The phylogeny of representative haemozoan species of the phylum Apicomplexa was reconstructed by cladistic analyses of ultrastructural and life-cycle characteristics. The analysis incorporated 4 apicomplexans previously not included in phylogenetic reconstructions: Haemogregarina clelandi from the Brisbane River tortoise (Emydura signata), Hepatozoon sp. from the slaty grey snake (Stegonotus cucullatus), Hepatozoon (Haemogregarina) boigae from the brown tree snake (Boiga irregularis), and Haemoproteus chelodina from the saw-shelled tortoise (Elseya latisternum). There was no apparent correlation between parasite phylogeny and that of their vertebrate hosts, but there appeared to be some relationship between parasites and their intermediate hosts, suggestive of parasite/vector co-evolution.

Experiments were designed to study the mating behaviour between the Schistosoma haematobium [male ]×S. intercalatum [female] hybrid and the 2 parental species S. haematobium and S. intercalatum. Individual worms were identified by electrophoretic analysis of glucose-6-phosphate dehydrogenase, which was characteristic for each isolate. Analysis of the data obtained showed that both heterospecific and homospecific pairs formed between the hybrids and S. haematobium and S. intercalatum. S. haematobium and the hybrid are better than S. intercalatum in forming pairs, and S. haematobium showed a greater homospecific mate preference compared with the hybrid. Analysis of the data using the Mantel-Haenszel test suggests that mating competition does exist between the schistosomes, with the hybrid being dominant over both the parental species and S. haematobium being dominant over S. intercalatum. The hybrid males showed a greater ability than S. intercalatum and S. haematobium males in taking away S. haematobium and S. intercalatum females from their homospecific males when introduced into a pre-established S. haematobium or S. intercalatum infection. They were able to take females from S. intercalatum homospecific pairs more easily compared with females from S. haematobium homospecific pairs. The significance of the results is discussed in relation to the epidemiological changes of schistosomiasis in Cameroon, where hybridization between S. haematobium and S. intercalatum has taken place, with S. haematobium and the hybrid managing to replace the endemic S. intercalatum over the last 30 years.

The acanthocephalan parasite Pomphorhynchus laevis is transmitted by a suitable intermediate host such as the amphipod Gammarus pulex to its definitive fish host. Parasite-induced alterations in both appearance and behaviour concur to render infected gammarids more vulnerable to predation, thus promoting parasite transmission. Experimental infection of laboratory bred full- and half-sib groups of three-spined sticklebacks (Gasterosteus aculeatus) provided evidence that the parasite imposes a survival cost proportional to the severity of infection on its final host. Variation among sibships in the susceptibility to infection was consistent. When given a choice, fish consumed significantly more infected than uninfected prey. Overall, more resistant fish did not prey upon infected gammarids more often than did relatively susceptible fish. Only fish with a relatively high physical condition properly adjusted prey selection to the extent of their parasite susceptibility, thus exploiting the enhanced profitability of infected prey.

A PCR procedure for the detection of Opisthorchis viverrini in experimentally infected bithynid snails and cyprinoid fishes was developed. This procedure was based on primers designed from a pOV-A6 specific probe sequence giving a 330 base-pair product. The detection was accomplished in host tissue homogenates to which a single cercaria or metacercaria was introduced. PCR can detect as little as a single cercaria artificially inoculated in a snail or a single metacercaria artificially inoculated in a fish sample. The method gave a 100% positivity rate for all infected snails or fishes. The method did not yield a 330 base-pair amplified product with other digenean fluke DNAs such as Haplorchis taichui, Centrocestus spp., Echinostoma malayanum, Fasciola gigantica, animal schistosomes, Paragonimus heterotremus or Haplorchoides spp. The assay has great potential for application in epidemiological surveys of both snail and fish intermediate hosts as well as for investigation of foodborne parasites in freshwater fishes.

