Relapse of infection after drug treatment of trypanosome infections under conditions precluding re-infection has usually been ascribed to drug resistance on the part of the parasite or to under-dosage of the drug. With Trypanosoma brucei infection in mice we have obtained evidence of another type of relapse. In infections resulting from the inoculation of 1 × 105 trypanosomes, derived from a stabilate T. brucei TREU 667, treatment with diminazene aceturate (Berenil) at 40 mg/kg at either 3 or 7 days after infection elicited a permanent cure. If, however, treatment was delayed later than 14 days after infection, then all the mice relapsed. These relapses generally occurred between 20 and 50 days after treatment, but some mice remained aparasitaemic for up to 60 days. The relapsed infections were apparently not due to the survival of ‘drug-resistant’ trypanosomes, as infections derived from a stabilate isolated from a relapsed Berenil-treated mouse were also permanently cured with Berenil if treated 3 days after infection; however, if treatment was delayed until 21 days post-infection, all the mice relapsed. The cause of relapse was not related to the number of parasites inoculated, as infection resulting from initial inocula of 1 × 105 to 1 × 108 trypanosomes were all cured if treated at 3 days after infection, and all eventually relapsed if treatment was delayed until day 21. This type of relapse phenomenon was not confined to T. brucei TREU 667 but also occurred with 5 other stabi-lates of T. brucei after Berenil treatment. Treatment of T. brucei TREU 667 infections with Ethidium and Prothidium at dose levels of 7.5 and 10 mg/kg respectively was also followed by relapse if treatment was delayed for 3 weeks after infection. The possible causes of relapse under these conditions, and its implications in the study of the natural disease, are discussed.

Young schistosomes collected after penetration through isolated mouse skin (3 h schistosomula) were cultured in medium containing immune rhesus monkey serum with a high titre of antibody known to kill schistosomula in the presence of complement. Morphological signs of damage in electron micrographs were confined to the surface tegument of the schistosomula. Antibodies in immune rhesus serum were shown to bind to the surface membrane of 3 h schistosomula using an antibody-enzyme bridge technique involving labelling with horseradish peroxidase and histochemical localization of the enzyme at the ultrastructural level. Schistosomula recovered from the lungs of mice 4 days after infection did not bind monkey antibodies at the surface and these 4-day schistosomula are not susceptible to damage by immune serum in vitro. Mouse erythrocyte antigens were detected on the surface of 4-day schistosomula using an appropriate antibody-enzyme bridge but these host antigens could not be found on 3 h schistosomula. This correlation between the presence of mouse host antigens on the surface of schistosomula and the inability of immune monkey antibodies to bind to the surface membrane is consistent with the hypothesis that host antigens are acquired by young schistosomes and serve to protect the surface membrane against antibody-mediated damage.

We have obtained 5 specific DNA probes for African trypanosomes of the subgenera Trypanozoon and Nannomonas. Each probe consists of one repeat unit of the major repetitive DNA (satellite DNA) of each species or intra-specific group. One probe hybridized with all members of subgenus Trypanozoon (except T. equiperdum which was not tested). In subgenus Nannomonas, one probe recognized T. simiae, but 3 probes were needed to identify all stocks of T. congolense available. Each of the 3 latter probes recognized trypanosomes from one of the 3 major groups of T. congolense previously defined by isoenzyme characterization, i.e. savannah, forest and Kenya coast types. As few as 100 trypanosomes could be unequivocally identified by dot blot hybridization and individual trypanosomes could be identified by in situ hybridization. We show how this simple methodology can be used in the field for the identification of immature and mature trypanosome infections in tsetse.

The current trend of antimony (Sb) unresponsiveness in the Indian subcontinent is a major impediment to effective chemotherapy of visceral leishmaniasis (VL). Although contributory mechanisms studied in laboratory-raised Sb-R parasites include an up-regulation of drug efflux pumps and increased thiols, their role in clinical isolates is not yet substantiated. Accordingly, our objectives were to study the contributory role of thiols in the generation of Sb unresponsiveness in clinical isolates. Promastigotes were isolated from VL patients who were either Sb responsive (n=2) or unresponsive (n=3). Levels of thiols as measured by HPLC and flow cytometry showed higher basal levels of thiols and a faster rate of thiol regeneration in Sb unresponsive strains as compared with sensitive strains. The effects of antimony on generation of reactive oxygen species (ROS) in normal and thiol-depleted conditions as also their H2O2 scavenging activity indicated that in unresponsive parasites, Sb-mediated ROS generation was curtailed, which could be reversed by depletion of thiols and was accompanied by a higher H2O2 scavenging activity. Higher levels of thiols in Sb-unresponsive field isolates from patients with VL protect parasites from Sb-mediated oxidative stress, thereby contributing to the antimony resistance phenotype.

