inhibition of nucleoside uptake, as observed in living worms using radio-isotope labelled adenosine and uridine. This effect is clearly seen in schistosomes but is absent in mammalian cells in culture. Moreover it is a specific pharmacological effect seen exclusively with the active levo-R(−)stereo isomer of the drug, and is shared by at least one benzodiazepine having antischistosomal activity. This novel effect acquires significance given that schistosomes cannot synthesize purine nucleosides de novo. A possible relationship between this novel effect and the known action of PZQ on calcium channels is discussed, since adenosine is known to bind to specific receptors and to behave as an indirect antagonist of calcium release in mammalian cells. If calcium channels were correlated with adenosine receptors also in schistosomes, as they are in mammals, this would support the hypothesis that PZQ-induced calcium influx may be correlated to adenosine receptor blockade.

Species of Protopolystoma are monogenean flukes that only infect allopolyploid hosts in the anuran genus Xenopus. Multivariate analyses of morphometric sclerite characters in the nominal species Protopolystoma simplicis suggest that morphologically distinguishable populations occur in the tetraploid host, Xenopus laevis victorianus, and in each of the octoploid hosts, X. vestitus and X. wittei. The species-level divergence of a lineage specific to X. laevis is supported by sequence variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene. Protopolystoma simplicis from X. laevis is redesignated P. microsclera n. sp., with P. simplicis being retained for populations in octoploid hosts. This division is consistent with large differences in egg hatching schedule, fixed differences at the mannose-6-phosphate isomerase and fumarate hydratase loci, and host-specificity in experimental analyses. Although the respective P. simplicis populations in X. vestitus and X. wittei also show significant diversity in allozyme expression, morphometrics and egg hatching schedule, they are retained in the same species because their level of mitochondrial DNA divergence is similar to that found within other Protopolystoma species. The consequences of splitting P. simplicis for a recent interpretation of the origin of Protopolystoma faunas in octoploid Xenopus spp. is discussed.

Monogenean communities of fish have generally been considered non-interactive as negative interspecific interactions have rarely been reported. Most of the earlier studies on monogenean communities, however, have been conducted not only in systems with relatively low parasite abundances but, more importantly, at study scales where microhabitat-level interactions between the parasites are easily overlooked. We examined the communities of 3 abundant Dactylogyrus (Monogenea) species on the gills of crucian carp (Carassius carassius) by analysing the interactions at the scale of individual gill filaments, where interactions between the species, if any, should most likely take place. Contrary to our expectations, we did not find evidence for competitive exclusion between the species, which suggests that monogenean communities are non-interactive even in high parasite abundances. At the species level, individual parasites were highly aggregated within the filaments, essentially showing a strong tendency to occur at either end of a filament. This, together with the result of differences in the distribution of juvenile parasites within the filaments compared to adults, suggests that these parasites are able to actively seek out their conspecifics in small-scale microhabitats during maturation, which again could enhance their mate-finding.

The infection biology of Branchotenthes octohamatus (Monogenea: Hexabothriidae) from the gills of the southern fiddler ray, Trygonorrhina fasciata (Rhinobatidae), was studied using the fluorescent dye, 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE). This is the first use of this technique on a monogenean species with unciliated larvae and the first for any monogenean larva infecting an elasmobranch host. CFSE-labelled post-larvae were recovered from gills of T. fasciata within 30 min of exposure to the host, providing strong evidence that larvae invade host gills directly and do not migrate after initial attachment elsewhere. The rapidity with which larvae settled suggests that the mode of infection may deliver larvae directly to the gills via the host's inhalant respiratory current. The specificity of B. octohamatus was investigated by exposing a sympatric rhinobatid host species, the western shovelnose ray, Aptychotrema vincentiana, to B. octohamatus larvae newly emerged from eggs laid by adult parasites from gills of T. fasciata. Experimental exposure of A. vincentiana to freshly hatched B. octohamatus larvae resulted in a persistent infection, indicating that B. octohamatus may not be strictly host specific. Post-larval development charted on these experimentally infected A. vincentiana specimens was slow. Parasites appeared to be sexually mature at 91 days at 21–25°C. Branchotenthes octohamatus larvae bear only 4 pairs of hooklets on the haptor whereas all other hexabothriid larvae described so far have 5 hooklet pairs. Ontogenetic changes to the haptor revealed that it is probably hooklet pair III that is lost from B. octohamatus prior to larval development.

The mode of action of bioactive plants on gastrointestinal nematodes remains obscure. Previous in vitro studies showed that exsheathment was significantly disturbed after contact with tannin-rich extracts. However, the role of important factors (extract concentration, parasite species) has not been assessed and no information is available on the occurrence in vivo. These questions represent the objectives of this study. The model incorporated the parasites Haemonchus contortus and Trichostrongylus colubriformis with sainfoin as the bioactive plant. A set of in vitro assays was performed, measuring the changes observed, after 3 h of contact with increasing concentrations of sainfoin, on the rate of artificial exsheathment. The results indicated that sainfoin extracts interfered with exsheathment in a dose-dependent manner and the process overall was similar for both nematodes. The restoration of control values observed after adding PEG to extracts confirms a major role for tannins. A second study was performed in vivo on rumen-cannulated sheep fed with different proportions of sainfoin in the diet to verify these in vitro results. The consumption of a higher proportion of sainfoin was indeed associated with significant delays in Haemonchus exsheathment. Overall, the results confirmed that interference with the early step of nematode infection might be one of the modes of action that contributes to the anthelmintic properties of tanniniferous plants.