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

Although previous studies have highlighted the inflammatory responses of fish infected with parasites and exposed to pollutants, very little is known about how these two stressors interact within the fish. In this review, which also contains original data, the effect of these two parameters on the fish inflammatory response is assessed and, in particular, the role of apoptosis and the acute phase protein, C reactive protein, is evaluated. In Cyprinus carpio exposed to 0·5 mg NH4+ l−1 or 0·1 mg Cd2+ l−1 and experimentally infected with the blood fluke, Sanguinicola inermis, the pollutant type and the order in which the fish experiences the parasite and toxicant, significantly affects the ultrastructural appearance and cellular content of the pronephros and thymus. This is reflected in the intensity of infection where the pollutant appears to have less effect on an established infection. Both stressors, pollutant and infection, may mediate their effects via the endocrine system. Studies have revealed that cortisol at 100 ng ml−1 is able to induce apoptosis in pronephric cells of carp and that an increase in apoptosis is associated with an increase in phagocytosis in this immune organ. In addition, C reactive protein, which is used as a biomarker of the inflammatory response in humans and other mammals, is evaluated as a possible indicator of physiological states in fish exposed to pathogens and pollutants.

The gene coding for α tubulin has been isolated by the polymerase chain reaction and sequenced from 2 isolates of Neospora caninum (Nc-Liverpool and Nc-SweB1)The nucleotide sequence data reported in this paper are available from the GenBank database under the accession numbers AF508031 (N. caninum) and AY169962 (H. heydorni).. The data show that the gene, as in Toxoplasma gondii, is single copy and contains 3 exons and 2 introns and is identical in sequence in the 2 isolates studied. Comparison of the predicted protein sequence shows it to be identical to the α tubulin protein encoded by the T. gondii gene. The majority of the nucleotide substitutions that have occurred during the evolution of the T. gondii and N. caninum genes from their common ancestor have occurred in the third codon position. A partial coding sequence for α tubulin was also obtained from Hammondia heydorni and compared to other α tubulin sequences from Apicomplexa. The results show the sequences of the T. gondii, N. caninum and H. heydorni α tubulin genes to be similar but not identical in sequence, thereby providing new evidence that N. caninum and H. heydorni are genetically distinct species.

Many parasite species are known to show high levels of genetic diversity, yet the consequences of this diversity for host–parasite interactions are not well understood. Variation in phenotypic traits such as growth rates and the ability to form transmission stages are raw material for natural and artificial selection to act upon with consequences for the evolution of the parasite species and disease control. In order to study genetic and phenotypic diversity amongst Theileria annulata parasites, a collection of 52 parasite clones was generated from cattle isolates and tick material recently collected in Tunisia. Genetic diversity was assessed using PCR-RFLP and monoclonal antibody markers, and genetically distinct clones selected for further study. Clones varied significantly in their growth rates in culture at 37 °C, their viability after a period of culture at 41 °C and their differentiation rates into transmission stages after culturing at 41 °C. The viability of a clone after culturing at 41 °C could not be predicted from its growth rate at 37 °C, but across clones, differentiation rates were positively correlated with growth rates at 37 °C. All 3 in vitro measures are likely to have relevance to parasite–host interactions in animals with clinical theileriosis, and should be acted on by within-host and between-host selection.

Echinostome larval stages in the snail have a great potential as effective competitors for the control of schistosomes and adult worms can cause painful intestinal diseases in humans. Ecology and transmission of the larval stages of trematodes are poorly understood, especially because their identification in field-collected samples by microscopy is difficult. We cloned, sequenced and analysed a 192 bp tandem repreated DNA sequence of Echinostoma caproni (EcSau3A), an often discussed antagonist of Schistosoma mansoni in Biomphalaria snails. PCR primers against this sequence can detect less than 10 fg of E. caproni DNA, 2 miracidia in snails 1 day p.i., 1 metacercaria in 50 mg snail tissue and 1 cercaria in 50 mg plankton with high specificity. Methods described in this study can support the discovery of fundamental ecological principles on distribution, host specificity and epidemiology of E. caproni larvae under field conditions.

