In Guatemalan villages people commonly rear pigs, and both hosts may be infected with Ascaris. This study was designed to ask whether both humans and pigs are potential hosts in a single parasite transmission cycle in such villages, or alternatively, if there are two separate transmission cycles, one involving pigs and one involving human hosts. Parasites were collected from both host species from locations in the north and south of Guatemala. Allelic variation in the nuclear genome of Ascaris was measured using enzyme electrophoresis, while mitochondrial DNA (mtDNA) sequence variation was quantified using restriction mapping. Low levels of enzyme polymorphism were found in Ascaris, but allele frequencies at two loci, mannose phosphate isomerase and esterase, suggest that there is little gene exchange between parasite populations from humans and pigs. MtDNA haplotypes fall into two distinct clusters which differ in sequence by 3–4%; the two clusters broadly correspond to worms collected from humans and those collected from pigs. However, some parasites collected from humans have mtDNA characteristic of the ‘pig Ascaris’ haplotype cluster, while some parasites collected from pigs have mtDNA characteristic of the ’ haplotype cluster. These shared haplotypes are unlikely to represent contemporary cross-infection events. Patterns of phylogenetic similarity and geographical distribution of these haplotypes suggest, instead, that they are the result of two historical introgressions of mtDNA between the two host-associated Ascaris populations. The results clearly demonstrate that Ascaris from humans and pigs are involved in separate transmission cycles in Guatemala.

Ookinetes have been cultured in vitro using modifications to the method of Weiss & Vanderberg (1977). Significant improvements in technique were produced by culture in medium at pH 8.4 and at a blood dilution at or over 1/10. Ookinetes produced were infective to mosquitoes by membrane feeding techniques. Ultrastructural analyses were made of nuclear, cytoskeletal, crystalloid and microneme development. The first intranuclear division in the zygote has been recognized as meiosis. Chromosome condensation during prophase follows the classical stages of leptotene, zygotene and pachytene. Diplotene and diakinesis are not present-the synaptonemal complexes persist into metaphase 1. Chromosomes separate at anaphase and rapidly de-condense prior to telophase. We have not recognized a second meiotic division in the ookinete. The implication of these findings to the molecular and Mendelian organization of the parasite genome are discussed.

The Leishmania donovani complex is considered to be composed of 3 species; L. donovani, L. infantum and L. chagasi, although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene (mspC) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum, which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum/L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

Nucleotide sequences were obtained for the second internal transcribed spacer of the ribosomal gene repeat and for part of the mitochondrial cytochrome c oxidase subunit I gene from geographical isolates of Paragonimus westermani from Japan, China, Korea, Taiwan, the Philippines, peninsular Malaysia and Thailand. Sequences were obtained from several other species of Paragonimus for comparative purposes. Two groups were recognized within P. westermani: an NE group (China, Japan, Korea, Taiwan) which was relatively uniform and included both diploid and triploid forms, and a southern group (Malaysia, Thailand, Philippines), members of which were genetically distant from one another. According to both ITS2 and COI data, genetic distances among P. westermani isolates equalled or exceeded those between some distinct species of Paragonimus. The ITS2 sequences were conserved relative to COI sequences. Substitutions among the latter may be approaching saturation within the genus Paragonimus.

Two major cytoplasmic inclusions, multilaminate vesicles and discoid granules, are present in the tegument of Schistosoma mansoni. These are produced at separate Golgi apparatuses in the tegument cell bodies and move up to the surface by a combination of diffusion and fluid flow. The discoid granules contain neutral mucopolysaccharide and are believed to break down to form the ground substance of the tegument. The multilaminate vesicles are rich in phospholipid and the contents, at least superficially, resemble unit membranes. The multilaminate vesicles are believed to contribute their contents to the multilaminate surface of the worm which appears to be continually replaced. These observations are related to current ideas on membrane turnover and the ability of the worm to acquire a disguise of host erythrocyte glycolipid.