A deterministic, dynamic simulation model is developed to account for the interactions between gastrointestinal parasitism and host nutrition, and predict their consequences on performance and level of parasitism of sheep. Larval intake and established adult worms are assumed to result in nutrient loss for the host. In order to reduce this loss the host will mount an immune response, which will affect the establishment rate of incoming larvae, mortality rate of adult worms, and fecundity of female worms, as well as nutrient loss caused by larval intake per se. Host anorexia is modelled as a function of worm mass. Parasitism is also assumed to affect the allocation of ingested nutrients to the host's bodily functions, with maintenance getting absolute priority, and protein allocated to immunity and production proportionally to their requirements. Inputs to the model include the expected growth attributes of the animal, feed quality, various parasitological parameters and daily larval intake. Outputs include feed intake, growth rate and body composition, as well as worm burden and faecal egg counts. The model allows exploration of the consequences of gastrointestinal parasitism on sheep of different growth characteristics, kept under environments that vary in the provision of nutrients and exposure to parasites.

This paper describes sensitivity analyses and expectations obtained from a mathematical model developed to account for the effects of host nutrition on the consequences of gastrointestinal parasitism in sheep. The scenarios explored included different levels of parasitic challenge at different planes of nutrition, for hosts differing only in their characteristics for growth. The model was able to predict the consequences of host nutrition on the outcome of parasitism, in terms of worm burden, number of eggs excreted per gram faeces and animal performance. The model outputs predict that conclusions on the ability of hosts of different characteristics for growth to cope with parasitism (i.e. resistance) depend on the plane of nutrition. Furthermore, differences in the growth rate of sheep, on their own, are not sufficient to account for differences in the observed resistance of animals. The model forms the basis for evaluating the consequences of differing management strategies and environments, such as breeding for certain traits associated with resistance and nutritional strategies, on the consequences of gastrointestinal parasitism on sheep.

A parasitological survey in the Bay of Fundy, Canada, resulted in the recovery of mature specimens from 5 species of phyllobothriid tapeworms (Cestoda: Tetraphyllidea) from 4 rajid skates: Echeneibothrium canadensis and E. dubium abyssorum specimens from Amblyraja radiata; E. vernetae and Pseudanthobothrium n.sp. from Leucoraja erinacea and L. ocellata; and P. hanseni from A. radiata and Malacoraja senta. Partial sequence data of a variable region (D2) from the large subunit ribosomal DNA (LSU) were used here to determine the host distribution of immature specimens for 4 of these 5 species (E. d. abyssorum was not included in the analyses). Immature specimens from both Pseudanthobothrium spp. were identified in the same hosts as recorded previously for mature specimens, thus suggesting that there are mechanisms that prevent the attachment of the parasite in an ‘unsuitable’ host species. Immature E. canadensis specimens were recovered exclusively from A. radiata, whereas immature E. vernetae specimens were recovered from L. erinacea and A. radiata, despite the latter host species not harbouring mature E. vernetae specimens. Their presence in the latter host species may be explained by host restriction or resistance, which allows the attachment of the parasites in the ‘wrong’ host species, but not establishment or development.

Ascariasis is an important infection in humans (Ascaris lumbricoides) and pigs (Ascaris suum) and individuals appear to be predisposed to either heavy or light worm burdens. These extremes of susceptibility and resistance are represented in a mouse model by 2 strains of mice, CBA mice showing high resistance to infection and C57BL/6 which are highly susceptible, as reflected in worm burdens in the lungs 6–7 days after infection. In an attempt to identify the point at which the difference between these 2 strains is first manifested, we quantified worm burdens at key stages during infection leading up to the pulmonary stage of development. Thus mice were inoculated with fully embryonated A. suum eggs and larval burdens were enumerated in the large intestine and rectum, liver and lungs of the 2 strains at 6 h post-inoculation (p.i.) and on each of days 1–8 p.i. inclusively. A higher percentage of the total inoculum was recovered from the intestine/rectum of C57BL/6j mice in contrast to CBA/Ca mice at 6 h p.i. Larvae were recovered from the intestinal contents and also whilst actively migrating through the large intestinal wall. The number of larvae recovered was significantly reduced in CBA/Ca mice in contrast to C57BL/6j mice between the phase of migration from the liver and arrival in the lungs. The combined results of the inoculation of mice with corticosteroids and the examination of the change in profile and number of leukocytes present in bronchoalveolar lavage fluid suggested that the pulmonary inflammatory immune response was not prominently involved in primary protection of mice to A. suum infection in the latter days of infection in the lungs. The susceptible C57BL/6j mice produced a BAL response almost twice as intense as that of resistant CBA/Ca mice with stronger neutrophil, lymphocyte and eosinophil but not macrophage components, suggesting that the difference in worm burdens between the strains was generated earlier in the course of infection. These results were further corroborated by a histological examination of the lung tissues which showed that the passage of the larval stages of A. suum through the mouse lungs was associated with a marked inflammatory response in both strains. Again, C57BL/6j mice exhibited increased inflammation relative to CBA/Ca mice. Hence some hepatic/post-hepatic factor that varies between the 2 strains, but exerts its effect before the lung phase plays a critical role in determining the success of larvae through the host tissues. The possible sites of this host defence are reviewed.