Toxocara species infect a wide range of companion, domestic and wild animals as definitive and paratenic hosts, via multiple routes of transmission, producing long-lived tissue-inhabiting larvae and resistant eggs that can survive in the external environment. Therefore Toxocara and the disease it causes in humans, toxocariasis, represents an ideal aetiological agent for the development of the one health approach. However, despite increasing awareness of the public health significance of toxocariasis, gaps in our understanding of certain key aspects of the parasite's biology and epidemiology remain. These gaps hinder our ability to integrate research effort within the veterinary, medical and environmental disciplines. This review will highlight key deficits in our understanding of nine dimensions of Toxocara epidemiology and discuss a potential scenario to develop a more integrated, one health approach to improve our understanding of the prevention and control of this complex and cryptic zoonosis.

The efficiency of treatment of human infections with the unicellular eukaryotic pathogens such as fungi and protozoa remains deeply unsatisfactory. For example, the mortality rates from nosocomial fungemia in critically ill, immunosuppressed or post-cancer patients often exceed 50%. A set of six systemic clinical azoles [sterol 14α-demethylase (CYP51) inhibitors] represents the first-line antifungal treatment. All these drugs were discovered empirically, by monitoring their effects on fungal cell growth, though it had been proven that they kill fungal cells by blocking the biosynthesis of ergosterol in fungi at the stage of 14α-demethylation of the sterol nucleus. This review briefs the history of antifungal azoles, outlines the situation with the current clinical azole-based drugs, describes the attempts of their repurposing for treatment of human infections with the protozoan parasites that, similar to fungi, also produce endogenous sterols, and discusses the most recently acquired knowledge on the CYP51 structure/function and inhibition. It is our belief that this information should be helpful in shifting from the traditional phenotypic screening to the actual target-driven drug discovery paradigm, which will rationalize and substantially accelerate the development of new, more efficient and pathogen-oriented CYP51 inhibitors.

Resistance against anthelmintics is widespread, particularly in parasitic nematode populations of small ruminants. Several new techniques or supplements have been developed or are under investigation. Biological control (BC) is one of these new methods. The net-trapping predacious fungus Duddingtonia flagrans produces thick walled resting spores, chlamydospores, which are able to survive passage through the gastrointestinal tract of cattle, horses, sheep and pigs. Under Danish climatic conditions it has been shown that the number of parasite larvae on pasture and the worm burden of the grazing animals is significantly reduced when animals are fed spores during the initial 2–3 months of the grazing season. Work with D. flagrans in France, Australia, USA, and Mexico has confirmed the strong BC potential of this fungus. Today much work is going into development of suitable delivery systems for grazing livestock worldwide. Ultimately, BC should be implemented in integrated parasite control strategies, both in conventional and organic livestock production.

Praziquantel is a broad-spectrum anthelmintic active against schistosome species which are major parasites of man. Two major effects on Schistosoma mansoni have been demonstrated; (i) spastic paralysis of the parasite musculature, possibly arising as a consequence of an influx of Ca2+ into the worm (Pax, Bennett & Fetterer, 1978; Coles, 1979) and (ii) vacuolation and degeneration of the worm tegument (Becker, Mehlhorn, Andrews, Thomas & Eckert, 1980). These events may contribute to the elimination of schistosomes in vivo, but this elimination may partly be dependent on the host immune response as infected T-cell-deprived mice are less able than immunologically intact animals to reduce their worm burdens following drug treatment (Doenhoff, Harrison, Sabah, Murare, Dunne & Hassounah, 1982). This latter observation raises the possibility that praziquantel may lower the ability of the parasite to evade host immunity by increasing the exposure of parasite antigens capable of acting as targets for host antibody, or antibody-armed cells at the worm surface. Consistent with this idea is the observation that adult schistosomes in praziquantel-treated mice are invaded a few hours after treatment by host granulocytes (Mehlhorn, Becker, Andrews, Thomas & Frenkel, 1981).

