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

An epidemiological survey was conducted during a 4-month period of intense malaria transmission in Dielmo, a holoendemic Senegalese village. Two thick blood smears per inhabitant were collected weekly. The sex ratio of Plasmodium falciparum gametocytes (gamete precursors) was studied in 50 gametocyte carriers. All age classes were represented (mean 19·7 years; range: 2 months–75 years); 42 (84%) of them did not receive antimalarial treatment. Overall 668 thick smears were examined until 100 gametocytes had been counted or for 40 min. A total of 11 204 gametocytes were observed with a mean sex ratio of 0·346 (95% CI 0·317–0·374), i.e. 2·89 females per 1 male. Among the 284 thick smears in which at least 10 gametocytes were observed, the mean percentage of male gametocytes was 27·8%, with a range of 0–82%. Great variability was observed between gametocyte carriers and also between thick smears from the same gametocyte carrier. A multivariate analysis was performed which highlighted the fact that only 2 variables had a significant effect on the sex ratio. Anaemia was associated with an increased percentage of males (Prevalence Rate Ratio [PPR] of male gametocytes was multiplied by 1·65 if haematocrit rate <32%) and a wave of gametocytes was associated with an increased percentage of female gametocytes (PRR was multiplied by 0·48 during the peak of gametocytaemia and for the 2 weeks following this peak). The variables without significant effect on sex ratio were: age, sex, clinical status and sickle cell trait status of the gametocyte carrier, density of asexual parasites, quinine treatment, and gametocyte density (when taking account of its waves). These results are discussed in regard of possible differential production, mortality or sequestration of one gametocyte sex and selective advantages for the transmission of parasites.

We studied infection dynamics of Plasmodium chabaudi in mice infected with 3 genetically distinct clones – 1 less virulent than the other 2 – either on their own or in mixtures. During the acute phase of infection, total numbers of asexual parasites in mixed-clone infections were equal to those produced by the 3 clones alone, suggesting strong in-host competition among clones. During the chronic phase of the infection, mixed-clone infections produced more asexual parasites than single-clone infections, suggesting lower levels of competition than during the acute phase, and indicating that a genetically diverse infection is harder to control by the host immune system. Transmission potential over the whole course of infection was lower from mixed-clone infections than from the average of the 3 single-clone infections. These results suggest that in-host competition reduces both growth rate and probability of transmission for individual parasite clones.

We have studied the genomic organization and transcription of the histone H2A genes in the protozoan parasite Leishmania infantum. In the parasite genome 2 gene clusters exist, each containing 3 H2A gene copies. Sequence analysesNucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers AJ419625, AJ419626 and AJ419627. showed the existence of significant sequence divergence among the H2A genes, mainly in their 5′- and 3′-untranslated regions (UTRs). Also, the existence of allelic alternatives has been evidenced. Based on the divergence in the 3′UTR regions, we have defined 3 classes of H2A transcripts, which are present at different levels in L. infantum promastigotes. However, transcription of the 3 classes of H2A genes occurs at similar levels, as measured by nuclear run-on assays, indicating that their abundance is regulated post-transcriptionally. Also, differences in regulation were observed among the H2A transcripts: the levels of transcripts with 3′-UTR type I and type III are affected by growth phase whereas transcripts with 3′-UTR type II, that are barely detected, remain constant. It is likely that the complexity, in both gene organization and differential expression exhibited by the L. infantum H2A genes, is imposed by the nature of the post-transcriptional mechanisms of regulation operating in this parasite.

