Using surface immunofluorescence isolate-specific antigens were detected on the membrane of erythrocytes infected with Babesia parasites. In addition, the strains reacted differently with Plasmagel in that the European isolate (B.c. canis) could be purified on Plasmagel effectively, whereas infected erythrocytes of the South-African isolate (B.c. rossi) could not. Experimental infection of dogs with Babesia canis isolates from geographically different areas revealed different pathology. The European isolate obtained from France exhibited transient parasitaemia, usually below 1%, associated with low PCV values and congestion of internal organs. Clinical disease was correlated with an effect on the coagulation system, and not with peripheral parasitaemia. Infection of dogs with South-African-derived isolate induced high parasitaemia usually much higher than 1%, which required chemotherapeutic treatment. In these animals clinical disease was correlated with peripheral parasitaemia and not with parameters of the coagulation system. The results show that the etiology of disease caused by these isolates of B.c. canis and B.c. rossi is different. This might have implications for the development of vaccines against these infections.

In a previous investigation, five Sarcocystis species were described from Norwegian red deer and believed to be conspecific with species occurring in either reindeer or moose based on sarcocyst morphology and nucleotide sequences of the nuclear ribosomal DNA unit. The aim of the present study was to characterize numerous isolates of these sarcocyst types at the mitochondrial cytochrome c oxidase subunit I gene (cox1) in order to corroborate or refute previous species designations of Sarcocystis in red deer. The Sarcocystis tarandi- and Sarcocystis rangiferi-like taxa in red deer and reindeer, respectively, were thoroughly compared by sequencing 14–27 isolates of each type. Sequence comparisons revealed four distinct sequence types, which by phylogenetic analyses were placed in four monophyletic groups according to host origin, and they were therefore considered to represent four separate species. The two taxa of this type in red deer were named Sarcocystis elongata and Sarcocystis truncata, respectively. Sequencing of many isolates of Sarcocystis hjorti and Sarcocystis ovalis from red deer and moose confirmed that these species occur in both hosts. A revised description of the two new species is given and the current knowledge concerning all six Sarcocystis species in red deer is reviewed.

Disruption of the endocrine system has been shown to occur in wild freshwater fish populations across the globe. Effects range from subtle changes in the physiology and sexual behaviour of fish to permanently altered sexual differentiation, impairment of gonad development and/or altered fertility. A wide variety of adverse environmental conditions may induce endocrine disruption, including sub-optimal temperatures, restricted food supply, low pH, environmental pollutants, and/or parasites. Furthermore, it is conceivable that any/all of these factors could act simultaneously to cause a range of disparate or inter-related effects. Some of the strongest evidence for a link between an adverse health effect, as a consequence of endocrine disruption, and a causative agent(s) is between the condition of intersex in wild roach (Rutlius rutilus) in UK rivers and exposure to effluents from sewage treatment works. The evidence to indicate that intersex in roach (and other cyprinid fish living in these rivers) is caused by chemicals that mimic and/or disrupt hormone function/balance in treated sewage effluent is substantial. There are a few parasites that affect the endocrine system directly in fish, including the tape worm Ligula intestinalis and a few parasites from the micropsora phylum. L. intestinalis acts at the level of the hypothalamus restricting GnRH secretion (resulting in poorly developed gonads) and is one of the very few examples where an endocrine disrupting event has been shown to result in a population-level effect (reducing it). It is well established that many parasites affect the immune system and thus the most common effect of parasites on the endocrine system in fish is likely to be an indirect one.

The Eucoilid parasitoid Leptopilina boulardi is able to suppress its host Drosophila melanogaster immune reaction. Some strains, however, are non-immune suppressive to that host. Virus-like particles (VLPs) responsible for the immune suppressive ability were investigated in different strains of L, boulardi with histochemical and ultrastructural techniques. Membrane-bound particles containing vesicles were observed in the reservoir of the long gland and also in the egg canal of the ovipositor. These particles are homologous with the immune suppressive VLPs already described in the reservoir of L. heterotoma. Similarities were also observed with the L2 particles described previously around the chorion of the parasitoid egg after infestation. A weak positive DNA specific histochemical reaction was observed inside the reservoir and at the ultrastructural level. Feulgen-derived techniques demonstrated that the reaction was localized inside the particles. The morphology of the particles as well as the immune suppressive ability varied between strains. Two morphotypes of VLPs are described; the ‘Is’ morphotype (always observed in immune suppressive or Is strains) and the ‘NIs’ morphotype (observed in the non-immune suppressive or NIs strain). The hybrids between Is and NIs strains produce a third type of particle, the ‘His’morphotype with half-immune suppressive ability and intermediate morphology. The origin of the particles' immune suppressive activity against D. melanogaster is discussed within the scope of host specificity.

