To study the prevalence of the polymorphism in position 200 of the beta-tubulin gene in the mechanism of benzimidazole (BZ) resistance in cyathostomes of horses, an allele-specific PCR was used to detect the genotype of individuals of BZ-susceptible and BZ-resistant populations. The molecular analysis of 100 adults recovered from an anthelmintic-naïve horse revealed 80% homozygous TTC/TTC individuals, 17% heterozygous TTC/TAC and 3% homozygous TAC/TAC. A naturally infected horse was treated with increasing fenbendazole (FBZ) dosages to select a BZ-resistant population of cyathostomes. The PCR based analysis of 3rd-stage larvae (L3) during the experiment revealed a decrease of the homozygous TTC/TTC genotype and an increase in heterozygous TTC/TAC and homozygous TAC/TAC individuals. After treatment 42·3% of the adults (n=104) were homozygous TTC/TTC, 55·8% were heterozygous TTC/TAC and only 1·9% showed the homozygous genotype TAC/TAC. The results of the molecular analysis lead to the proposal that polymorphism within codon 200 is not the only reason for the development of BZ resistance in small strongyles.

Experiments were conducted to investigate the potential for transmission of Orientia tsutsugamushi, the aetiological agent for scrub typhus, when naturally infected mite larvae were co-feeding with uninfected larvae. Larvae from colonies of Leptotrombidium deliense and L. imphalum infected with O. tsutsugamushi were used. Transmission of O. tsutsugamushi to previously uninfected L. deliense and Blankaartia acuscutellaris co-fed with infected L. deliense was shown to occur. The overall minimum rate of acquisition was 1·6% (4/258) for L. deliense and 2·5% (3/119) for B. acuscutellaris. When individual infected L. deliense were co-fed with B. acuscutellaris acquisition of O. tsutsugamushi was not detected. However, when 4 and 8 infected larvae were co-fed with B. acuscutellaris acquisition of O. tsutsugamushi was detected. Transmission of O. tsutsugamushi was not observed when uninfected L. deliense were co-fed with infected L. imphalum. This novel transmission route may explain the occurrence of rickettsiae in genera other than Leptotrombidium spp, which are considered to be the main vectors of O. tsutsugamushi.

Vole tuberculosis (TB; Mycobacterium microti) is an understudied endemic infection. Despite progressing slowly, it causes severe clinical pathology and overt symptoms in its rodent host. TB was monitored for 2 years in wild field voles in Kielder Forest, UK. The prevalence of characteristic cutaneous TB lesions was monitored longitudinally at 4 sites, with individuals live-trapped and repeatedly monitored. A prevalence of 5·2% of individuals with lesions was recorded (n=2791). In a cross-sectional study, 27 sites were monitored bi-annually, with TB assessed by post-mortem examination for macroscopic lesions, and by culture and histopathology. Seventy-nine voles (10·78%; n=733) were positive for mycobacteria, with the highest prevalence in spring (13·15%; n=327). TB prevalence varied, with between 0% and 50% of voles infected per site. Prevalence increased with age (mass), and apparent seasonality was due to a higher proportion of older animals in spring. Survival analysis supported this result, with cutaneous lesions only manifesting in the advanced stages of infection, and therefore only being found on older voles. The body condition of individuals with lesions declined at the time when the lesion was first recorded, when compared to individuals without lesions, suggesting there may be an acute phase of infection during its advanced stage. Although predicted survival following the appearance of a cutaneous lesion was lower than for uninfected individuals, this was not significant.

Climate change, in particular rising temperature, is suspected to be a major driver for the emergence of many wildlife diseases. Proliferative kidney disease of salmonids, caused by the myxozoan Tetracapsuloides bryosalmonae, was used to evaluate how temperature dependence of host–parasite interactions modulates disease emergence. Brown trout (Salmo trutta fario) kept at 12 and 15 °C, were experimentally infected with T. bryosalmonae. Parasite development in the fish host and release of spores were quantified simultaneously to unravel parasite transmission potential from the vertebrate to the invertebrate host. A change to a stable plateau in infection intensity of the kidney coincided with a threshold at which spore shedding commenced. This onset of parasite release was delayed at the low temperature in accordance with reaching this infection intensity threshold, but the amount of spores released was irrespective of temperature. The production of parasite transmission stages declined with time. In conclusion, elevated temperature modifies the parasite transmission opportunities by increasing the duration of transmission stage production, which may affect the spread and establishment of the parasite in a wider range of rivers.

