Factors which may be important in the large-scale extraction of coccidial oocysts from faeces have been investigated with Eimeria tenella. Age of bird, inoculum, feeding status at the time of inoculation, period of collection, feeding status during collection, collection medium, homogenization and sieving, flotation, washing, sporulation and further purification have all been considered. The aim has been to establish a method to produce the maximum number of oocysts of a required degree of purity and viability, with the expenditure of the minimum amount of physical effort, time, animals and chemicals. In our method, groups of chickens 3–4 weeks of age are inoculated with 5000 oocysts of E. tenella and food is supplied ad lib. Over the period 5–8 days after inoculation, faeces are collected in trays containing 2% (w/v) potassium dichromate solution, while food intake is restricted. The faecal material is homogenized, passed once through 40 and 100 mesh sieves, centrifuged and the oocysts recovered from the sediment by 3 flotations in saturated salt solution. Following washing, oocysts are sporulated by forced aeration at 30°C and may be further purified by hypochlorite treatment, or passage in 5% Tween 80 solution through a glass bead column followed by sucrose density gradient centri-fugation. Routine passages along these lines over a 5 year period have given a recovery of 46% of the oocysts excreted by over 7000 birds.

The objectives of this study were (1) to examine risk factors for Schistosoma mansoni infection among schoolchildren living in western Côte d'Ivoire, and (2) to carry forward spatial risk prediction and mapping at non-sampled locations. First, demographic and socio-economic data were obtained from 3818 children, aged 6–16 years, from 55 schools. Second, a single stool sample was examined from each child by the Kato-Katz technique to assess infection status of S. mansoni and its intensity. Third, remotely sensed environmental data were derived from satellite imagery and digitized ground maps. With these databases a comprehensive geographical information system was established. Bayesian variogram models were applied for spatial risk modelling and prediction. The infection prevalence of S. mansoni was 38·9%, ranging from 0% to 89·3% among schools. Results showed that age, sex, the richest wealth quintile, elevation and rainfall explained the geographical variation of the school prevalences of S. mansoni infection. The goodness of fit of different spatial models revealed that age, sex and socio-economic status had a stronger influence on infection prevalence than environmental covariates. The generated risk map can be used by decision-makers for the design and implementation of schistosomiasis control in this setting. If successfully validated elsewhere, this approach can guide control programmes quite generally.

There is growing interest and commitment to the control of schistosomiasis and other so-called neglected tropical diseases (NTDs). Resources for control are inevitably limited, necessitating assessment methods that can rapidly and accurately identify and map high-risk communities so that interventions can be targeted in a spatially-explicit and cost-effective manner. Here, we review progress made with (1) mapping schistosomiasis across Africa using available epidemiological data and, more recently, climate-based risk prediction; (2) the development and use of morbidity questionnaires for rapid identification of high-risk communities of urinary schistosomiasis; and (3) innovative sampling-based approaches for intestinal schistosomiasis, using the lot quality assurance sampling technique. Experiences are also presented for the rapid mapping of other NTDs, including onchocerciasis, loiasis and lymphatic filariasis. Future directions for an integrated rapid mapping approach targeting multiple NTDs simultaneously are outlined, including potential challenges in developing an integrated survey tool. The lessons from the mapping of human helminth infections may also be relevant for the rapid mapping of malaria as its control efforts are intensified.

In this paper the different options for the diagnosis of gastrointestinal nematode infections are discussed. Diagnostic tests have a role in confirming the clinical diagnosis of parasitic gastroenteritis, but are more important for herd health monitoring of nematode infections, in particular for cattle. Therefore, emphasis is placed on discussing the available diagnostic parameters on their usefulness for that purpose. For clinical diagnosis the clinical signs, combined with the history of the animals is usually sufficient and a laboratory confirmation is not required. Faecal egg counts are, with two exceptions, not suitable for confirmation of the clinical diagnosis, because correlation between faecal egg counts and infection levels is usually low. These exceptions are the diagnosis of haemonchosis in small ruminants and the detection of anthelmintic resistance. This also limits the value of DNA-based tests of faecal material; even quantitative tests of nematode species specific DNA will have little value for diagnosis and monitoring. Pasture larval counts and worm counts are useful parameters for basic epidemiological studies on nematode infections. However, they are too laborious to be used for either routine diagnosis or monitoring. Blood parameters, such as gastrin and pepsinogen and serology are valuable tools for diagnosis. Pepsinogen and ELISAs based on recombinant proteins show most promise as parameters for herd health monitoring. However, extensive epidemiological studies are still needed before these parameters can be implemented in routine herd health monitoring schemes for parasitic gastroenteritis.

