1. The formation of Opalina cysts in Rana temporaria was compared, during September to December, in groups of untreated frogs exposed to normal seasonal fluctuations in temperature, and treated frogs kept at room temperature. Cyst formation was enhanced, to the same degree, in frogs injected with saline or chorionic gonadotrophin (Pregnyl), and, to a higher degree, in frogs injected with frog pituitaries.

2. Observations were made, during September to December, on the state of the testes in Rana temporaria infected with Opalina. The presence of Opalina cysts was most closely correlated with spermiation.

3. Possible factors affecting the formation of opalinid cysts are discussed. It is concluded that the seasonal production of cysts by Opalina is not due to cyclical changes inherent in the protozoa. The factor or factors provoking encystation appear to be correlated with the reproductive cycle of the host, and may be the gonadal hormones.

In Trypanosoma brucei brucei, an invariant surface glycoprotein of molecular weight 75 kDa (ISG75) is uniformly distributed over the surface of a trypanosome and is specific for bloodstream-form parasites. For the other taxa of the Trypanozoon subgenus no data about this surface molecule are available. Therefore, we investigated the ISG75 in the genomes of several pathogenic Trypanozoon by Southern blot, PCR and RT-PCR and sequence analysisNucleotide sequence data reported in this paper are available in the GeneBank™, EMBL and DDBJ databases under the Accession numbers DQ200175-DQ200256.. This study reveals that (i) all members of the Trypanozoon subgenus, i.e. T. b. brucei, T. b. gambiense, T. b. rhodesiense, T. evansi and T. equiperdum, harbour ISG75 as multiple gene copies with at least 4–16 copies per genome; (ii) ISG75 gDNA and cDNA sequences are distributed in 2 groups that share at least 75% and 77% identity respectively; (iii) sequences from both groups are transcribed in all species and subspecies of the Trypanozoon subgenus; (iv) the main differences between group I and group II are located in the variable region at the amino-terminus of the putative proteins; (v) however, all the sequences in both groups have some well-conserved features, such as the cysteine residues, an amino-terminal cleavable signal peptide, a single α-helix transmembrane domain and a cytoplasmic domain at the carboxy-terminus.

Two behaviour patterns which could bring about clumping in N. brasiliensis, sexual attraction and thigmokinesis, were investigated. A T-tube choice-chamber to facilitate an in vitro study of sexual attraction is described. A method for studying aggregation is also outlined. Male N. brasiliensis were shown to be attracted to female worms, positive sexual attraction. Both sexes were shown to exhibit strong thigmokinetic responses, and possible functions of such behaviour are discussed.

I am grateful to Professors J. Brough and D. Bellamy for providing the facilities during the course of this study. I should also like to thank Dr E.. A. Hammond for his supervision, help and criticism throughout the project. This work was carried out during the tenure of a Scientific Research Council Studentship.

Neospora caninum is a commonly diagnosed cause of reproductive losses in farmed ruminants worldwide. This study examined 495 and 308 samples (brain, heart and placenta) which were collected from 455 and 119 aborted cattle and sheep fetuses, respectively. DNA was extracted and a nested Neospora ITS1 PCR was performed on all samples. The results showed that for bovine fetuses 79/449 brain [17.6% (14.2–21.4)], 7/25 heart [28.0% (12.1–49.4)] and 5/21 placenta [23.8% (8.2–47.2)] were PCR positive for the presence of Neospora DNA. Overall 82/455 [18.0% (14.6–21.7)] of the bovine fetuses tested positive for the presence of N. caninum DNA in at least one sample. None (0/308) of the ovine fetal samples tested positive for the presence of Neospora DNA in any of the tissues tested. The results show that N. caninum was associated with fetal losses in cattle (distributed across South-West Scotland), compared to sheep in the same geographical areas where no parasite DNA was found. Neospora is well distributed amongst cattle in South-West Scotland and is the potential cause of serious economic losses to the Scottish cattle farming community; however, it does not appear to be a problem amongst the Scottish sheep flocks.