Many parasites, including important species that affect humans and livestock, must survive the harsh environment of insect guts to complete their life-cycle. Hence, understanding how insects protect themselves against such parasites has immediate practical implications. Previously, such protection has been thought to consist mainly of mechanical structures and the action of lectins. However, recently it has become apparent that gut infections may interact with the host immune system in more complex ways. Here, using bumble bees, Bombus terrestris and their non-invasive gut trypanosome, Crithidia bombi, as a model system we investigated the effects of parasitic infection, host resources and the duration of infections on the host immune system. We found that infection doubled standing levels of immune defence in the haemolymph (the constitutive pro-phenoloxidase system), which is used as a first, general defence against parasites. However, physical separation of the parasite from the haemolymph suggests the presence of a messenger system between the gut and the genes that control the pro-phenoloxidase system. Surprisingly, we found no direct effect of host resource-stress or duration of the infection on the immune system. Our results suggest a novel and tactical response of insects to gut infections, demonstrating the complexity of such host–parasite systems.

The factors contributing to the similarity between digenean parasite assemblages parasitizing the black-headed gull were investigated. Thirteen different digenean species from the digestive tract and the bursa of Fabricus were found. We predicted that the structure of parasite communities is not random at either component community or infracommunity levels. We searched for nested patterns in the digenean community, and explored the possible factors contributing to nestedness. We found that digenean species which occupy a narrow range of intermediate hosts are placed out of order in the nested matrix. The influence of several variables related to the water reservoir, geographical distances, and the abundance of intermediate hosts on the species diversity and similarity of parasite communities were tested as well. Because of the complexity in bird digenean life-cycles we supposed that intermediate hosts and ecological characters of their environment could play a major role. We showed that the presence of intermediate hosts is the factor limiting the qualitative and quantitative similarity of parasite communities among different host populations as well as influencing the digenean species diversity in the definitive host. The similarity in abundance of intermediate hosts between different localities was facilitated by the presence of vegetation in water reservoir and more diversified type of water bottom. Digenean species diversity was higher when the water reservoir was exposed to temporal drying. We hypothesized that this factor could facilitate the inclusion of molluscs infected by digeneans in the diet of birds. Both species diversity and dominance were influenced by the bottom type of water reservoir and temporal drying up. Geographical distances between localities influenced the quantitative similarity of digenean communities as well as species richness.

Using indirect immuno- and enzyme-cytochemical techniques, interfaced with confocal scanning laser microscopy and standard optical microscopy, neuronal pathways have been demonstrated in whole-mount preparations of the unpaired diporpae and freshly paired juvenile stages of Eudiplozoon nipponicum (Monogenea: Diplozoidae). All 3 main classes of neuronal mediators, cholinergic, aminergic and peptidergic, were identified throughout both central and peripheral elements of a well-differentiated orthogonal nervous system. Neural mapping revealed considerable overlap and similarity in staining of the nervous systems of the diporpa and adult worm. The main differences in the diporpa relate to the innervation of the temporary ventral sucker and dorsal papilla, structures which are unique to the larva and which enable fusion between worms but then disappear. Branches from the longitudinal nerve cords innervate these structures and appear to be involved in the process of somatic fusion, probably giving rise to the inter-specimen connections that later link the 2 central nervous systems in paired adult parasites. In the hindbody, there is extensive haptoral innervation associated with the developing clamps and small central hooks. Reactive neuronal components were found associated with the early stages of clamp development prior to connections being made with the extrinsic adductor muscle bundles. The muscle systems of the diporpa and juvenile stages comprise a lattice-like arrangement of circular, longitudinal and diagonal fibres that make up the body wall, together with buccal suckers, haptoral clamps and associated adductor muscles, and the transient ventral sucker. All have obvious importance to diporpae when they migrate over the gill and undertake body contact, torsion and fusion during the process of pairing. Behaviour during the pairing of diporpae is described.