Despite years of investigation into triclabendazole (TCBZ) resistance in Fasciola hepatica, the genetic mechanisms responsible remain unknown. Extensive analysis of multiple triclabendazole-susceptible and -resistant isolates using a combination of experimental in vivo and in vitro approaches has been carried out, yet few, if any, genes have been demonstrated experimentally to be associated with resistance phenotypes in the field. In this review we summarize the current understanding of TCBZ resistance from the approaches employed to date. We report the current genomic and genetic resources for F. hepatica that are available to facilitate novel functional genomics and genetic experiments for this parasite in the future. Finally, we describe our own non-biased approach to mapping the major genetic loci involved in conferring TCBZ resistance in F. hepatica.

Three hymenolepidid tapeworms, Hymenolepis diminuta, H. nana and H. microstoma, are commonly maintained in laboratory rodents and used in many experimental model systems of tapeworm infections. We examined partial sequences from the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences to infer phylogenetic relationships of the 3 hymenolepidid species. Parts of the CO1 gene and ITS2 were amplified by PCR and sequenced directly. The CO1 gene sequence obtained was the same in length (391 bp) among all specimens. In the case of ITS2, however, several insertions and deletions were detected (671–741 bp) not only among species but also between an American isolate and a Japanese isolate of H. diminuta. Percentage nucleotide differences between H. diminuta and H. microstoma, or H. diminuta and H. nana were 16·6–18·2% for the CO1 gene and 21·3–22·9% for ITS2. The differences in both sequences between H. microstoma and H. nana were about 14%. Phylogenetic trees inferred from both of the nucleotide sequences showed similar topology, and suggest that H. diminuta may have diverged from the common ancestral line the earliest, and that H. nana is closer to H. microstoma than to H. diminuta.

The human pathogenic nematode Strongyloides stercoralis infects approximately 30–100 million people worldwide. Analysis of the adaptive immune response to S. stercoralis beyond descriptive studies is challenging, as no murine model for the complete infection cycle is available. However, the combined employment of different models each capable of modelling some features of S. stercoralis life cycle and pathology has advanced our understanding of the immunological mechanisms involved in host defence. Here we review: (i) studies using S. stercoralis third stage larvae implanted in diffusion chambers in the subcutaneous tissue of mice that allow analysis of the immune response to the human pathogenic Strongyloides species; (ii) studies using Strongyloides ratti and Strongyloides venezuelensis that infect mice and rats to extend the analysis to the parasites intestinal life stage and (iii) studies using S. stercoralis infected gerbils to analyse the hyperinfection syndrome, a severe complication of human strongyloidiasis that is not induced by rodent specific Strongyloides spp. We provide an overview of the information accumulated so far showing that Strongyloides spp. elicits a classical Th2 response that culminates in different, site specific, effector functions leading to either entrapment and killing of larvae in the tissues or expulsion of parasitic adults from the intestine.

The uptake in vitro of various substances by Brugia pahangi was investigated using infective larvae obtained from Aedes aegypti and worms removed from Meriones unguiculatus at 2, 3, 10, 20 and 90 days post-infection. Worms incubated in growth medium 199 containing 1% Trypan blue possessed demonstrable dye in the oral orifice, the anterior oesophageal lumen and the external openings of the vulva and the cloaca or anus but the dye was not found in the gut lumen even after incubation for 24 h. No uptake of ferritin particles into the intestine of the worms was found and no fluorescence could be demonstrated in the gut lumen of worms incubated in medium containing 50% (v/v) fluorescein isothiocyanate-conjugated calf serum for up to 24 h. Trypan blue uptake by the gut of Aspiculuris tetraptera was clearly observed after incubation for several hours. The uptake of D-glucose and L-leucine by B. pahangi was demonstrated using autoradiographic and scintillation counting techniques and incorporation into worm tissues was detected. Glucose was found to be readily incorporated in the apical, glycogen-rich areas of the myocytes of worms of all ages studied and in the uterine epithelium of the adult female. In contrast, a lower incorporation of D-glucose was found in the eggs, embryos and vas deferens and especially in the gut. The incorporation of L-leucine occurred throughout the tissue of the worms during a 30 mm incubation. Labelling was also located over the surface of the cuticle of the worms, when incubated for a period of 15 to 60 mm in L-[3H]leucine. Scintillation counting techniques demonstrated that there was no uptake of 14C-labelled L-glucose or sucrose by B. pahangi. The data presented on the uptake in vitro of nutrients or other compounds by infective larvae and adult stages of B. pahangi did not demonstrate an intestinal route of uptake but indicated that the transcuticular route of uptake may be employed.