Babesia microti (Apicomplexa: Piroplasmida) has historically been considered a common parasite of Holarctic rodents. However, human babesiosis due to this species has generally been limited to the northeastern seaboard of the United States and Minnesota and Wisconsin. The absence of reports of B. microti babesiosis from sites where the agent is enzootic, such as in western Europe, remains unexplained. Previous work focusing on the 18S rDNA demonstrates little sequence diversity among samples from allopatric host populations across a wide geographical area. It may be that genetic diversity is underestimated due to sample size or the gene analysed. Accordingly, we collected blood or spleen samples from American or Eurasian animals with parasites that were morphologically consistent with B. microti, amplified the 18S rDNA and beta-tubulin gene, and conducted phylogenetic analysis. Surprisingly, what was considered to be ‘B. microti’ by microscopy appears to be a diverse species complex. We identify 3 distinct clades within this complex, including parasites from non-rodent hosts. Rodent parasites comprise 2 clades, one representing zoonotic isolates, and the other apparently maintained in microtine rodents, and therefore their morphological detection within animals from a site does not necessarily imply a risk to public health.

The prophylactic efficacy of autoclaved Leishmania donovani (ALD) and autoclaved L. major (ALM) – a heterologous vaccine developed against cutaneous leishmaniasis (used as a reference vaccine), along with BCG – was evaluated against L. donovani in hamsters (Mesocricetus auratus). Animals were immunized with triple doses (21 days apart) of either ALD or ALM (1·0 mg) with or without BCG (0·1 mg) and challenged 21 days later with 1×106L. donovani amastigotes intracardially. Animals immunized with ALM+BCG and ALD+BCG yielded 94·3% and 86·1% parasite inhibition respectively in comparison to the BCG only and unvaccinated controls. Fifty and 33·3% of the vaccinated animals (ALM+BCG and ALD+BCG respectively) were completely devoid of parasites when tested on day 45 post-challenge (p.c.) and survived till the experiment was terminated. The mean survival of ALM+BCG and ALD+BCG groups (animals harbouring parasites) was longest (168 and 139 days respectively). No significant increase in anti-leishmanial antibody level (ELISA) was noticed in ALD+BCG and ALM+BCG groups whereas it increased progressively in the rest of the experimental groups. The lymphoproliferative responses to PHA and Con A, of the 2 vaccinated groups were comparable to that of normal controls on day 45 p.c. The study suggests that ALD along with BCG can offer substantial protection against visceral leishmaniasis in hamsters.

Studies of invertebrate–parasite interactions frequently report that infection reduces host fecundity. The extent of the reduction is likely to be determined by a wide range of host and parasite factors. We conducted a laboratory experiment to evaluate the role of parasite genetics and infection genetic diversity on the fecundity of mosquitoes carrying malaria parasites. The malaria vector Anopheles stephensi was infected with either of 2 different genotypes of the rodent malaria parasite Plasmodium chabaudi, or by a mixture of both. Mixed genotype infections reduced mosquito fecundity by 20%, significantly more than either of the 2 single genotype infections. Mixed genotype infections were associated with high gametocyte densities and anaemia in mice, both of which were correlated with reduced bloodmeal size in mosquitoes. Bloodmeal size was the most important predictor of mosquito fecundity; the presence and number of parasites had no direct effect. Parasite density influenced the propensity of mosquitoes to feed on infected mice, with a higher percentage of mosquitoes taking a meal as asexual parasite and gametocyte density increased. Thus mosquitoes may preferentially feed on hosts who will most impair their fecundity.

Riboprinting is one of several molecular methods that can generate comparative data independently of the complexity of the organism's morphology. Restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Leishmania spp. yields a typical ‘riboprint’ profile that can vary intraspecifically. A selection of 76 stocks of L. major and L. tropica, isolated from patients with cutaneous leishmaniasis, was analysed by riboprinting to assess divergence within and between species. L. major and L. tropica could be easily differentiated from each other. Analysis of PCR–RFLP profiles indicated that stocks of Leishmania spp. could be broadly partitioned into 2 species corresponding to L. major and L. tropica. To test if ribosomal 18S sequences were homogeneous within each species, several isolates of each of the Leishmania spp. were digested. Interpretation of the riboprint profiles of the 18S independently amplified by PCR, there would appear to be one restriction pattern present within each Leishmania spp. Homogeneity within copies of the ribosomal 18S within a single genome has, therefore, been demonstrated. The species designation established by riboprinting results were in agreement with the zymodeme analysis of the same isolates. The restriction patterns produced were simple, reproducible and easy to interpret.