This paper reviews the occurrence and impact of threadworms, Strongyloides spp., in companion animals and large livestock, the potential zoonotic implications and future research. Strongyloides spp. infect a range of domestic animal species worldwide and clinical disease is most often encountered in young animals. Dogs are infected with Strongyloides stercoralis while cats are infected with different species according to geographical location (Strongyloides felis, Strongyloides tumefaciens, Strongyloides planiceps and perhaps S. stercoralis). In contrast to the other species, lactogenic transmission is not a primary means of infection in dogs, and S. stercoralis is the only species considered zoonotic. Strongyloides papillosus in calves has been linked to heavy fatalities under conditions of high stocking density. Strongyloides westeri and Strongyloides ransomi of horses and pigs, respectively, cause only sporadic clinical disease. In conclusion, these infections are generally of low relative importance in livestock and equines, most likely due to extensive use of macrocyclic lactone anthelmintics and/or improved hygiene. Future prevalence studies need to include molecular typing of Strongyloides species in relation to different hosts. More research is urgently needed on the potential zoonotic capacity of Strongyloides from dogs and cats based on molecular typing, information on risk factors and mapping of transmission routes.

A general method of investigating parasite population genetics is presented and used to investigate the evolution of drug resistance in Plasmodium. The most important biological factor is the nature of the control, presumably through host immunity, of the malarial infection. Two models are examined: a ‘generalized immunity’ (GI) model in which immunity regulates the overall level of infection, and a ‘specific immunity’ (SI) model in which each clone within the infection is regulated independently. These models are used to investigate 3 critical factors in the evolution of resistance: (i) the frequency of resistant alleles in the population prior to drug use, (ii) the dynamics of resistance following drug application and (iii) the magnitude of threshold frequencies below which resistance will not evolve. These analyses also identify the implicit assumptions made in several previous models, reconcile their differing conclusions and allow a more informed debate about the practical application of drugs.

We examined the parasite-induced cataract formation in rainbow trout (Oncorhynchus mykiss) using slit-lamp microscopy to determine the relationship between cataract intensity and number of Diplostomum spathaceum parasites that were established in the lens. Cataract intensity increased significantly with parasite burden, but was also affected by the pattern of exposure to the parasite cercariae. The slit-lamp methodology proved useful in scoring the cataracts since it provides a 3-dimensional view into the lens and gives an actual picture of the location and intensity of the cataracts, which allows detailed investigations of mechanisms underlying cataract formation in various fish species. Potential applications of the method in fish farming and parasitological studies are discussed.

1. A 12·8% incidence of hydatid infections occurred in 709 horses examined in England. By contrast, hydatid cysts do not occur in horses in New Zealand where Echinococcus granulosus granulosus is common in sheep, cattle and swine.

2. Experimentally, scolices from hydatid cysts from English horses reached a gravid state in laboratory dogs. It was not possible to infect horses with E. g. granulosus of sheep-dog origin, and only one of two sheep became infected with two cysts following the ingestion of eggs of English horse-dog origin.

3. Our observations, together with those of others on secondary hydatid cyst formation, indicate that the two types are not only biologically but morphologically distinct.

4. In the horse-dog worms, the cirrus sac tilts anteriorly at an acute angle; the testes (about 35) are distributed throughout the proglottid with two or three rows posterior to the vitelline gland, and persist in proglottids when some mature eggs are present; the average length of the rostellar hooks is greater than in E. g. granulosus.

5. It is proposed that the horse-dog material be named E. g. equinus subsp. nov.

6. Rostellar hooks from cysts in British cattle resembled morphologically those from horses, while those from a British sheep and from man were like the hooks from hydatid cysts in man and livestock in New Zealand.

7. A red deer, hedgehog and European rabbit were not infected artificially following the ingestion of eggs of E. g. equinus.