East Africa is a global hot spot for the diversity of ixodid ticks. As ectoparasites and as vectors of pathogens, ticks negatively affect the well-being of humans, livestock and wildlife. To prevent tick infestations, livestock owners and managers typically treat livestock with acaricides that kill ticks when they attempt to feed on livestock hosts. Because of the costs of preventing and mitigating tick parasitism, predicting where and when ticks will be abundant is an important challenge in this region. We used a 7-year monthly record of tick abundance on large experimental plots to assess the effects of rainfall, wildlife and cattle on larvae, nymphs and adults of two common tick species, Rhipicephalus pulchellus and Rhipicephalus praetextatus. Nymphal and adult ticks were more abundant when there had been high cumulative rainfall in the prior months. They were less abundant when cattle were present than when only large wild mammals were. Larval abundance was not affected by the presence of cattle, and larvae did not appear to be sensitive to rainfall in prior months, though they were less abundant in our surveys when rainfall was high in the sampling month. The challenges of managing ticks in this region are being exacerbated rapidly by changes in rainfall patterns wrought by climate change, and by overall increases in livestock, making efforts to predict the impacts of these drivers all the more pressing.

Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

Bioactive molecules isolated from plants are promising sources for the development of new therapies against leishmaniasis. We investigated the leishmanicidal activity of cariphenone A (1), isouliginosin B (2) and uliginosin B (3) isolated from Hypericum species. Promastigotes and amastigotes of Leishmania amazonensis were incubated with compounds 1–3 at concentrations 1–100 µm for 48 h. The anti-promastigote effect of compounds was also tested in combinations. The cytotoxicity against macrophages and human erythrocytes were determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method and hemolysis assay, respectively. The compounds 1–3 showed high leishmanicidal activity against promastigotes, IC50 values of 10.5, 17.5 and 11.3 µm, respectively. Synergistic interactions were found to the associations of compounds 1 and 2 [Σ fractional inhibitory concentration (FIC) = 0.41], and 2 and 3 (ΣFIC = 0.28) on promastigotes. All Hypericum compounds induced mitochondrial hyperpolarization and reactive oxygen species production in promastigotes. The compounds showed low cytotoxicity toward mammalian cells, high selectivity index and killed intracellular amastigotes probably mediated by oxidative stress. These results indicate that these compounds are promising candidates for the development of drugs against leishmaniasis.

Echinococcus granulosus sensu latu (s.l.) and Taenia hydatigena are common parasites of ruminant intermediate hosts in the Balkans. Transmission is linked mainly to home slaughtering and the feeding of infected organs to dogs. In Kosovo, many old sheep are slaughtered particularly during Eid al-Adha (Feast of Sacrifice). To determine whether this tradition could affect parasite transmission, we compared the probability of 504 dogs to contract taenid infections after deworming during one period before Eid al-Adha and a similar period beginning with this event. Initially, taeniid eggs were detected in 6·2% (CI 4·2–8·6) of the dogs. The prevalence before Eid al-Adha was significantly lower (1·2%, CI 0·4–2·6) as compared with the prevalence after the event (4·3%, CI 2·6–6·3). A comparable trend was apparent at species level for T. hydatigena and E. granulosus. These results indicate that the pronounced increase of taeniid infections, including E. granulosus s.l., after Eid al-Adha is linked to traditional home slaughtering that occurs during this celebration. This particular epidemiological situation provides an opportunity for implementing focussed control activities.