We evaluated the growth inhibitory efficacy of the imidazole derivatives, clotrimazole (CLT) and ketoconazole (KC), and the herbicide clodinafop-propargyl (CP), in in vitro cultures of Babesia bovis and B. bigemina. Clotrimazole was effective in a dose range of 15 to 60 μM (IC50: 11 and 23·5 μM), followed by KC (50 to 100 μM; IC50: 50 and 32 μM) and CP (500 μM; IC50: 265 and 390 μM). In transmission electron microscopy, extensive damage was observed in the cytoplasm of drug-treated parasites. Combinations of CLT/KC, CLT/CP and CLT/KC/CP acted synergistically in both parasites. In contrast, the combination of KC/CP was exclusively effective in B. bovis, but not in B. bigemina.

A female-specific sequence was isolated from a cDNA library of Schistosoma mansoni and further characterizedSequences reported here are available from DDBJ/EMBL/GenBank under the Accession number Z27402.. Expression of the corresponding gene (p19) depends on pairing with a male. In situ hybridization and immunohistology experiments revealed exclusive expression of the gene in the cells of the vitellarium, suggesting a function in egg formation. In addition, experimental evidence for cross-linking of the protein under oxidative conditions supports the assumption that the p19 gene may code for an egg-shell precursor protein.

Echinococcosis is one of the world's most geographically widespread parasitic zoonoses, with transmission occurring in tropical, temperate and arctic biomes. Most human infections are due to Echinococcus granulosus transmitted between domestic dogs and livestock, but this cosmopolitan species also cycles between wild carnivores (principally canids) and wild ungulates. The other species with significant zoonotic potential is E. multilocularis that occurs naturally in fox definitive hosts and small mammal intermediate hosts. These two species cause human cystic or alveolar echinococcosis respectively, which may be considered serious public health problems in several regions including developed countries. This review provides an introductory overview to the Supplement and summarises the biology and epidemiology of these two related cestodes with an emphasis on applied aspects relating to detection, diagnosis and surveillance in animal and human populations, and includes aspects of transmission ecology, and also considers aspects of community epidemiology and potential for control.

The sex ratios of malaria and related Apicomplexan parasites play a major role in transmission success. Here, we address 2 fundamental issues in the sex ratios of the rodent malaria parasite, Plasmodium chabaudi. First we test the accuracy of empirical methods for estimating sex ratios in malaria parasites, and show that sex ratios made with standard thin smears may overestimate the proportion of female gametocytes. Secondly, we test whether the mortality rate differs between male and female gametocytes, as assumed by sex ratio theory. Conventional application of sex ratio theory to malaria parasites assumes that the primary sex ratio can be accurately determined from mature gametocytes circulating in the peripheral circulation. We stopped gametocyte production with chloroquine in order to study a cohort of gametocytes in vitro. The mortality rate was significantly higher for female gametocytes, with an average half-life of 8 h for female gametocytes and 16 h for male gametocytes.