Totipotent somatic stem cells (neoblasts) are key players in the biology of flatworms and account for their amazing regenerative capability and developmental plasticity. During recent years, considerable progress has been made in elucidating molecular features of neoblasts from free-living flatworms, whereas their role in parasitic species has so far merely been addressed by descriptive studies. Very recently, however, significant advances have been made in the in vitro culture of neoblasts from the cestode Echinococcus multilocularis. The isolated cells proved capable of generating mature metacestode vesicles under laboratory conditions in a manner that closely resembles the oncosphere-metacestode transition during natural infections. Using the established neoblast cultivation protocols, combined with targeted manipulation of Echinococcus genes by RNA-interference, several fundamental questions of host-dependent parasite development can now be addressed. Here, I give an overview of current cultivation techniques for E. multilocularis neoblasts and present experimental approaches to study their function. Furthermore, I introduce the E. multilocularis genome sequencing project that is presently in an advanced stage. The combined input of data from the E. multilocularis sequencing project, stem cell cultivation, and recently initiated attempts to genetically manipulate Echinococcus will provide an ideal platform for hypothesis-driven research into cestode development in the next years.

In order to identify Echinococcus multilocularis DNA in fox faeces for epidemiological purposes, we have developed a new method to prepare DNA suitable for PCR amplification. DNA isolation from fox excrement was performed according to a novel procedure involving lysis in KOH, phenol–chloroform extraction and a purification step on a matrix (Prep-A-Gene®). The target sequence for amplification was the E. multilocularis U1 snRNA gene. PCR products were indistinguishable for 32 different E. multilocularis isolates and no signal was observed after ethidium bromide staining with DNAs from other tapeworm species, including E. granulosus. The sensitivity of amplification was monitored by the addition of E. multilocularis DNA or eggs to faeces free of E. multilocularis and was estimated to be 1 egg per 4 g of faeces. PCR products were blotted onto nylon membranes and hybridized with an internal oligonucleotide probe in order to confirm the results. Twenty nine faecal samples from foxes shot in Franche-Comté (East France) were tested. Out of 10 samples from foxes in which no E. multilocularis adult worms could be observed after necropsy, 7 were PCR positive, showing that the PCR test is more sensitive than microscopical examination. Out of 19 samples from foxes harbouring E. multilocularis adult worms, 18 were PCR positive. The remaining PCR-negative sample could be due either to the misidentification of the species of adult worm (E. granulosus and E. multilocularis), or to DNA variation between different isolates of E. multilocularis. Further work in the field should be initiated in order to confirm these results.

A comparison has been made of the responses of random-bred CFLP and inbred NIH mice to infection with Trichuris muris. Random-bred mice showed greater variation in worm burdens and less uniformity in worm expulsion. Irradiation prior to infection reduced variation, but did not increase the mean level of infection above that shown by the most susceptible unirradiated mice. In NIH mice, however, irradiation raised the level of infection in all mice. The factors responsible for variation between CFLP mice and for the level of infection in NIH mice came into play after the fifth day of infection and were inactivated by cortisone acetate. It is suggested that these factors are immunologically mediated and under direct genetic control. Uniformity of infection and expulsion in NIH mice is therefore seen as a consequence of genetic uniformity; variability in CFLP mice as a consequence of genetic variation.

The time of worm expulsion was found to differ markedly between inbred strains of mice. Hybrid progeny showed the expulsion time characteristic of the parental strain with the most rapid expulsion; greater resistance was therefore inherited as a dominant characteristic. The genetic control of immunity to T. muris is discussed in the context of the antibody- and cell-mediated components of the expulsion process.

Metabolic studies in vitro and studies on chemical composition indicate basic biochemical differences between the horse and sheep strains of Echinococcus granulosus and between these and the closely related species, E. multilocularis. The horse strain of E. granulosus has a similar level of DNA, but significantly more polysaccharides and lipids, with less RNA and protein than the sheep strain. E. multilocularis has significantly more lipids and DNA but less polysaccharides than the horse and sheep strains of E. granulosus, more RNA and protein than the horse strain but similar protein to and less RNA than the sheep strain. Incubations under air and under 95% N2-5% CO2 for 3 h show that only E. multilocularis takes up glucose, that all three forms consume different amounts of oxygen and endogenous glycogen and produce different concentrations of lactate, succinate, acetate, malate, pyruvate, propionate and ethanol as end products of carbohydrate metabolism.