Available data in the literature pertaining to the life-history characteristics of all known species of mammalian schistosomes have been gathered, and correlations between such variables as length of pre-patent period, adult worm size, rate of progeny production and progeny size have been explored. Accommodation of the schistosome life-cycle to the constraints imposed by certain host characteristics such as life-expectancy and size is discussed. Of the 23 known species of mammalian schistosomes, 20 species apparently rely to a major extent on relatively large-bodied and long-lived mammals such as primates, ungulates and proboscideans for their transmission. Only 1 species, Schistosomatium douthitti, is exclusively dependent on rodents for its transmission. S. douthitti attains maturity within its definitive host faster than any other mammalian schistosome, and is the only species known to be capable of producing viable eggs by facultative parthenogenesis. For all species of mammalian schistosomes, adult worm size, as estimated by female length, is positively correlated with the number of uterine eggs contained within the female (r = 0·682). For the 7 species for which data exist, rate of egg production/worm pair/day is positively correlated with uterine egg counts (r = 0·873) and inversely correlated with egg length (r = −0·787) and miracidium length (r = −0·953). Length of the pre-patent period is positively correlated with egg length (r = 0·503). With respect to the molluscan host, the number of cercariae produced by snails is positively correlated with the shell size of the snail (r = 0·657). For the 5 species for which data exist, the rate of egg production is inversely correlated with shell size of the intermediate host (r = −0·955) and the common logarithm of the number of cercariae produced (r = −0·893). Comparisons between species suggest that exceptionally low rates of cercariae production in the intermediate host may be compensated for by rapid rates of egg production in the definitive host, implying a degree of integration in the schistosome life-cycle not previously appreciated. Most species of mammalian schistosomes have long-lived definitive hosts, and snail hosts capable of producing many cercariae; compensatory relationships are therefore less obvious in such species. Additional quantitative data on all aspects of schistosome life-histories, particularly rate and duration of egg production, are needed to confirm or refute the relationships discussed above.

The mitochondrial genomes of the genus Echinococcus have already been sequenced for most species and genotypes to reconstruct their phylogeny. However, two important taxa, E. felidis and E. canadensis G10 genotype (Fennoscandian cervid strain), were lacking in the published phylogeny. In this study, the phylogeny based on mitochondrial genome sequences was completed with these taxa. The present phylogeny highly supports the previous one, with an additional topology showing sister relationships between E. felidis and E. granulosus sensu stricto and between E. canadensis G10 and E. canadensis G6/G7 (closely related genotypes referred to as camel and pig strains, respectively). The latter relationship has a crucial implication for the species status of E. canadensis. The cervid strain is composed of two genotypes (G8 and G10), but the present phylogeny clearly suggests that they are paraphyletic. The paraphyly was also demonstrated by analysing the complete nucleotide sequences of mitochondrial cytochrome c oxidase subunit 1 (cox1) of E. canadensis genotypes from various localities. A haplotype network analysis using the short cox1 sequences from worldwide isolates clearly showed a close relatedness of G10 to G6/G7. Domestic and sylvatic life cycles based on the host specificity of E. canadensis strains have been important for epidemiological considerations. However, the taxonomic treatment of the strains as separate species or subspecies is invalid from a molecular cladistic viewpoint.

Benzimidazoles (BZ) are widely used to treat parasitic nematode infections of humans and animals, but resistance is widespread in veterinary parasites. Several polymorphisms in β-tubulin genes have been associated with BZ-resistance. In the present study, we investigated β-tubulin isotype 1 sequences of 18 Haemonchus contortus isolates with varying levels of resistance to thiabendazole. The only polymorphism whose frequency was significantly increased in the resistant isolates was TTC to TAC at codon 200. Real-time PCR (using DNA from 100 third-stage larvae, L3s) and pyrosequencing (from DNA from 1000–10 000 L3s) were used to measure allele frequencies at codon 200 of these isolates, producing similar results; drug sensitivity decreased with increasing TAC frequency. Pyrosequencing was also used to measure allele frequencies at positions 167 and 198. We showed that such measurements are sufficient to assess the BZ-resistance status of most H. contortus isolates. The concordance between real-time PCR and pyrosequencing results carried out in different laboratories indicated that these tools are suitable for the routine diagnosis of BZ-resistance in H. contortus. The molecular methods were more sensitive than the ‘egg hatch test’, and less time-consuming than current in vivo- or in vitro-anthelmintic resistance detection methods. Thus, they provide a realistic option for routine molecular resistance testing on farms.