The last 30 years have seen an impressive use of ultrasonography (US) in many fields of veterinary and clinical medicine and the technique is being increasingly applied to a wide variety of parasitic infections including the cestode zoonoses Echinococcus granulosus and E. multilocularis. US provides real-time results which are permanently recordable with a high resolution and diagnostic accuracy. These properties, coupled with the clinical value of the images obtained and the non-invasive nature of the test which is safe, require no special patient preparation time; it is easy to operate and this has resulted in the establishment of US as the diagnostic technique of choice for cystic (CE) and alveolar (AE) echinococcosis. The lack of ionizing radiation and side-effects mean that examination times are not restricted. The hand-held probes facilitate what amounts to a rapid, bloodless non-invasive laparotomy, enabling a search from an infinite number of angles for lesions producing information on their number, size and type of cysts, their location and clinical implications. Such clinical information has facilitated the development of treatment protocols for different cyst types. Less invasive surgical techniques, such as US guidance for PAIR (Puncture, Aspiration, Injection, Re-aspiration), PAIRD (PAIR plus Drainage) or PPDC (Percutaneous Puncture with Drainage and Curettage) are also possible. Longitudinal US studies have facilitated monitoring the effects of the outcome of treatment and chemotherapy. Portable ultrasound scanners which today weigh as little as a few pounds, powered by battery or generators have facilitated the use of the technique in mass community-based screening studies. The majority of these studies have been conducted in remote, low socio-economic areas where there were few, if any, hospitals, veterinary facilities, schools or trained personnel. The surveys led to the discovery of unexpectedly high prevalences of CE and AE in asymptomatic individuals of endemic areas and especially amongst transhumant or nomadic pastoralists living in various parts of the world. Screening for CE and AE is justified as an early diagnosis leads to a better prognosis following treatment. The application of US in field and clinical settings has led to a better understanding of the natural history of CE and AE and to the development of a WHO standardized classification of cyst types for CE. This classification can be used in helping define the treatment options for the different cysts found during the surveys, which in turn can also be used to calculate the public health cost of treating the disease in an endemic community. The case mix revealed can also influence the specificity (particularly proportions of cyst types CE4 and CE5 and cystic lesions – CL) of US as a diagnostic test in a particular setting. Community based US surveys have provided new insights into the public health importance of CE and AE in different endemic settings. By screening whole populations they disclose the true extent of the disease and reveal particular age and sex risk factors. Through the treatment and follow-up of all infected cases found during the mass screening surveys a drastic reduction in the public health impact of the disease in endemic communities can be achieved. Educational impacts of such surveys at the national, community and individual levels for both professional and lay people are beginning to be appreciated. The translation of the information gained into active control programmes remains to be realized. In areas where intermediate hosts, such as sheep and goats, are not slaughtered in large numbers mass US screening surveys to determine the prevalence of CE in livestock has proved possible. Longitudinal studies in such intermediate hosts would reveal changes in prevalence over time, which has been used as a marker for control success in other programmes. Mass US screening surveys in an ongoing control programme in Argentina has demonstrated the early impact of control in the human population and identified breakthroughs in that control programme. Mass US screening surveys must adhere to the highest ethical standards and the outcome of surveys should result in the application of appropriate WHO recommended treatment options for different cyst types. Follow-up strategies have to be in place prior to the implementation of such surveys for all infected individuals who do not require treatment and for all suspected, but not confirmed, cases found during the surveys. The use of US in community screening surveys has revealed the complexity of ethical issues (informed consent, confidentiality, follow-up, detection of lesions that are not the focus of the study etc) and also provided real solutions to providing the most ethical guidelines for the early detection and treatment of CE and AE.

Previous experimental gametocyte infections of Anopheles arabiensis on 3 volunteers naturally infected with Plasmodium falciparum were conducted in Senegal. They showed that gametocyte counts in the mosquitoes are, like macroparasite intakes, heterogeneous (overdispersed). They followed a negative binomial distribution, the overdispersion coefficient seeming constant (k=3·1). To try to explain this heterogeneity, we used an individual-based model (IBM), simulating the behaviour of gametocytes in the human blood circulation and their ingestion by mosquitoes. The hypothesis was that there exists a clustering of the gametocytes in the capillaries. From a series of simulations, in the case of clustering the following results were obtained: (i) the distribution of the gametocytes ingested by the mosquitoes followed a negative binomial, (ii) the k coefficient significantly increased with the density of circulating gametocytes. To validate this model result, 2 more experiments were conducted in Cameroon. Pooled experiments showed a distinct density dependency of the k-values. The simulation results and the experimental results were thus in agreement and suggested that an aggregation process at the microscopic level might produce the density-dependent overdispersion at the macroscopic level. Simulations also suggested that the clustering of gametocytes might facilitate fertilization of gametes.