Habitat fragmentation can adversely impact biodiversity, although where remnant fragments of natural vegetation provide favourable conditions the negative effects of fragmentation may be mitigated. Host-parasite systems in fragmented areas have only recently been examined, with parasites generally showing higher prevalence and richness in fragments, mediated by changes in host density. However, the effect of fragmentation on parasite body size and fecundity remains poorly investigated. Thus, here we compared the body size and condition of a generalist rodent host, Rhabdomys pumilio and the body size of 2 common flea species between pristine natural areas and remnant fragments within agriculture areas. Host body length, weight and body condition values were significantly larger in fragments than in pristine natural vegetation. Listropsylla agrippinae fleas showed the same pattern, being significantly larger in fragments, while Chiastopsylla rossi fleas did not differ in size between fragments and natural areas. The differential response of the 2 flea species may reflect the strength of association between the host and parasite, with the former spending a greater proportion of its lifespan on the host. Therefore, in this study agriculture fragments provide better conditions for both an opportunistic rodent and a closely associated flea species.

This study provides a morphological and phylogenetic characterization of two novel species of the order Haplosporida (Haplosporidium carcini n. sp., and H. cranc n. sp.) infecting the common shore crab Carcinus maenas collected at one location in Swansea Bay, South Wales, UK. Both parasites were observed in the haemolymph, gills and hepatopancreas. The prevalence of clinical infections (i.e. parasites seen directly in fresh haemolymph preparations) was low, at ~1%, whereas subclinical levels, detected by polymerase chain reaction, were slightly higher at ~2%. Although no spores were found in any of the infected crabs examined histologically (n = 334), the morphology of monokaryotic and dikaryotic unicellular stages of the parasites enabled differentiation between the two new species. Phylogenetic analyses of the new species based on the small subunit (SSU) rDNA gene placed H. cranc in a clade of otherwise uncharacterized environmental sequences from marine samples, and H. carcini in a clade with other crustacean-associated lineages.

In the last issue of Parasitology a description is given by Wenyon (5) of the developmental cycle of Leucocytozoon canis in the tick Rhipicephalus sanguineus. Wenyon refers to the description of the development given by me (2), but rightly points out that one stage, that of the large compound cysts, was overlooked. The surmise, however, that these large and easily ruptured cysts were overlooked owing to their having been broken up in the preparation of films is only part of the truth. A full explanation involves facts in regard to the development of this parasite which have not been touched upon by Wenyon; it is possible, as will be seen shortly, that Wenyon's description of the cycle, like mine, involved a considerable error. In this respect, as a result of work not yet published, I am able to supplement what has been written on the subject.

The technique of Random Amplification Polymorphic DNA allows fragments of the genome to be amplified by means of polymerase chain reaction (PCR) without previous knowledge of their sequences. The protozoa of the genus Leishmania present great genetic variability, making it difficult to characterize the different species. A method is developed with a single 10-mers long primer, which allows the species L. braziliensis, L. mexicana, L. infantum, L. tropica, L. chagasi, L. amazonensis and L. major to be differentiated. These products amplified by RAPD have also facilitated the design of some primers that amplify L. braziliensis DNA exclusively.

Anthropogenic activities are having profound impacts on species interactions, with further consequences for populations and communities. We investigated the influence that anthropogenic eutrophication has on the prevalence of the parasitic tapeworm Schistocephalus solidus in threespine stickleback Gasterosteus aculeatus populations. We caught stickleback from four areas along the coast of Finland, and within each area from one undisturbed and one eutrophied habitat. We found the prevalence of the parasite to be lower in the eutrophied habitats at the start of the breeding season, probably because of fewer piscivorous birds that transmit the parasite. However, while the prevalence of the parasite declined across the season in the undisturbed habitat, it did less so in eutrophied habitats. We discuss different processes that could be behind the differences, such as lower predation rate on infected fish, higher food availability and less dispersal in eutrophied habitats. We found no effect of eutrophication on the proportion of infected stickleback that entered reproductive condition. Together with earlier findings, this suggests that eutrophication increases the proportion of infected stickleback that reproduce. This could promote the evolution of less parasite resistant populations, with potential consequences for the viability of the interacting parties of the host–parasite system.