Experimental work has established that a sexual process can occur in African trypanosomes (Jenni, Marti, Schweizer, Betschart, Le Page, Wells, Tait, Paindavoine, Pays & Steinert, 1986; Paindavoine, Zampetti-Bosseler, Pays, Schweizer, Guyaux, Jenni & Steinert, 1986; Tait, personal communication). However, the role of the process in natural populations of trypanosomes is poorly understood. This paper considers what information can be gained from analyses of isoenzyme polymorphism. A cladistic approach is used to help determine whether trypanosome diversity could have been produced by mutation alone. When applied to three East African populations of Trypanosoma brucei it provides evidence that some diversity has arisen through a sexual process; this explains the variation observed within a locality and can account for the evolution of differences between localities. However, the extent to which genetic exchange currently operates is less clear. Analysis of genotype frequencies indicates that agreements with Hardy-Weinberg expectations can be obtained even if genetic exchange exerted no influence over genotype frequencies. Moreover, analysis of joint locus frequencies reveals disequilibrium between loci and that trypanosome populations may be lacking several genotype combinations. Thus, genetic exchange may not occur sufficiently frequently, or in such a way as to break up associations between loci. The relevance of these observations to the evolution of strain differences within T. brucei is discussed.

Steinernema carpocapsae (Rhabditida: Steinernematidae) host recognition behaviour was assessed and compared with 2 measures of host suitability. Previous research showed that S. carpocapsae infective juveniles respond to host cues in a hierarchical order, with attraction to Galleria mellonella volatiles being stimulated by contact with G. mellonella cuticle. We measured host recognition behaviour by calculating the percentage response of S. carpocapsae infective juveniles to volatiles produced by G. mellonella last instars after the nematodes were exposed to the cuticle of 11 candidate arthropod hosts and 2 control surfaces. Host suitability was measured by nematode-induced mortality to candidate hosts at 2 nematode doses and the level of reproduction supported by each host. The highest recognition response was scored for Agrotis ipsilon (Lepidoptera: Noctuidae). This insect also incurred nearly 100% mortality due to nematode infection and supported the highest level of reproduction. Non-insect arthropods tested (Chilopoda and Isopoda) stimulated no behavioural response and were not susceptible to nematode infection. Other insect species elicited intermediate levels of the recognition response. There were significant correlations between behavioural response and nematode-induced mortality at the lower dose. The level of reproduction supported by the candidate hosts was also correlated with S. carpocapsae behavioural response.

A low molecular weight factor in a basal medium essential for erythrocytic Plasmodium falciparum development in a serum-free medium using a cell growth-promoting factor derived from adult bovine serum was detected. The factor was hypoxanthine. The optimal hypoxanthine concentration for parasite growth was between 15 and 120 μM. The contribution of hypoxanthine to increased parasite growth was clearly evident in cultures on day 4. Among various low molecular weight supplements tested, adenine, adenosine, AMP, ATP, cyclic AMP, guanine, guanosine, inosine, inosine mono-phosphate, xanthine, NAD, NADH, NADP, NADPH and deoxyguanosine triphosphate showed a similar effect to that of hypoxanthine in the serum-free culture system. On the other hand, the addition of uric acid, FAD, thymidine, uridine, orotic acid, deoxythymidine triphosphate, deoxycytidine triphosphate, deoxyadenosine triphosphate, ribose-1-phosphate, or ethanolamine was not beneficial to the parasite growth. The results presented here will not only be of practical value, but will provide important information about the developmental requirements of the parasite.

Available primary structural information suggests that the FMRFamide-related peptides (FaRPs) from parasitic and free-living nematodes are different, and that free-living forms may not represent appropriate models for the study of the neurochemistry of parasitic forms in the laboratory. However, here we report the isolation and unequivocal identification of AF2 (originally isolated from the parasite, Ascaris suum) from acidified alcoholic extracts of the free-living species, Panagrellus redivivus. While reverse-phase HPLC analysis of extracts revealed FMRFamide-immunoreactivity to be highly heterogeneous, AF2 was the predominant FMRFamide-immunoreactive peptide present (at least 26 pmol/g wet weight of worms). This peptide was also the major immunoreactant identified by an antiserum raised to the conserved C- terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), which has been used previously to isolate neuropeptide F (NPF). These observations were confirmed by radioimmunoassay and chromatographic fractionation of an acidified alcoholic extract of A. suum heads. The FMRFamide-related peptides present in a nematode extract may be highly dependent on the extraction medium employed, and these data would suggest that this complement of neuropeptides may not be as different between parasitic and free-living nematodes as initial studies have suggested. Finally, all of the evidence suggests that NPF is not present in nematodes and that the PP-immunoreactant previously demonstrated immunochemically is probably AF2.