Mr J. T. Briggs, Chief Public Health Inspector, Public Health Department, Bently, Doncaster, England, was responsible for the inspection of the British horses reported in this paper. We are indebted to Mr Briggs for this courtesy and for sending us fresh hydatid cysts from horses for experimental purposes. Mr N. Aitken was responsible for maintaining the experimental horses in an area of New Zealand known to be free of E. granulosus. Our thanks are also due to Messrs G. D. Page, D. V. Weston, Miss L. A. Duncan and Mrs B. R. Clarke for their able technical assistance.

1. The reproductive cycle of the female fly was worked out at an average temperature of 24° C. The lowest egg of the right ovary develops first, and is ripe in 7 days. Ovulation never occurs earlier than the 8th day, and frequently later; it bears some relation to fertilization, for eggs in virgin females failed to ovulate for weeks. The first larva was produced in a variable time after fertilization (because of delayed ovulation), but the succeeding larvae were produced very regularly, with an average period of 9·9 days between them.

2. Pupae produced in the laboratory usually weigh less than those collected in the field. The reason for this is nutritional, and flies must take large meals at the right stage in gestation to produce large pupae. It is impossible to ensure that this happens with all flies in the laboratory.

3. Abortions are caused in captivity by flies obtaining too little blood. They may be induced in pregnant females by allowing them to take only small meals, when either an egg or a small larva is extruded. When these flies were properly fed later, they produced normal larvae.

4. A constant temperature of 30° C. causes sterility in females. Ovaries develop abnormally, embryos fail to hatch from eggs, but young larvae (in the uteri) are not adversely affected.

5. A constant temperature below 22° C. slows down the rate of development of the eggs in the ovaries and lengthens the larval period. Experiments at this temperature are complicated by inactivity of males, which leave many females unfertilized. The length of the combined embryonic and larval periods is 17½ days.

6. Experiments performed outside in the laboratory compound suggest that the period between each larva in the field (during December in Uganda) will be about 13½ days (min. temp. 16° C.; max. temp. 27° C.).

7. Flies appear to be able to breed equally well with relative humidities of 47, 88 and 100 per cent. In the drier air care must be taken with feeding, or the flies die from desiccation.

8. A temperature of 20° C. can cause pregnant females to retain their larvae for an extra 12 hours, but not longer.

In this study, we investigated the presence of the larval stage of the tapeworm Echinococcus granulosus in wild ungulates in Estonia, genetically characterized E. granulosus isolates using mitochondrial gene sequences and used the sequence data, together with those available in a public database, to infer the phylogenic relationships of E. granulosus ‘genotypes’ G5-G10. While 0·8% of the 2038 moose (Alces alces) examined were found to be infected with E. granulosus, the parasite was not detected in other wild ungulates, such as roe deer (Capreolus capreolus: 1044 specimens examined) and wild boar (Sus scrofa: 442 specimens). Genetic analyses of concatenated atp6, nad1 and cox1 gene (1028 bp) sequences revealed that 2 novel E. granulosus haplotypes, namely E8 (11 samples: 69%) and E10 (5 samples: 31%), grouped with E. granulosus G8 and G10, respectively, are present in Estonia. This is the first record of an E. granulosus G8 in Eurasia. Phylogenetic analyses, using 4 different methods, demonstrated with considerable statistical support that E. granulosus G6/7 forms a subgroup together with G10, whereas G8 is a sister taxon to G6/7-G10.

Baseline data from an immuno-epidemiological study of hookworm infection in a rural village in Madang Province, Papua New Guinea are reported. Necator americanus was found to be the commonest helminth infection, with a prevalence of near 100% and intensity of 40 worms per host in adults. Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura were also present, at prevalences of 53, 10 and 3% respectively; Ancylostoma duodenale was absent. The frequency distribution of N. americanus was highly over-dispersed, and was well described by a negative binomial distribution with aggregation parameter, k, of 0·370. Intensity of infection was significantly related to host age, but did not differ between the sexes. Haemoglobin levels and haematocrit values were indicative of anaemia in the community, but were unrelated to hookworm infection. Levels of antibodies (IgG, IgA and 1gM combined) against adult Necator cuticular collagen and excretory-secretory (ES) products were determined.