Resistance to the benzimidazole class of anthelmintics in nematodes of veterinary importance has a long history. Research into the mechanisms responsible for this resistance is subsequently at a more advanced stage than for other classes of anthelmintics. The principal mechanism of resistance to benzimidazoles is likely to involve changes in the primary structure of β-tubulins, the building blocks of microtubules. Specifically, point mutations in the β-tubulin isotype 1 gene leading to amino acid substitutions in codons 167, 198, and 200 of the protein have been associated with resistance in nematodes. These single nucleotide polymorphisms offer a means of detecting the presence of resistance within populations. In this mini-review, we focus on the prevalence and importance of these polymorphisms in three groups of nematodes: trichostrongylids, cyathostomins, and hookworms. A brief overview of existing strategies for genotyping single nucleotide polymorphisms is also presented. The CARS initiative hopes to exploit these known polymorphisms to further our understanding of the phenomenon of BZ resistance.

RNA interference (RNAi) has emerged as an invaluable gene-silencing tool for functional analysis in a wide variety of organisms, particularly the free-living model nematode Caenorhabditis elegans. An increasing number of studies have now described its application to plant parasitic nematodes. Genes expressed in a range of cell types are silenced when nematodes take up double stranded RNA (dsRNA) or short interfering RNAs (siRNAs) that elicit a systemic RNAi response. Despite many successful reports, there is still poor understanding of the range of factors that influence optimal gene silencing. Recent in vitro studies have highlighted significant variations in the RNAi phenotype that can occur with different dsRNA concentrations, construct size and duration of soaking. Discrepancies in methodology thwart efforts to reliably compare the efficacy of RNAi between different nematodes or target tissues. Nevertheless, RNAi has become an established experimental tool for plant parasitic nematodes and also offers the prospect of being developed into a novel control strategy when delivered from transgenic plants.

The biosynthesis and fate of 4 different dense granule proteins of Toxoplasma gondii were studied with 3 monoclonal antibodies raised against tachyzoites and 1 polyclonal antibody raised against a recombinant protein. These proteins have the following molecular weights: 27 kDa (GRA 1), 28 kDa (GRA 2), 30 kDa (GRA 3) and 40 kDa (GRA 4). All four proteins were found in dense granules by immunoelectron microscopy; in T. gondii-infected cells, they were found in the vacuolar network but, in addition, GRA 3 was also detected on the parasitophorous vacuole membrane. Therefore, dense granule contents undergo differential targeting when exocytosed in the parasitophorous vacuole. Metabolic labelling and immunoprecipitation showed that GRA 2 and GRA 3 were processed from lower molecular weight precursors, and that GRA 2 and GRA 4 incorporated [3H] glucosamine and are thus likely to be glycosylated.

Throughout evolution, enzymes and their metabolites have been highly conserved. Parasites are no exception to this and differ most markedly by the absence of metabolic pathways that are present in the mammalian host. In general, parasites are metabolically lazy and rely on the metabolism of the host both for a supply of prefabricated components such as purines, fatty acids, sterols and amino acids and for the removal of end-products. Nonetheless, parasites are metabolically highly sophisticated in that (1) they retain the genetic capacity to induce many pathways, when needed, and (2) they have developed complex mechanisms for their survival in the host. Certain unique features of the metabolism of trypanosomes, leishmania, malaria and anaerobic protozoa will be discussed. This will include (1) glycolysis and electron transport with reference to the unique organelles: the glycosome and the hydrogenosome, (2) purine salvage, pyrimidine biosynthesis and folic acid metabolism and (3) polyamine and thiol metabolism with special reference to the role of the unique metabolite of trypanosomes and leishmanias, trypanothione.