Characteristics of four species of Eimeria isolated from Apodemus sylvaticus are described. From oocyst morphology, three are identified as E. apionodes, E. hungaryensis and E. uptoni, the fourth being unnamed. Cloning demonstrated that oocysts of E. hungaryensis were polymorphic. Previous literature relating to the taxonomy is discussed and discrepancies outlined. Species isolated from A. sylvaticus were not transmissible to Clethrionomys glareolus, but both E. hungaryensis and E. apionodes have been passaged through immunosuppressed laboratory mice, the former species more than 16 times. In both A. sylvaticus and immunosuppressed laboratory mice, endogenous development of E. hungaryensis occurred mainly in enterocytes near the tips of the villi in the first half of the small intestine, with a few parasites in the rest of the small intestine and into the large intestine. The pre-patent period was 2 days in both hosts but oocyst output was higher in the natural hosts. E. apionodes parasitized enterocytes on the sides or at the base of the villi, mainly in the last 90% of the small intestine with a few parasites in the large intestine. The pre-patent period was 7 days. Parasites, probably E. hungaryensis and E. apionodes, were also isolated from A. flavicollis.

Neospora caninum is a protozoan parasite, primarily associated with bovine abortion. The only definitive hosts discovered to date are carnivores. This study aimed to identify the role of mammalian carnivores in the epidemiology of bovine neosporosis. A sample bank of serum, fecal and brain samples was established: American mink (Mustela vison), red foxes (Vulpes vulpes), pine martens (Martes martes), badgers (Meles meles), stoats (Mustela erminea), otters (Lutra lutra) and feral ferrets (Mustela putorius). Approximately 1% of mink and 1% of fox samples were positive by IFAT. According to PCR analysis of DNA extracted from brain tissue, 3% of the mink, 4% of the otters and 6% of the foxes examined were infected with N. caninum. All fecal samples tested negative for N. caninum DNA (n = 311), suggesting that the species that tested positive were intermediate not definitive hosts. This is the first time that tissues from mustelids have tested positive for N. caninum. The need to test 2 relatively large (∼200 mg) targeted parts of the brain to avoid false negatives was also identified. The relatively low prevalence of N. caninum in Irish carnivores suggests that the local ecology of a species has an important influence on its epidemiological role.

In Ticks (Part ii. p. 314) various cases are cited by me in which Ixodes in the larval, nymphal and adult stages have been found lodged beneath the skin of the host: Dubreuilh (1838), Van Beneden (1883) and Blanchard (1891) record cases in man where ticks were found either in a pustule or in cysts beneath the skin. In Blanchard's case a living I. ricinus (♀), 8 mm. in length, was excised from the patient, in whom it caused a subcutaneous tumour about the size of a nut which had appeared some weeks before. Mégnin (1867 and 1892) found (?) ricinus nymphs beneath the scabs covering pustules on the legs of a horse at Versailles. Kossel, Schütz, Weber and Miessner (1903) state that ricinus nymphs and larvae occasionally bore themselves beneath the skin of cattle.

Infection of the desert toad, Scaphiopus couchii, by the monogenean Pseudodiplorchis americanus involves 2 principal sites: post-invasion juveniles reside in the respiratory tract for 1 month before migrating to the urinary bladder where they reach sexual maturity and may live up to 4 years. While previous work has demonstrated the long-term impact on host condition of the blood-feeding adults, this study assesses pathological effects of the short-term pulmonary infection. Lung ultrastructure was compared in toads (i) maintained in captivity for 1 year without invasion, and (ii) experimentally infected with 50–300 juveniles/host, equivalent to burdens in the wild, and examined 23–44 days p.i. Typically, the alveolar lining of S. couchii is composed of a single cell type with characteristics of both Type I and Type II pneumocytes. However, infected lung tissue exhibited an inflammatory reaction with epithelial cell vacuolation, interstitial oedema, and an increase of alveolar exudate, leucocytes and fibrous tissue. Accompanying a post-infection increase in host immune cells in the lungs, there was evidence of reciprocal tegumental damage to the parasites. Lung epithelium of toads free of infection for 1 year exhibited scar tissue representing a residual effect of past infection. The pathological consequences of P. americanus infection therefore have 2 components. Acute lung infection coincides with the host's brief activity season: impaired respiratory function could compromise feeding and accumulation of reserves and hence ability to survive following a 10 month period of hibernation. Additionally, adult toads are normally exposed annually to re-infection and may accumulate chronic lung damage with extended effects on host survival.