Echinococcus granulosus cysts were cultured in vitro from the oncosphere, and cyst morphogenesis is described. Cysts grew to a mean diameter of 16 mm in 120 days, with a maximum of 20 mm. An amorphous layer was apparent around the larvae during early post-oncospheral re-organization. Organisms became vesicular at 6 days and laminations appeared in the amorphous layer at 10 days. Confluence of proliferating cells to form the syncytium of the germinal membrane was observed.

To assess the potential role of IgA antibody in expulsion of the nematode of the genus Trichinella from the intestine, a panel of IgA monoclonal antibodies (mAbs) were produced from the mesenteric lymph node cells from BALB/c mice orally vaccinated with irradiated muscle larvae of Trichinella britovi. One IgA mAb, HUSM-Tb1, formed immunoprecipitates on the surface of live muscle larvae, and by immunohistochemistry reacted with their stichocytes and cuticular surface, but not with those tissues of the adult stage or newborn larvae. Intraperitoneal injection of BALB/c mice with this mAb 5 h before challenge conferred a high level of protection (more than 95%) against T. britovi infection, when 2·0 mg of specific IgA/20 g body weight was given to a mouse. The same treatment produced a similar effect in SCID mice lacking functional T- and B-cells, indicating no requirement of synergistc T-cell factors for the effect. Passive transfer of the mAb at the time of challenge or later showed less or no effect upon worm expulsion. It is concluded that the mucosal IgA response, when adequately induced, can impede the establishment of infective Trichinella parasites in the mouse intestine.

Although parasites are reported to alter host responses to predators, little is known about the neurochemical mechanisms involved. Using an odour preference test, we examined the effects of an acute, subclinical infection with the naturally occurring, single host, enteric protozoan parasite, Eimeria vermiformis, on the responses of male laboratory mice, Mus musculus, to a predator. Uninfected mice avoided the odour of a predatory cat, spending a minimal amount of time in a Y-maze in the vicinity of the cat odour. In contrast, mice infected with E. vermiformis, spent a significantly greater amount of time in the proximity of the cat odour, showing a reduced avoidance of the cat odour and a reduction in predator-induced fear or anxiety. This was not related to augmented opioid activity and decreased pain sensitivity in the infected mice, as neither treatment with the exogenous opiate, morphine, nor restraint stress-induced augmentation of endogenous opioid activity, had any significant effects on the responses of uninfected mice to cat odour. The altered responses of the infected mice to the cat odour were reduced by peripheral administration of the gamma-aminobutyric A (GABAA) antagonists, bicuculline and picrotoxin, but were not significantly affected by either the benzodiazepine antagonist, Ro 15–1788, the opiate antagonist, naloxone, or the excitatory amino acid, N-methyl-D-aspartate (NMDA) antagonist, MK-801. These results indicate that infection with E. vermiformis in mice reduces the avoidance of predator odour through neurochemical systems associated with anxiety involving, at least in part, GABAA receptor mechanisms.

A comparison is given of the ultrastructure of the vitelline cell in Schistosoma mansoni, S. haematobium, S. japonicum and S. mattheei. Four stages in development of the vitelline cell have been categorized as follows: Stage 1, the undifferentiated cell; Stage 2, the developing cell showing the beginning of synthetic activity; Stage 3, the developing cell showing active protein synthesis; Stage 4, the fully mature vitelline cell. These stages in development have been defined morphologically and Stages 1, 2 and 3 are very similar in all 4 species. Lipid is present in the Stage 4 cells of all species but appears earlier at Stage 3 in S. haematobium and S. mattheei. There are several differences as to the intercellular inclusions of the Stage 4 cells, the most marked of these being the absence of calcareous corpuscles from S. japonicum as compared with the other 3 species.