Circulating microRNAs (miRNAs) have received considerable attention as a novel class of biomarkers for the diagnosis of cancer and as signalling molecules in mediating intercellular communication. Schistosomes, the causative agents of schistosomiasis, live in the blood vessels of a mammalian host in the adult stage. In the present study, we characterized schistosome-specific small RNA populations in the plasma of rabbits infected with Schistosoma japonicum (S. japonicum) using a deep sequencing method and then identified five schistosome-specific miRNAs, including four known miRNAs (Bantam, miR-3479, miR-10 and miR-3096), and one novel miRNA (miR-0001, miRBase ID: sja-miR-8185). Four of the five schistosome-specific miRNAs were also detected by real-time RT–PCR in the plasma of S. japonicum-infected mice. In addition, our study indicated that schistosome Argonaute 2/3 may be an excretory-secretory (ES) protein. In summary, our findings are expected to provide useful information for further development of novel biomarkers for the diagnosis of schistosomiasis and also for deeper understanding of the mechanism of host–parasite interaction.

Actinomyces rhodnii has been isolated regularly from Rhodnius prolixus reared in the laboratory. It is not transmitted through the egg but is taken up by the young nymph from the environment; sometimes from the contaminated surface of the egg; more often perhaps from the dry excreta of other members of the species.

Rhodnius has been reared free from Actinomyces by sterilizing the surface of the egg and feeding with suitable precautions.

These sterile insects grow and moult normally until the 4th or 5th instar. Moulting is then delayed or may fail entirely in spite of repeated feeds of blood. Very few insects without Actinomyces become adult, and those few are almost certainly incapable of reproduction. Normal growth and moulting and egg production are resumed if the insects are re-infected with Actinomyces.

Populations of Bulinus globosus and Biomphalaria pfeifferi were studied in a river habitat in Zimbabwe over a period of 12 months. Data were obtained on the prevalences of infections of Schistosoma haematobium (also S. mattheei) and S. mansoni respectively. Population parameters showed the following patterns for both snail species. (1) A patchy distribution correlated with the distributions of aquatic plants. (2) Life-expectancies of only a few weeks. (3) Recruitment rates correlated with water temperature and showing a distinct seasonal peak. (4) Spatial variation in recruitment. (5) A redistribution of snails during the rainy season. Epidemiological parameters showed the following patterns. (1) Large seasonal variations in the prevalence of patent infections. (2) Evidence from size-prevalence curves that suggests a variable force-of-infection from man to snail, correlated with water temperature. (3) Prevalences of infection that were higher in the vicinity of (±60 m from) major water contact sites. Local prevalences of infection for B. globosus sometimes exceeded 50% and may have approached 100% if pre-patent infections are included. Snail numbers may limit transmission at these locations. Attention is drawn to the need to make field observations at an appropriate spatial scale and also to the implications for the effectiveness of focal snail control as a means of reducing transmission.

Two groups of H-2 congenic strains of mice were compared for their susceptibility to a primary infection with the nematode Trichuris muris. Mice of the BALB genetic background were markedly more resistant than mice of the BIO genetic background, as reflected by the rate of expulsion of T. muris from the large intestine. Within each of the two groups of H-2 congenic strains mice possessing the H-2k haplotype (BALB/k, B10.BR) were more susceptible to infection than mice expressing other haplotypes; B10 background strains expressing H-2b (B10) or H-2q (B10.G) alleles were the most resistant of the four congenic strains studied. Differential resistance was observed within three of the four BIO congenic strains and this is discussed in terms of rate of development of the protective immune response in relation to worm development. The results support the conclusion that both H-2-linked and non-H-2 genes play important roles in controlling the immune response which expels worms from the gut.

Neospora caninum is a recently identified coccidian parasite which was, until 1988, misdiagnosed as Toxoplasma gondii. It causes paralysis and death in dogs and neonatal mortality and abortion in cattle, sheep, goats and horses. The life-cycle of Neospora has not yet been elucidated. The only two stages identified so far are tissue cysts and intracellularly dividing tachyzoites. Very little is known about the biology of this species. We have set up a fluorescence-based adhesion/invasion assay in order to investigate the interaction of N. caninum tachyzoites with bovine aorta endothelial (BAE) cells in vitro. Treatment of both host cells and parasites with metabolic inhibitors determined the metabolic requirements for adhesion and invasion. Chemical and enzymatic modifications of parasite and endothelial cell surfaces were used in order to obtain information on the nature of cell surface components responsible for the interaction between parasite and host. Electron microscopical investigations defined the ultrastructural characteristics of the adhesion and invasion process, and provided information on the intracellular development of the parasites.