Branchiobdellidans or crayfish worms are clitellate annelids and ectosymbionts of freshwater crayfish. An investigation of branchiobdellidan infestation was undertaken in a population of endangered white-clawed crayfish (Austropotamobius pallipes) in the river Aire, UK. Thirty two percent of animals were infested either by the adult parasite or their cocoons (n=107). Parasite burden increased with host size, but did not differ with sex. Observations of crayfish gill tissue revealed a strong positive relationship between melanization of filaments and parasite prevalence and burden. Taxonomic identification revealed that 1 species of branchiobdellidan was present, Branchiobdella astaci. The first sequences were generated for this species and phylogenetically analysed alongside published sequences for 5 other branchiobdellidan species in Europe. The position of B. astaci within the genus Branchiobdella was confirmed, and it was found to cluster as a sister group to B. parasita.

The simple cylindroid pharynx of Ascaris lumbricoides has been shown to be characterized by a pharyngo-intestinal valve which is composed of two sets of valves. This is the only valve structure in the pharynx of this nematode. The pharynges of Oxyuris equi, Aplectana brevicaudata and Panagrellus silusiae have a series of valve structures along the lengths of the organs. All these types have a valve at the anterior end of the corpus, corrugated ‘valves’ in the posterior bulb, and pharyngo-intestinal valves. A valve between the corpus and the isthmus has been shown in Oxyuris.

In all the nematodes studied the pharyngo-intestinal valve is characterized by an overlapping of the muscle fibres associated with the valve and the main musculature of the pharynx that immediately precedes them. It has been suggested that the pharyngo-intestinal valves of the types studied may have evolved from the type of valve found in the Enoplina.

The marginal tissues of the pharynx of Ascaris lumbricoides have been shown to be composed of three types of tissues. It has been suggested that the marginal tissues have a suspensory and skeletal function. The suspensory strands subserve the first function, while the investing strands and the associated fibrous layer provide a longitudinal skeletal structure in the pharynx. In Oxyuris no fibrous layer is associated with the marginal tissues.

I would like to thank Professor J. E. Harris and Dr H. D. Crofton for their help and criticism during the course of this work, which was carried out during the tenure of a Postgraduate Studentship awarded by the Agricultural Research Council.

LACK (Leishmania analogue of the receptor kinase C) is a conserved protein in protozoans of the genus Leishmania which is associated with the immunopathogenesis and susceptibility of BALB/c mice to L. major infection. Previously, we demonstrated that intranasal immunization with a plasmid carrying the LACK gene of Leishmania infantum (LACK-DNA) promotes protective immunity in BALB/c mice against Leishmania amazonensis and Leishmania chagasi. In the present study, we investigated the protective immunity achieved in hamsters intranasally vaccinated with 2 doses of LACK-DNA (30 μg). Compared with controls (PBS and pCI-neo plasmid), animals vaccinated with LACK-DNA showed significant reduction in parasite loads in the spleen and liver, increased lymphoproliferative response and increased nitric oxide (NO) production by parasite antigen-stimulated splenocytes. Furthermore, hamsters vaccinated with LACK-DNA presented high IgG and IgG2a serum levels when compared to control animals. Our results showed that intranasal vaccination with LACK-DNA promotes protective immune responses in hamsters and demonstrated the broad spectrum of intranasal LACK-DNA efficacy in different host species, confirming previous results in murine cutaneous and visceral leishmaniasis.