This review of the safety of the co-administration regimens to be used in programmes to eliminate lymphatic filariasis (albendazole+ivermectin or albendazole+diethylcarbamazine [DEC]) is based on 17 studies conducted in Sri Lanka, India, Haiti, Ghana, Tanzania, Kenya, Ecuador, the Philippines, Gabon, Papua New Guinea, and Bangladesh. The total data set comprises 90635 subject exposures and includes individuals of all ages and both genders. Results are presented for hospital-based studies, laboratory studies, active surveillance of microfilaria-positive and microfilaria-negative individuals, and passive monitoring in both community-based studies and mass treatment programmes of individuals treated with albendazole (n=1538), ivermectin (9822), DEC (576), albendazole+ivermectin (7470), albendazole+DEC (69020), or placebo (1144). The most rigorous monitoring, which includes haematological and biochemical laboratory parameters pre- and post-treatment, provides no evidence that consistent changes are induced by any treatment; the majority of abnormalities appear to be sporadic, and the addition of albendazole to either ivermectin or DEC does not increase the frequency of abnormalities. Both DEC and ivermectin show, as expected, an adverse event profile compatible with the destruction of microfilariae. The addition of albendazole to either single-drug treatment regimen does not appear to increase the frequency or intensity of events seen with these microfilaricidal drugs when used alone. Direct observations indicated that the level of adverse events, both frequency and intensity, was correlated with the level of microfilaraemia. In non microfilaraemic individuals, who form 80–90% of the ‘at risk’ populations to be treated in most national public health programmes to eliminate lymphatic filariasis (LF), the event profile with the compounds alone or in combination does not differ significantly from that of placebo. Data on the use of ivermectin+albendazole in areas either of double infection (onchocerciasis and LF), or of loiais (with or without concurrent LF) are still inadequate and further studies are needed. Additional data are also recommended for populations infected with Brugia malayi, since most data thus far derive from populations infected with Wuchereria bancrofti.

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.

Captive-bred Mus musculus (house mice) and Apodemus sylvaticus (field mice) were each infected with 50 oocysts of Toxoplasma gondii M1 strain per os and infection in them and their offspring was assessed by polymerase chain reaction (PCR) amplification of the T. gondii B1 gene in brain tissue and by serology, using the modified agglutination test (MAT). The chronically infected female A. sylvaticus (n=10) and M. musculus (n=23) were mated at least 6 weeks after infection (and subsequently to produce up to 6 litters) and their pups examined 3 weeks after weaning at 6 weeks of age. By PCR, in offspring of A. sylvaticus and M. musculus respectively, vertical transmission was demonstrated in 82·7% (n=83) and 85·0% (n=207) of all pups (N.S., P>0·05), 95% (n=21) and 100% (n=30) of all litters (N.S., P>0·05), with a mean (±S.E.) proportion of each litter infected of 0·87 (0·06) and 0·86 (0·04) (N.S., P>0·05). There was no change in any of these variables between first and subsequent litters. By serology, whilst MAT suggested 100% vertical transmission in A. sylvaticus, it under-estimated rates of infection in offspring of M. musculus. A limited series of bioassays from M. musculus tissues confirmed the good correlation of PCR and the poor correlation of MAT with mouse inoculation. These results indicate that vertical transmission in A. sylvaticus and M. musculus is extremely efficient and probably endures for the life of the breeding female. This mechanism favours parasite transmission and dispersion by providing a potential reservoir of infection in hosts predated by the cat.

Hosts exert selection pressures on their parasites and it is often assumed that host–parasite coevolution with each host is less intense in a generalist parasite than for a parasite with a narrow host range. Selection pressure on the parasite, however, is rather determined by host specificity, i.e. the relative importance of each host, than simply by the range of hosts. The determination of host specificity requires an assessment of the prevalence and intensity of parasite infestation within each host's nests, as well as the local abundance of each host species. Since the hen flea, Ceratophyllus gallinae, is a rather generalist parasite of birds it could be concluded that there has been weak coevolution with each of its hosts. By reviewing the literature on the prevalence and intensity of hen flea infestations in bird nests we estimated the number of individuals produced in the nest of each host species. The comparative analysis shows (1) that the prevalence of infestation is highest in hole-nesting avian families, (2) that prevalence and intensity of infestation among bird families are highly correlated, and (3) that hole-nesting Paridae have the highest intensities of infestation and harbour the majority of the flea population. These results underline the fleas' potential for coevolution with Paridae despite their extensive host range.