Few studies have described the combined effect of age, gender, management and control programmes on helminth prevalence and egg shedding in grazing equines. Here, fecal samples collected from 1221 Thoroughbred horses, residing at 22 studs in the UK, were analysed. The distribution of strongyle eggs amongst individuals in relation to age, gender and management practices was investigated. Fecal worm egg counts (FWECs), described as the number of eggs per gramme (epg) of feces, were determined using a modification of the salt flotation method. The FWEC prevalence (mean%) of strongyles, Parascaris equorum, tapeworm spp. and Strongyloides westeri was 56, 9, 4 and 8%, respectively. Strongyle, P. equorum, tapeworm spp. and S. westeri infections were detected on 22 (100%), 11 (50%), 9 (41%) and 8 (36%) of studs, respectively. Within all age and gender categories, strongyle FWECs were highly over-dispersed (arithmetic mean = 95 epg, aggregation parameter k=0·111) amongst horses. Animal age, last anthelmintic type administered and management practices (for example, group rotation on grazing) most strongly influenced strongyle prevalence and level of egg shedding (P < 0·05). Overall, 11% of equines (range: 234–2565 epg) were responsible for excreting 80% of the strongyle eggs detected on FWEC analysis. The results confirm that the judicious application of targeted treatments has potential to control equine strongyle populations by protecting individual horses from high burdens, whilst promoting refugia for anthelmintic susceptible genotypes.

The anthelmintic properties of tanniferous plants and of their secondary metabolites represent one possible alternative to chemotherapy that is currently being explored as a means of achieving sustainable control of gastrointestinal nematodes in ruminants. Previous in vivo and in vitro results suggest that tanniferous plants can have direct anti-parasitic effect against different stages of nematodes. However, the mode of action of the bioactive plant compounds remains obscure. The objectives of the current study were (1) to examine the hypothesis that extracts of tanniferous plants might interfere with the exsheathment of third-stage infective larvae (L3); (2) to assess the role of tannins in the process by examining the consequence of adding an inhibitor of tannins (polyethylene glycol: PEG) to extracts. The effects of 4 tanniferous plant extracts on exsheathment have been examined on L3 of Haemonchus contortus and Trichostrongylus colubriformis. Artificial exsheathment was induced in vitro by adding hypochloride solution to larval suspension. The evolution of exsheathment with time was measured by repeated observations at 10-min interval for 60 min. The selected plants were: genista (Sarothamnus scoparius), heather (Erica erigena), pine tree (Pinus sylvestris), and chestnut tree (Castanea sativa), with tannin contents ranging from 1·5 to 24·7% of DM. Extracts of a non-tanniferous plant (rye grass, tannin content: 0·3% of DM) were included in the assay as negative controls. The extracts were tested at the concentration of 600 μg/ml and the effects were compared to the rate of exsheathment of control larvae in PBS. No statistical differences in the pattern of exsheathment was observed after addition of rye grass or genista extracts for both nematode species and with heather extracts for T. colubriformis. In contrast, pine tree extracts on larvae of both species and heather extracts with H. contortus induced a significant delay in exsheathment. Last, contact with chest nut extracts led to a total inhibition of the process for both nematodes. These results suggest that extracts of tanniferous plants might affect a key process in the very early stages of larval invasion of the host. In most cases, the addition of PEG led to a total or partial restoration towards control values. This suggests that tannins are largely involved in the inhibitory process. However, other secondary metabolites may also interfere with the process that would help to explain some of the differences in response observed between the two nematode species.