As the most primitive metazoan phylum, the Platyhelminthes occupies a unique position in nervous system evolution. Centrally, their nervous system consists of an archaic brain from which emanate one or more pairs of longitudinal nerve cords connected by commissures; peripherally, a diverse arrangement of nerve plexuses of varying complexity innervate the subsurface epithelial and muscle layers, and in the parasitic taxa they are most prominent in the musculature of the attachment organs and egg-forming apparatus. There is a range of neuronal-cell types, the majority being multi- and bipolar. The flatworm neuron is highly secretory and contains a heterogeneity of vesicular inclusions, dominated by densecored vesicles, whose contents may be released synaptically or by paracrine secretion for presumed delivery to target cells via the extracellular matrix. A wide range of sense organ types is present in flatworms, irrespective of life-styles. The repertoire of neuronal substances identified cytochemically includes all of the major candidate transmitters known in vertebrates. Two groups of native flatworm neuropeptides have been sequenced, neuropeptide F and FMRFamide-related peptides (FaRPs), and immunoreactivities for these have been localised in dense-cored neuronal vesicles in representatives of all major fiatworm groups. There is evidence of co-localisation of peptidergic and cholinergic elements; serotoninergic components generally occupy a separate set of neurons. The actions of neuronal substances in flatworms are largely undetermined, but FaRPs and 5-HT are known to be myoactive in all of the major groups, and there is immuno-cytochemical evidence that they have a role in the mechanism of egg assembly.

We report experimental data comparing the effects of Mesostigmatid mites and Ischnoceran lice on the reproductive performance of a single group of captive rock doves (Columba livia). Several components of host reproductive success were compared for the two groups, including number of eggs laid, hatching success, nestling growth rates, fledging success, post-fledging body mass and survival. Adult body mass and survival were also compared. There was a dramatic difference in the effects of the mites and lice. The former drove host reproductive success to zero, mainly by agitating adults and causing them to incubate eggs less faithfully. Nestling growth rates and post-fledging survival were also significantly reduced by mites. Lice showed no effect on reproductive success whatsoever, even though the feather damage they cause is known to have energetic consequences (Booth, Clayton & Block, 1993). Neither parasite had a significant effect on adult birds. Although Ischnocera are found on most species of birds, our results for lice constitute the first experimental test of the impact of Ischnocera on avian reproductive success (preliminary report by Clayton & Tompkins, 1994). We discuss reasons for the different effects of mites and lice, including the relationship of horizontal (mites) and vertical (lice) transmission to the evolution of virulence.

The anisakid nematodes morphologically corresponding with Pseudoterranova decipiens sensu lato (s.l.) (Krabbe, 1878) from different seal or sea lion hosts and geographical origins, previously identified as Pseudoterranova krabbei, P. decipiens (s.s.), P. bulbosa, P. azarasi and P. cattani by multilocus enzyme electrophoresis, were characterized using a DNA approach. Also a population of P. decipiens (s.l.) from Chaenocephalus aceratus, the blackfin icefish, from Antarctica and another from Osmerus eperlanus, the European smelt, from Germany were included in the study. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by PCR from individual nematodes and analysed by single-strand conformation polymorphism (SSCP), followed by selective sequencing. While no variation in single-stranded ITS-1 and ITS-2 profiles was detected among samples representing each of the species or populations (with the exception of slight microheterogeneity), SSCP analysis of the ITS-2 amplicons allowed the unequivocal differentiation of all of the 5 sibling species of P. decipiens (s.l.) examined, which was supported by sequence differences in ITS rDNA. Samples representing the P. decipiens (s.l.) population from O. eperlanus had the same SSCP profile as those of P. decipiens (s.s.), which was supported by a lack of nucleotide difference in the ITS between them, suggesting that the former represented P. decipiens (s.s.). Based on SSCP results and ITS sequence data, P. decipiens (s.l.) from C. aceratus was genetically most distinct with respect to all other members of Pseudoterranova examined, which indicated that it may represent P. decipiens E (based on geographical origin) or a distinct species. These findings and the molecular approach taken should have important implications for studying the life-cycles, transmission patterns, epidemiology and population genetics of these anisakid nematodes, and the diagnosis of their infections.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