The domestic dog is the reservoir host of Leishmania infantum, the causative agent of zoonotic visceral leishmaniasis endemic in Mediterranean Europe. Targeted control requires predictive risk maps of canine leishmaniasis (CanL), which are now explored. We databased 2187 published and unpublished surveys of CanL in southern Europe. A total of 947 western surveys met inclusion criteria for analysis, including serological identification of infection (504, 369 dogs tested 1971–2006). Seroprevalence was 23 2% overall (median 10%). Logistic regression models within a GIS framework identified the main environmental predictors of CanL seroprevalence in Portugal, Spain, France and Italy, or in France alone. A 10-fold cross-validation approach determined model capacity to predict point-values of seroprevalence and the correct seroprevalence class (<5%, 5–20%, >20%). Both the four-country and France-only models performed reasonably well for predicting correctly the <5% and >20% seroprevalence classes (AUC >0 70). However, the France-only model performed much better for France than the four-country model. The four-country model adequately predicted regions of CanL emergence in northern Italy (<5% seroprevalence). Both models poorly predicted intermediate point seroprevalences (5–20%) within regional foci, because surveys were biased towards known rural foci and Mediterranean bioclimates. Our recommendations for standardizing surveys would permit higher-resolution risk mapping.

We report the results of experimental work on (a) the influence of temperature on the age-dependent survival and infectivity of the mira-cidia of Schistosoma mansoni and (b) the relationship between snail age, snail size and susceptibility to infection. The death rate of miracidia declined exponentially with age where life-expectancy was maximal (approximately 16 h) at 15 °C. Infectivity also declined rapidly with larval age but, in contrast to larval survival, the rate of infection was at a maximum at 25 °C. Snail susceptibility was shown to be more closely correlated with host size rather than host age. Susceptibility declined exponentially with increased host size. Size-dependent susceptibility was shown to generate concave age-prevalence curves for infection within snail populations, where the maximum prevalence is generated in snails of intermediary age. Simple mathematical models are developed to aid estimation of larval survival and infection rates and experimental results are discussed in relation to the overall transmission success of the parasite from man to snail.

This study compared the biological and genetic properties of a bovine (NC-SweB1) and a canine (NC-Liverpool) isolate of Neospora caninum. A mouse model for CNS infection demonstrated marked differences in pathogenicity between the isolates. NC-Liverpool induced severe clinical signs of neosporosis in 57/58 mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss. In contrast NC-SweB1 induced similar but less severe symptoms in a much smaller proportion of mice over the same time-period. Statistically significant differences were observed between the isolates in the response (mean weight loss) of mice through time to the different doses inoculated. Histopathological effects on brain tissue reflected the isolate-based differences described above. NC-Liverpool infection resulted in intense inflammatory infiltrates and highly necrotic lesions whereas NC-SweB1 induced a milder meningoencephalitis. Passage in cell-culture over a period of 14 months did not affect the pathogenicity of NC-Liverpool. Immunoblots showed that antibodies to N. caninum appeared earlier in mice inoculated with NC-Liverpool than with NC-SweB1. Finally, RAPD–PCR analysis of NC-Liverpool DNA generated profiles distinct from that observed with DNA from NC-SweB1 or Toxoplasma gondii. In summary this study provides evidence for significant biological and genetic differences between 2 isolates of N. caninum.

Infestations of post-smolt sea trout (Salmo trutta L.) by the salmon louse (Lepeophtheirus salmonis Krøyer) were characterized in 42 estuaries over a 5 year period in Ireland. Spatial variation in infestation was more significant than temporal trends and existed at 3 levels; between regions (regions >100 km of coastline), between bays within regions (bays <50 km in length) and between estuaries within bays (distance between estuaries <10 km). The observed spatial structure in infestations inferred that production of the infective larvae varied between regions and bays and that there was limited movement of fish and infective larvae between regions and bays. In addition the different levels of infestation recorded between estuaries in the same bay indicated short spatial scale variability in parasite transmission. Significantly higher infestations occurred in bays that contained lice-infested farmed salmon. Lice-infested wild spring salmon, which were present in estuaries of some systems, did not have a significant positive impact on infestations.