The Kato-Katz method is widely used for diagnosing helminth infections in epidemiological surveys, but is known to have a low sensitivity. In the case of Schistosoma mansoni, statistical methods have been developed to compensate for the poor sensitivity, but the same is not true of any other helminth parasite, or infections with multiple-helminth species. We screened 101 schoolchildren from a rural area of Côte d'Ivoire over 5 consecutive days and made 5 Kato-Katz readings from each stool specimen. We estimated single and dual-species infections with S. mansoni and hookworm based on raw egg count data and after developing a latent-class model. The cumulative prevalence of co-infections was estimated at 9·9% after reading slides on the first day, and 57·0% after reading all 25 slides per person. The latent class model yielded a co-infection prevalence estimate of 79·6%, with marginal prevalence estimates for hookworm and S. mansoni infections of 83·9% and 91·6% respectively. The sensitivities of a single Kato-Katz thick smear for detection of S. mansoni alone, hookworms alone, or S. mansoni plus hookworms were 22·4%, 8·0% and 17·7%, respectively. In the current setting this could be attributable to low infection intensities of both parasites, combined with intra-specimen and day-to-day variation in egg output. If confirmed in other settings, these findings have implications for estimating the prevalence of multiple species helminth infections, and hence the design and implementation of efficacious and cost-effective control programmes.

This study reports the development of a novel multiplex PCR assay based on SCAR (Sequence-Characterised Amplified Region) markers for the simultaneous diagnosis of the 7 Eimeria species that infect domestic fowl. Primer pairs specific for each species were designed in order to generate a ladder of amplification products ranging from 200 to 811 bp. Sensitivity tests for each species were carried out, showing a detection threshold of 1–5 pg, which corresponds approximately to 2–8 sporulated oocysts. Distinct isolates of the 7 Eimeria species from different geographical sources were tested and successfully detected by the assay. All the species were amplified homogeneously, whether or not one of them was present in a high quantity, indicating that there was no cross-interference. The assay was also tested with different sources of Taq DNA polymerase and thermocycler models, confirming the high reproducibility of the reaction. The economy of consumables and labour represented by a single-tube reaction greatly facilitates the molecular diagnosis of a large number of samples, making it appropriate for field epizootiological surveys. We propose the use of this multiplex PCR assay as a rapid and cost-effective diagnostic method for the detection and discrimination of the 7 Eimeria species that infect domestic fowl.

There is no published account which allows the morphological discrimination of microfilariae of Onchocerca volvulus and M. ozzardi from each other. However, they occur together in parts of Brazil and Venezuela, and presumably there is always the possibility that migration could establish new sympatric populations in the future. The objective of this study was to evaluate simple morphological characters that might be used for species-diagnosis of microfilariae. The conclusions were that the location of microfilariae in the blood or skin, the body size and the nucleation of the nerve ring are expected to be useful first indications of species identity, but cannot be used for confident diagnosis. The structure of the cephalic armature (stained with alcian blue) seems to be species specific, but is of limited application because it is often difficult to see. However, the pattern of nucleation of the tail (as expressed by the ratio of the length of the terminal nucleus compared with the length of the tail space) is distinctive and is expected to be diagnostic.

The northern biotype of Echinococcus granulosus occurs in North America and northern Eurasia in life-cycles involving cervids. Previously, cervid isolates of E. granulosus from North America have been characterized using molecular genetic techniques as the G8 genotype. In this study, 5 isolates of E. granulosus were collected from 4 reindeer and 1 moose in north-eastern Finland. DNA sequences within regions of mitochondrial cytochrome c oxidase I (COI) and NADH dehydrogenase I (NDI) genes and the internal transcribed spacer 1 (ITS-1) fragment of the ribosomal DNA were analysed. The mitochondrial nucleotide sequences were identical in all isolates, but high sequence variation was found in the ITS-1 region. Mitochondrial and nuclear sequences of the Finnish cervid E. granulosus and the camel strain (G6) of E. granulosus resembled closely each other. According to phylogenetic analyses, the Finnish isolates have close relationships also with the pig (G7) and cattle (G5) strains. Although some similarities were found with the previously published North American cervid strain (G8), particularly in the NDI sequence and some of the ITS-1 clones, the Finnish E. granulosus form represents a distinct, previously undescribed genotype of E. granulosus. The novel genotype is hereby named as the Fennoscandian cervid strain (G10).