Two important recent advances in Leishmania immunology are: (i) the demonstration of a dramatic dichotomy in T helper 1 versus T helper 2 subset expansion leading to protection versus disease exacerbation; and (ii) analysis of the macrophage activation pathways leading to enhanced intracellular killing of parasites, in particular the tumour necrosis factor α (TNFα)-dependent sustained induction of the inducible nitric oxide synthase gene (Nos2) leading to the generation of large amounts of nitric oxide (NO). Given the broad spectrum of disease phenotypes in human leishmaniasis, one might predict that a genetic defect at any key point in this macrophage activation pathway and/or in pathways leading to activation of different T cell subsets, and the latter may be a pleiotropic effect of the former, will contribute to disease susceptibility. By studying disease in genetically-defined inbred mouse strains, it has been possible to identify 5 regions of the murine genome carrying leishmanial susceptibility genes. The genes include: (i) Scl-2 (mouse chromosme 4/human chromosome 9p; candidate Janus tyrosine kinase 1) controlling a unique no lesion growth resistance phenotype to Leishmania mexicana; (ii) Scl-1 (distal mouse chromosome 11/human 17q; candidates Nos2, Sigje, MIP1α, MIP1β) controlling healing versus non-healing responses to L. major; (iii) the ‘T helper 2’ cytokine gene cluster (proximal murine chromosome 11/human 5p; candidates IL4,5,9) controlling later phases of L. major infection; (iv) the major histocompatibility complex (MHC: H-2 in mouse, HLA in man: mouse chromosome 17/human 6p; candidates class II and class III including TNFα/β genes); and (v) Nramp1, the positionally cloned candidate for the murine macrophage resistance gene Ity/Lsh/Bcg (mouse chromosome 1/human 2q35). This review examines these 5 regions and the candidate genes within them, reflecting on their current status as candidates for human disease susceptibility genes.

CBA/T6 and Balb/c mice inoculated with Plasmodium berghei ANKA strain (PbA) died from cerebral malaria 6–8 days post-inoculation. DBA/2J mice similarly inoculated developed a non-fatal cerebral malaria, with mild temporary cerebral symptoms, and died between days 15 and 22 from other malaria-related complications. When inoculated with P. berghei K173 (Pb) these mouse strains did not develop a cerebral malaria but died between days 15 and 22 from other malaria-related complications. These mouse strain/parasite strain combinations allow for detailed examination of factors critical in the pathology of murine cerebral malaria. Monastral Blue, a colloid dye, when injected intravascularly between days 0 and 2 into PbA-inoculated CBA (PbA-CBA) or Balb/c (PbA-Balb/c) mice prevented death from cerebral malaria. There was no evidence of increased vascular permeability at this stage. When Monastral Blue was injected between days 5 and 8, there was increased vascular permeability in the kidney, liver, lung, spleen and brain of PbA-CBA and PbA-Balb/c mice. Injection of Monastral Blue into these animals at this time also precipitated cerebral symptoms and death, but not in Pb-infected mice. Endothelial and mononuclear cells phagocytosed, and were coated with, the Monastral Blue particles when the dye was injected between days 5 and 8 into PbA-CBA and PbA-Balb/c mice. Control, uninfected mice did not demonstrate either of these features. Pb-infected mice only demonstrated coated mononuclear cells. Mononuclear cell attachment to the endothelium, increased vascular permeability and increased association of Monastral Blue particles with monocytes and endothelial cells were correlated with cerebral symptoms and death. Monastral Blue is thus a useful agent for studying the roles of mononuclear cells and endothelium in murine cerebral malaria.

A typical postparturient rise of parasite egg output occurred in a group of lactating ewes. At parturition these animals carried few inhibited fourth-stage larvae and the rise was associated with a net increase in parasite populations, which did not occur in non-lactating ewes. Lactating ewes whose established parasite populations were effectively removed just prior to parturition showed a similar rise derived entirely from infection acquired during lactation, which was of much greater magnitude than that seen in similarly treated non-lactating animals. Circumstantial evidence of increased fecundity of T. colubriformis females in lactating ewes was also obtained.

It was concluded that the postparturient rise of egg output may derive from an increase in adult parasite populations by an increased rate of establishment of newly ingested infective larvae which develop to the adult stage without inhibition, as well as by the resumption of development of previously inhibited fourth-stage larvae. In addition, both newly acquired and established adult female parasites may show increased fecundity.

Ewes whose lambs were removed at birth or after a period of lactation showed a rapid fall in faecal egg counts associated with rejection of part of their existing worm burden and a pronounced increase in resistance to new infection.