We are developing Tetrahymena thermophila as a delivery system for recombinant vaccines against parasitic protozoa, including the common fish parasite, Ichthyophthirius multifiliis. T. thermophila cell lines expressing I. multifiliis genes under the control of a cadmium-inducible metallothionein gene promoter conferred strong protection against a lethal parasite challenge when administered parenterally to naïve fish. Nevertheless, given that heavy metals can be toxic to parasites, a question arose as to whether protection resulted from Cd residues carried over with the vaccine, rather than acquired immunity per se. To address this issue, we examined the sensitivity of I. multifiliis to Cd in vitro and determined Cd concentrations in different host tissues following i.p. injection of juvenile channel catfish with the recombinant vaccine. We found that CdCl2 at concentrations [ges ]50 ppb were lethal to I. multifiliis theronts in vitro. Furthermore, Cd concentrations were clearly elevated in fish tissues and reached levels equivalent to 74 ng/g wet weight (74 ppb) in the skin within 14 days of injection with recombinant T. thermophila. Nevertheless, fish injected with non-transformed Tetrahymena grown in the presence or absence of CdCl2 showed no significant difference in either relative survival or parasite load following direct challenge with I. multifiliis.

Psoroptes ovis, the causative agent of sheep scab, is an important ectoparasitic mite infecting sheep, goats and cattle. Infection is characterized by an extensive dermatitis, scab formation and intense itching. Initial focal lesions spread outwards, coalesce and may extend over the whole body. The host response to infestation has all the characteristics of an immediate-type hypersensitivity reaction but the mite antigens and allergens which initiate this response are almost completely undefined. Here, 507 randomly selected cDNAs derived from a mixed population of P. ovis were sequenced and the resultant nucleotide sequences subjected to Cluster analysis and Blast searches. This analysis yielded 280 clusters of which 49 had >1 sequence with 24 showing significant Blast X homology to another protein in the databases. There were 231 sequences which appeared on one occasion and 109 of these showed significant Blast X homology to other sequences in the databases. This analysis identified homologues of 9 different types of allergens which have been characterized in other allergic conditions such as responses to house dust mites. It also identified a number of cysteine proteases which may contribute to lesion development as well as several free-radical scavenging enzymes which may protect the mite from host immune effector responses.

The parasitic female of Strongyloides venezuelensis keeps invading the epithelial layer of the host intestinal mucosa. Upon invasion, it adheres to the surface of the intestinal epithelial cells with adhesion molecules secreted from the mouth. It has been demonstrated that S. venezuelensis are expelled from the intestine because mucosal mast cells inhibit the attachment of adult worms to the mucosal surface. In the present study, we generated specific antibodies against secreted adhesion molecules to investigate their function in vivo, because these molecules have been demonstrated only in vitro in spite of the importance in the infection processes. A mouse monoclonal antibody specific to S. venezuelensis adhesion molecules inhibited the attachment of adult worms to plastic dishes and the binding of adhesion molecules to rat intestinal epithelial cells. Immunohistochemical study revealed that adhesion molecules were produced by oesophageal glands and were continuously secreted in vivo to line the wall of the tunnels formed by adult worms in the intestinal mucosa. Our findings indicate that adhesion molecules play essential roles in the infection processes of S. venezuelensis in the host intestine.

Groups of nymphal Rhipicephalus appendiculatus Muguga, having a mean of 1 or 9 Theileria parva Muguga-infected salivary gland acini per tick, were kept under quasi-natural conditions at an altitude of 1950 m or 20°C at a relative humidity of 85% in the laboratory and their survival and infection prevalence and abundance determined over time. Theileria parva infections for both categories of ticks survived in the nymphal ticks for 50 or 26 weeks post salivary gland infection under quasi-natural or laboratory conditions respectively. There was a distinct decline in infections in the more heavily infected nymphae under both conditions of exposure, reflecting an apparent density dependence in parasite survival. Nymphal ticks having an average infection level of 1 infected salivary gland acinus per tick, survived for up to 69 or 65 weeks post-repletion under quasi-natural or the laboratory conditions respectively. Nymphae having an average infection level of 9 infected salivary gland acini per tick survived for a similar duration under each of the 2 conditions. The infection level of 9 infected salivary gland acini per tick did not seem to significantly affect the survival of the tick vector compared to those having an average of 1 infected salivary gland acinus per tick.