Infections by larval stages of tapeworms of the genus Echinococcus (echinococcosis or hydatid disease) are zoonotic infections of major public health importance throughout much of the world. Humans become infected through accidental ingestion of eggs passed in faeces of canid definitive hosts. Tibetan populations of China have some of the highest documented levels of infections by both Echinococcus granulosus and E. multilocularis, the causes of cystic and alveolar echinococcosis, respectively. In this study we measured the prevalence of cystic (CE) and alveolar (AE) echinococcosis disease in Tibetan communities in Qinghai, Province, China, and identified putative risk factors for both infections in these communities. 3703 volunteers in three predominately Tibetan counties of Qinghai were surveyed between June 1997 and June 1998. Parasitic lesions were diagnosed by imaging of characteristic space-occupying lesions in abdominal organs (ultrasound) or the lungs (radiographs). Specific serodiagnostic assays (Dot-ELISA and Em2-ELISA) were performed on sera of positively imaged subjects to further distinguish the disease agent. All participants completed a questionnaire documenting age, sex, education level, occupation, lifestyle (nomadic or settled), slaughter practices, drinking water source, hygienic practice and association with dogs. Data were analyzed using SAS version 8. 6·6% of the volunteers had image-confirmed infection with E. granulosus (CE) and 0·8% had E. multilocularis (AE) infection. The significant univariate factors for echinococcal infection (both CE and AE) included livestock ownership, Tibetan ethnicity, female gender, low income, herding occupation, limited education, water source, age greater than 25 years old, poor hygienic practices, offal disposal practices and dog care. Multivariate analysis revealed that livestock ownership was a significant risk factor for both forms of the disease, as well as age greater than 25 years, female gender, herding occupation, and being nomadic (vs semi-nomadic or settled). No additional significant risk factors were identified among the 344 nomadic participants. Being female and being older than 25 years of age were significant factors among the 1906 semi-nomadic participants. Among the 1445 settled participants, allowing dogs to sleep indoors was statistically significant. Issues such as inadequate assessment of animal ownership, selection bias, disease misclassification, and loss of information may have led to reduction in strength of some risk factor associations and need to be addressed in future epidemiologic analysis of echinococcosis in this population.

Oocyst prevalence and intensity have been recorded in 349 laboratory infections of Anopheles stephensi with Plasmodium berghei. Intensity and prevalence of infection are shown to be predictably related. The structure and heterogeneity in the infections has been analysed with the objective of describing the biological mechanisms by which the observed negative binomial oocyst distributions are generated. The analysis has revealed that the most likely processes lie within the population dynamic events of malaria within the mosquito, namely gametogenesis, fertilization and mortality. The distribution is similar in all Plasmodium – mosquito combinations examined so far, whether they are of laboratory (P. gallinaceum in Aedes aegypti) or field (P. vivax in An. albimanus and P. falciparum in An. gambiae s.l. and An. funestus) origin. Further we conclude that there is competition between parasites in the vector. Oocyst frequency distribution analysis shows that under natural conditions of transmission intensity, and even under the best laboratory conditions, significant numbers (> 10%) of fully susceptible mosquitoes will not be infected under conditions where the mean infection is as high as 250 oocysts. Failure to infect is not therefore an absolute indicator of refractoriness. In assessing transmission data it is shown that sample sizes should not be less than 50, and ideally 100 mosquitoes, if reliable data are to be obtained. In the field it is suggested that difficulties in determining the low natural intensity of oocyst infections indicate that prevalence estimates are a useful and accessible parameter to measure. In determining the impact of transmission blocking mechanisms we predict that under conditions where high oocyst intensities prevail, large reductions in intensity will be required before a reduction in prevalence can be expected i.e. here it will be necessary to measure intensity of infection. Conversely, under conditions where low oocyst intensities prevail, a rapid reduction in prevalence will occur with little concurrent reduction in intensity i.e. prevalence determination will be the more sensitive estimate.

Continuous culture was achieved in several cell lines of a microsporidium obtained from the skeletal muscle of an AIDS patient. Development in COS-1 and RK13 cells was prolific. Spores from the original biopsy were also inoculated into athymic mice by i.m. and i.p. routes. Infection was found in several organs as well as in skeletal muscle after a few weeks. All stages were surrounded by an electron-dense surface coat. Meronts had 2–4 nuclei and divided by binary fission. In sporogony the surface coat became separated from the plasma membrane to form a sporophorous vesicle, within which division into sporoblasts was effected by repeated binary fissions. The number of sporoblasts (and later spores) within the sporophorous vesicles varied from 2 to > 32 and the sizes of the vesicles varied, according to the number of spores contained therein, from 5 μm diameter to 14·0 × 11·0 μm. Spores measured 4·0 × 2·4 μm and had a prominent posterior vacuole. The parasite differs from the genus Pleistophora in that it does not form multinucleate sporogonial plasmodia and that the sporophorous vesicle enlarges during sporogony and its wall is not a multilayered structure. It is proposed to place it in a new genus and species Trachipleistophora hominis n.g., n.sp.