All of the differences observed between lactating and non-lactating ewes in the behaviour of their parasite populations were found to be qualitatively the same as those that have been frequently reported in susceptible and resistant non-reproductive sheep. The hypothesis is suggested, therefore, that the postparturient rise derives from a pronounced but temporary relaxation of immunological control of gastro-intestinal nematode infection associated with the endocrine status of the lactating ewe.

The sexual phase of the malaria pathogen, Plasmodium falciparum, culminates in fertilization within the midgut of the mosquito and represents a crucial step in the completion of the parasite's life-cycle and transmission of the disease. Two decades ago, the first sexual stage-specific surface proteins were identified, among them Pfs230, Pfs48/45, and Pfs25, which were of scientific interest as candidates for the development of transmission blocking vaccines. A decade later, gene information gained from the sequencing of the P. falciparum genome led to the identification of numerous additional sexual-stage proteins with antigenic properties and novel enzymes that putatively possess regulatory functions during sexual-stage development. This review aims to summarize the sexual-stage proteins identified to date, to compare their stage specificities and expression patterns and to highlight novel regulative mechanisms of sexual differentiation. The prospective candidacy of select sexual-stage proteins as targets for transmission blocking strategies will be discussed.

Primary immune expulsion of Trichuris muris was markedly delayed by concurrent infection with Nematospiroides dubius. Maximum delay of expulsion was dependent on size and timing of N. dubius infection relative to T. muris infection. In NIH mice infection with 400 N. dubius larvae immediately before or after T. muris infection was found to be most effective in suppressing expulsion. Infection on day 8 of T. muris infection, when mice are sensitized to T. muris, also impaired expulsion. From this evidence it is suggested that the larvae of N. dubius are immunosuppressive and that the efferent role of the immune response to T. muris is inhibited. The results are discussed in terms of non-specific immunosuppression and their relevance to the tropical disease situation is emphasized.

Despite the fact that eflornithine was considered as the safer drug to treat human African trypanosomiasis (HAT) and has been freely available since 2001, the difficulties in logistics and cost burden associated with this drug meant that the toxic melarsoprol remained the drug of choice. The World Health Organization responded to the situation by designing a medical kit containing all the materials needed to use eflornithine, and by implementing a training and drugs distribution programme which has allowed a transition to this much safer treatment. The introduction of the combination of nifurtimox and eflornithine (NECT) has accelerated the shift from melarsoprol to the best treatment available, due to reduced dosage and treatment time for eflornithine that has significantly lessened the cost and improved the burden of logistics encountered during treatment and distribution. The decrease in the use of more dangerous but cheaper melarsoprol has meant a rise in the per patient cost of treating HAT. Although NECT is cheaper than eflornithine monotherapy, an unexpected consequence has been a continuing rise in the per patient cost of treating HAT. The ethical decision of shifting to the best available treatment imposes a financial burden on HAT control programmes that might render long-term application unsustainable. These factors call for continuing research to provide new safer and more effective drugs that are simple to administer and cheaper when compared to current drugs.

A method was developed, which enables substances to be injected into the feeding structure (syncytium) established by Heterodera schachtii in roots of Arabidopsis thaliana. The technique was used to study the uptake of nutrients by the feeding nematode. The fluorescent dye lucifer yellow CH (LYCH) and fluorescence-labelled dextrans of different molecular weights were injected into the thin and translucent roots of A. thaliana. Such roots are a feature of this plant and they provide optimal conditions for microinjection. Injected LYCH was taken up by feeding juveniles and adults, indicated by the staining of the alimentary duct and the digestive system. Fluorescent dextrans of 3, 10 and 20 kDa but not of 40 and 70 kDa were ingested, suggesting that molecules of a maximum Stokes radius of 3·2 to 4·4 nm are taken up. It is likely that the feeding tube, forming the interface between the plant cytosol and the nematode's digestive system, is responsible for this size exclusion effect. The injected fluorescent substances were not detected in plant cells adjacent to the syncytium or in the root vascular elements. Injections into parts of roots which were infested by several nematodes revealed that feeding H. schachtii individuals may share one syncytium.

Several lines of indirect evidence suggest that subjects with latent infection of the coccidian parasite Toxoplasma gondii have a higher concentration of testosterone than uninfected controls. Here, we searched for direct evidence of latent toxoplasmosis-associated differences in testosterone concentration among a population of 174 female and 91 male students screened for Toxoplasma infection. We have found Toxoplasma-infected men to have a higher concentration of testosterone and Toxoplasma-infected women to have a lower concentration of testosterone than Toxoplasma-free controls. The opposite direction of the testosterone shift in men compared to women can explain the observed gender specificity of behavioural shifts in Toxoplasma-infected subjects.