The presence of haemoparasites from the Order Piroplasmida and the genera Bartonella and Trypanosoma was assessed in the blood of 60 bats, belonging to 7 species, inhabiting sites across Cornwall in southwest England. DNA extracted from macerated heart tissue was incorporated into taxon-specific polymerase chain reactions (PCRs) and amplification products were sequenced as a means of identifying, or assigning an identity, to detected haemoparasites. A Piroplasmida species was detected in 6 Pipistrellus spp., whereas Bartonella infections were detected in 5 bats belonging to 4 different species. Trypanosoma dionisii was detected in 1 Pipistrellus spp. Phylogenetic inference from alignment of a partial 18S rRNA-encoding gene sequence of the pipistrelle-associated Piroplasmida species with homologous sequences available for other members of the Order indicated that this organism was unique but specifically related to members of the genus Babesia, a phylogeny that would be in keeping with the organism being Babesia vesperuginis. Alignment of partial citrate synthase gene sequences from the bat-associated bartonellae revealed 5 distinct genotypes that were probably derived from 2 distinct Bartonella species. The study demonstrates the utility of molecular methods for detecting haemoparasites in dead bats and provides, for the first time, tangible identities for bat-associated Babesia and Bartonella species.

Species- and subspecies-specific trypanosome DNA hybridization probes have been employed in the detection and identification of trypanosome infections in Glossina morsitans centralis. Several ways of sample preparation including the use of tsetse organ suspensions, proboscides and dissected midguts, as well as tsetse abdominal content touch-blots were explored. The results of hybridization of radio-isotope-labelled species-specific DNA probes to tsetse samples indicated that it was possible to detect trypanosomes in the organs where parasite development is known to characteristically occur for each subgenus. Duplicate slot-blots of samples prepared from midguts of tsetse infected with 2 strains of T. congolense and from non-infected fly controls show that it is not only possible to detect infection in tsetse but also to identify the strain of parasite present in a sample after hybridization with the DNA probes specific for each strain. The results, obtained after hybridization of sequential abdominal touch-blots from the same fly with the DNA probe specific for one strain of T. congolense, indicated that at least 8 positive signals can be observed after an overnight exposure. Because of their simplicity and potentially low cost, the techniques described here would be appealing for screening large numbers of tsetse samples from the field for the presence of any trypanosome residing in the guts or proboscis of the vector. In addition, the possibility of doing multiple touch-blots from the same fly gives the opportunity of detecting mixed trypanosome infections in the vector.

Snails and digeneans have been associated for at least 200 million years. Their inter-relationships over such a time-span must have been complex and varied. Few studies have attempted to explore these relationships in the light of knowledge of the phylogeny of both host and parasite groups. Here we focus on two important families of digeneans, the Schistosomatidae and the Paragonimidae, for which molecular phylogenies are available. We investigate the types of evolutionary relationships between host and parasite, operating at different phylogenetic depths, that might explain current host specificity and distributions of both associates. Both families of parasites utilise a number of highly diverged gastropod families, indicating that host extensions have featured in their histories. However, schistosomatids and paragonimids show different patterns of association with their snail hosts. As befits the apparently more ancient group, schistosomatids utilise snails from across a wide phylogenetic range within the Gastropoda. The genus Schistosoma itself has experienced one long-range host switch between pulmonates and caenogastropods. By contrast, paragonimids are restricted to two superfamilies of caenogastropods. Despite these differences, modern schistosomatid species appear to be more host specific than are paragonimids and host additions, at the level of host family, are far less common among species of schistosomatids than among paragonimids. Some species of Paragonimus exhibit remarkably low levels of host specificity, with different populations utilising snails of different families. Existing knowledge relating to the phenomenon will be presented in the context of phylogenies of schistosomatids, paragonimids, and their snail hosts. Discussion focuses on the usefulness of current theories of snail–digenean coevolution for interpreting these findings. In the past, much emphasis has been placed on the idea that digeneans engage in a one-to-one arms race with their snail host. We consider that phylogenetic tracking rather than an arms-race relationship might be a common alternative. Not being bound by the restrictions imposed by an arms race, some digeneans might be able to extend to new host species more easily than the literature suggests. Switches into related host taxa are most likely. However, ecologically equivalent but unrelated gastropod hosts may also be exploited. Given the right ecological setting, digeneans are able to switch across considerable phylogenetic distances. Examples from the Paragonimidae and Schistosomatidae are given.

A study of electrophoretic forms of a selection of enzymes has been made in strains of subspecies of the rodent malaria parasites Plasmodium berghei and P. vinckei. Considerable electrophoretic variation of these enzymes occurred among the strains studied. On the basis of the results it was possible to identify groups of parasites within each species. While generally substantiating the classical taxonomy the identification of groups provided further information on the relationships between the subspecies.