All three species of parasites found in sticklebacks from Priddy had overdispersed distributions, similar in shape to host–parasite distributions described by other authors.

The distributions of both Echinorhynchus clavula and Diplostomum gasterostei were fitted closely by the negative binomial. The values of k were similar for both species, being always less than unity; the Diplostomum distributions were more overdispersed and therefore had higher values of p.

The Schistocephalus distributions were mostly described by the log normal, but the high percentage infection and relatively low overdispersion meant that the fit was often not close. There was also some truncation at the upper end.

The relationship between the parameters of the negative binomial (p and k) and the shape and parameters of the original distribution have been investigated, both theoretically and with respect to the distributions of the stickleback parasites.In general p varied as the overdispersion and k as the percentage infection.

The advantages of an overdispersed distribution to the host and parasite populations are discussed and related to the life cycles of the parasites.

I am most grateful to Dr H. D. Crofton for his help and encouragement during this study, and to the staff of the Computer Unit, University of Bristol for their assistance with programming problems. This work was supported by a Science Research Council research studentship and a NATO studentship.

1. An account is given of the history, synonymy and host-range of Toxocara cati.

2. Eggs of T. cati were fed to various animals, and the second-stage larvae were found in the tissues of earthworms, cockroaches, chickens, mice, dogs, lambs and cats.

3. Cats were successfully infected by feeding eggs of T. cati and by feeding mice harbouring larvae in the tissues.

4. In egg-infected cats the larvae were found in the liver, lungs, muscles and tracheal washings as well as in the digestive tract, indicating that they migrate through the tissues.

5. In mice the larvae were found in the liver, lungs and muscles, but did not reach the alimentary tract. Changes occurred in the mouth region of the larvae in mouse tissues between 8 and 11 days after infection, but no actual moult was observed. No significant growth of the larvae was observed in mouse tissues.

6. In mouse-infected cats the larvae were mostly confined to the wall and contents of the digestive tract.

7. It appeared that the second moult occurred in the stomach wall of egg-infected cats at 3–10 days after infection; moulting larvae measuring 0·353–0·423 mm. in length; in a mouse-infected cat moulting larvae (length 0·459–0·765 mm.) were observed at 6 days after infection.

8. In both egg- and mouse-infected cats the third-stage larvae grew in the stomach wall and moulted for the third time at a length of 0·9–1·2 mm. This occurred at about 10 days in mouse-infected cats and at about 19 days in egg-infected cats.

9. The smallest fourth-stage larvae were found in the stomach contents whence they passed into the intestine. Probably they are at first attached to the wall and later become free in the intestinal contents.

10. The fourth-stage larvae have lips resembling the adult; sexual differentiation occurs during this stage, being first evident at 1·5 mm.

11. The fourth moult occurs in the intestine at a length of approximately 4·5–5·5 mm.

12. In the adult stage the lateral alae gradually disappear and the cervical alae reach the adult form at a length of approximately 45 mm. The smallest female observed containing eggs in the vagina measured 55 mm. in length. Eggs were first observed in the faeces at 56 days after infection by eggs.

13. The evidence from experimental and natural infection indicated that infection of cats with T. cati always takes place after birth.

14. No larvae were found in the alimentary tract of dogs following experimental infection either with eggs or with infected mice.

15. The relationship of the life history of T. cati to that of other ascaridid species, to the feeding habits of the definitive hosts, and in relation to the occurrence of human infection with this parasite is discussed.