We have investigated the development of the schistosome egg and its secretions in order to understand how it migrates through gut tissues and also initiates pathology in the liver. We show by electron microscopy that the subshell envelope is absent in the newly deposited egg, but appears very early and differentiates as development progresses. In the mature egg, this nucleated envelope contains extensive endoplasmic reticulum, suggestive of a protein synthetic capacity. Furthermore, Reynolds' layer only appears between the envelope and the egg-shell in the mature egg and may represent its accumulated secretions. We have biosynthetically labelled and collected the secretions (ESP) released by mature but not immature eggs during culture. Their fractionation by SDS–PAGE reveals a simple pattern of 6 bands, differing markedly in composition from soluble egg antigen preparations. Electrophoresis in casein substrate gels demonstrates the presence of 2 distinct proteases in the egg secretions. By immunocytochemistry, ESP localized predominantly to the envelope of the mature egg, suggesting that this layer rather than the miracidium is the source of egg secretions.

The successful cultivation of a variety of haemoflagellates in three different liquid media is reported. These media include medium 199, Grace's insect tissue-culture medium and Schneider's drosophila medium, each in combination with 30% (v/v) foetal calf serum. These media were used to cultivate Old and New World species of visceral and cutaneous human Leishmania, as well as Leishmania species isolated from sandflies, rodents, and reptiles. Four strains of Trypanosoma cruzi, an isolate of T. rangeli and an isolate of T. lewisi have also been cultivated in these media. One or more of these media have been used to cultivate 121 strains of haemoflagellates, including at least 14 different species (11 Leishmania and 3 Trypanosoma) and many geographic isolates or strains. The Leishmania include L. braziliensis, L. peruviana, L. mexicana, L. tropica, L. donovani, L. chagasi, L. enriettii, L. hertigi, L. hoogstraali, L. adleri, and L. agamae. Using the Schneider's based medium, we have obtained primary isolates of both cutaneous and visceral Leishmania of man and of experimentally infected laboratory rodents and canines. Freeze-dried preparations of the Schneider's based medium that were reconstituted with distilled water after 24 months of storage at ambient temperature have proven to be suitable cultivation media. This feature makes the media valuable field tools.

The various species of human Leishmania cultivated in these media have in our experience demonstrated no differences in growth rate, viability after liquid nitrogen preservation, or infectivity for laboratory animals and tissue-culture cells compared with promastigotes derived from blood-agar cultivation.

Anti-parasitic drugs may achieve their therapeutic effect either by direct activity against the pathogenic organism, or by altering host factors which lead to parasite killing. In this review, we discuss the evidence for an indirect mode of action for one major anti-filarial drug, diethylcarbamazine (DEC). The interpretation most consistent with existing data is that DEC alters arachidonic acid metabolism in microfilariae and in host endothelial cells. These changes may result in vasoconstriction and amplified endothelial adhesion leading to immobilization of microfilarial parasites, enhanced adherence and cytotoxic activity by host platelets and granulocytes. These events would represent activation of the innate, non-specific immune system, independent of the adaptive, antigen-specific, immune response. This model explains the paradox between rapid clearance in vivo and the lack of an in vitro effect, as well as the efficacy of DEC in non-immune animals.

The proportion of blood meals taken on humans which are infectious to mosquitoes in the Madang area, Papua New Guinea was estimated by two methods. In the first, laboratory reared Anopheles farauti were fed on individuals of all ages at village surveys. The results showed that 3·8% of people were infectious and that the mean percentage of mosquitoes which became infected by feeding on these people was 37·9%. From the average proportion of mosquitoes infected, the probability that a mosquito feeding on a human would pick up infection was 0·013±0·005. In the second approach mosquitoes were fed on identified Plasmodium falciparum, P. vivax and P. malariae gametocyte carriers. The results indicated that 46% of gametocyte carriers were infectious and that the mean probability of a mosquito becoming infected after feeding on a gametocyte carrier was 0·151±0·029. Gametocyte prevalence rates in all ages measured over 18 months in three villages averaged 3·3% P. falciparum, 4·0% P. vivax and 0·7% P. malariae, totalling 8·0±0·7%. Combining gametocyte prevalence rates with the probability of a mosquito becoming infected from a gametocyte carrier, the probability of a mosquito becoming infected following a blood meal on a member of the human population was estimated to be 0·012±0·003.