The amounts of ammonia excreted by two endoparasitic flatworms have been compared and, weight for weight, the sheep liver fluke Fasciola hepatica produces ten times as much ammonia as the rat tapeworm Hymenolepis diminuta. This is thought to reflect a difference in diet, the fluke feeding mainly on the blood and tissue proteins of its host, and the tapeworm mainly on carbohydrates from the food materials in the gut of the host. However, the addition of glucose to the saline in which the flukes are cultured produces a marked decrease in the ammonia and an increase in the lactic acid excreted, suggesting that flukes are not obligatory protein feeders but that they may utilize carbohydrate when it is available. The relatively low production of lactic acid when proteins are being metabolized suggests that the flukes are producing an alternative end-product of respiratory metabolism. It is possible that this is the neutral lipid found in the excretory system of Fasciola and the possibility of an alternative metabolic pathway for the production of such lipid is discussed. The respiratory metabolism appears to be independent of oxygen, and the possible role of oxygen in other aspects of the metabolism of the fluke is discussed.

I should like to express my sincere gratitude to Dr J. Llewellyn for his constant advice and encouragement during the course of this work and the preparation of the manuscript. I also thank Professor 0. E. Lowenstein, F.R.S., for allowing me the use of the facilities in his department during the tenure of an S.R.C. Research Studentship. Finally, my thanks are due to Mr E. Wilson, the Veterinary Officer at the Birmingham City Meat Market, for his constant co-operation in obtaining supplies of liver flukes.

In order to construct an analytical model of onchocerciasis transmission, it is necessary to elucidate the functional relationships of the various population rate processes taking place within the human and vector hosts. Two previous papers have explored the evidence for density-dependent regulation in relation to microfilarial intake by, and larval development within, the Simulium host. This paper investigates the survivorship of wild-caught blackfly samples fed on subjects with different intensities of Onchocerca volvulus microfilarial infection. Analyses were based on data for Guatemalan S. ochraceum s.l. (possessing a well-developed cibarial armature), West African S. damnosum s.l. (forest species), and South Venezuelan S. guianense (the latter two lacking a toothed cibarium). The mean survival times of samples of the 3 species, kept under laboratory conditions, decreased as parasite intake increased, the rate of mortality being dependent on the fly’s age (measured as time post-feeding) and on the worm load acquired. An empirical, timedependent hazard function was fitted to observed death rates/fly/day which rose very shortly after engorgement, declined subsequently, and rose again throughout the extrinsic incubation period of the parasite. The parameters of this hazard model were all positively correlated with the density of microfilariae in the bloodmeal. Expressions of survivorship and life-expectancy as explicit functions of time post-feeding and mean parasite intake were derived. The average expectation of life at engorgement for uninfected flies in the laboratory was estimated to be around 1 week for both, armed and unarmed blackflies. Residual life-expectancy decreased with time post-feeding and microfilarial load in both categories of vectors. This decline (resulting from age- and parasite-dependent mortality rates) was much more pronounced in those species lacking a toothed fore-gut. Whilst a fraction of heavily infected S. ochraceum was able to survive the latent period of the parasite, being therefore potentially capable of transmitting the infection, equivalent worm loads in S. guianense resulted in a drastic reduction of the expectation of infective life. These results provide additional evidence to support the hypothesis that, in the case of intrinsically susceptible vectors, unarmed simuliids are more efficient at low microfilarial loads, when the transmission rate from human to vector host is higher, and parasite-induced fly mortality is negligible. The opposite takes place in armed flies, which perform poorly at low parasite burdens and better at heavier loads, with little parasite-induced vector death.

Antigens were released in vitro from human red cells infected with Plasmodium falciparum only when parasites reached a late stage of schizogony (probably at rupture of infected red cells during reinvasion). Immune complexes formed if antibody was present. Serum from immune Aotus monkeys opsonized parasite debris from artificially disrupted infected Aotus red cells.

There is growing emphasis on the use of community treatment to reduce the level of morbidity caused by helminth infection. The design of chemotherapy programmes, in terms of frequency of treatment and proportion of the community treated, would be assisted by a quantitative framework which enabled the morbidity reduction achieved by different approaches to be compared. The present study describes a model developed for this purpose which embodies two innovative features. First, a quantitative score of morbidity (the proportion of individuals harbouring an intense infection) is used to rate the success of a programme and, second, the distribution of helminths in the host population is generated by a mechanism that allows the distribution to change dynamically as a function of both treatment and reinfection. The model behaviour, using values typical of Ascaris lumbricoides, indicates that the benefit derived from community chemo- therapy increases non-linearly with the coverage and efficacy of treatment.