Here we report on the action of the novel cyclo-depsipeptide anthelmintic, emodepside, on the body wall muscle of the parasitic nematode, Ascaris suum. Emodepside caused (i) muscle relaxation, (ii) inhibition of muscle contraction elicited by either acetylcholine (ACh), or the neuropeptide, AF2 (KHEYLRFamide) and (iii) a rapid relaxation of muscle tonically contracted by ACh. The inhibitory action of emodepside on the response to ACh was not observed in a denervated muscle strip, indicating that it may exert this action through the nerve cord, and not directly on the muscle. Electrophysiological recordings showed emodepside elicited a Ca++-dependent hyperpolarization of muscle cells. Furthermore, the response to emodepside was dependent on extracellular K+, similar to the action of the inhibitory neuropeptides PF1 and PF2 (SDPNFLRFamide and SADPNFLRFamide). Thus emodepside may act at the neuromuscular junction to stimulate release of an inhibitory neurotransmitter or neuromodulator, with a similar action to the PF1/PF2 neuropeptides.

The use of naturally infected hosts in studies attempting to identify parasite-induced changes in host biology is problematical because it does not eliminate the possibility that infection may be a consequence, rather than a cause, of host trait variation. In addition, uncontrolled concomitant infections may confound results. In this study we experimentally infected individual laboratory-bred female three-spined sticklebacks Gasterosteus aculeatus L. with the pseudophyllidean cestode Schistocephalus solidus [Müller], and compared the morphology and growth patterns of infected females with sham-exposed controls over a 16-week period. Fish were fed a ration of 8% body weight per day. Non-invasive image analysis techniques allowed the growth of individual plerocercoids to be tracked in vivo throughout the course of infection, and patterns of host and parasite growth were determined. Females that developed infections diverged morphometrically from unexposed control females and exposed-uninfected females at 6 weeks post-infection, with the width of the body at the pectoral fins giving the earliest indication of infection success. When including the plerocercoid, infected females gained weight more quickly than controls, but when plerocercoid weight was removed this trend was reversed. There was no effect of infection on the increase in fish length. Plerocercoids grew at different rates in individual hosts, and exhibited measurable sustained weight increases of up to 10% per day. Final estimates of plerocercoid weight from morphometric analysis prior to autopsy were accurate to within +/−17% of actual plerocercoid weight. At autopsy, infected female sticklebacks had significantly lower perivisceral fat reserves but had developed significantly larger ovaries than controls. The results are discussed in relation to previous studies examining natural infections, and the value of utilizing experimental infections to examine ecological aspects of host–parasite interactions is discussed.

Most sylvatic species of Trichinella are known to have poor infectivity to rats, but in the present study oral administration of bile from other hosts appeared to modify this infectivity. A total of 75 rats were inoculated in groups of 25 with 3 species of Trichinella (T. spiralis, T. nativa, and T. nelsoni) and each group of rats was given per os daily doses of bile from pig, sheep, chicken and fox respectively (4×5 rats). As a control 1 group of 5 rats was given daily doses of water. Whereas, the addition of bile did not increase the establishment of T. spiralis, fox bile had a significant positive effect on the establishment of muscle larvae of T. nativa and T. nelsoni. Addition of bile to cultures of the same Trichinella species had an overall negative effect on the in vitro survival of larvae. The present observation that carnivore bile favours the establishment of sylvatic Trichinella may explain why carnivores are equally receptive to all Trichinella species.