This review of protozoan and helminth infections in pregnancy focuses on the impact on the immune response in the newborn infant to maternal infection. Studies of protozoan and helminth infections in pregnant women and in their offspring have shown that children exposed to antigens or microorganisms during pregnancy often have a reduced immune response to these infections. The most common finding is a reduced IFNγ response to specific antigens regardless of specific infection studied. In some studies the impaired immune response disappeared before the age of one year, while in other studies the impaired immune response was present as much as two decades after birth. Data from chronic viral infections like Rubella, cytomegalovirus and hepatitis B also show that congenital or perinatal infections may result in a life-long inability to control the infections. Studies of both helminth and protozoan infections show that children exposed to antigens during gestation have a microorganism-specific impaired immune response which is characterized by reduced IFN-γ and stimulation of responses to specific antigens.

In this study, cDNA microarray analysis of a closely related species, Leishmania major, was used as a screening tool to compare antimonial-resistant and susceptible clinical isolates of Leishmania donovani in order to to identify candidate genes on the basis of antimony resistance. Clinically confirmed resistant isolate 39 and sensitive isolate 2001 were used in this study. Many differentially regulated genes were identified whose expression levels differ in sodium antimony gluconate (SAG)-treated patients. Interestingly, genes on the array, showing changes in expression of over 2-fold revealed the identity of ABC transporters, which are known determinants of drug resistance in laboratory mutants. The functionality of the transporters was validated by flow cytometry which, being biologically informative, provides direct clues to gene function. The results suggest that isolate 39 could have developed resistance by an increased multidrug resistance protein (MRP)-like pump. This study provides preliminary clues to the role of a thiol-dependent efflux system in antimonial resistant clinical isolates of Leishmania donovani.

The heteroxenous protozoan parasite Toxoplasma gondii is transmitted from the intermediate host (any warm-blooded animal) to the definitive host (members of the felidae) by carnivory. The infected intermediate hosts develop several specific behavioural changes that are usually considered products of manipulative activity of the parasite aimed to increase the probability of its transmission to the definitive host. Among other changes, the infected rodents were shown to have impaired learning capability. All previous studies were done 2–6 weeks after the infection. Therefore, it was difficult to resolve whether the observed impairment of learning processes was a result of acute or latent toxoplasmosis, i.e. whether it was a side-effect of the disease or a product of manipulation activity. Here we studied the learning capability of Toxoplasma-infected mice in the static rod test and 8-arm radial maze test and their spontaneous activity in the wheel running test 10 weeks after the infection. The infected mice achieved worse scores in the learning tests but showed higher spontaneous activity in the wheel running test. However, a detailed study of the obtained results as well as of the data reported by other authors suggested that the differences between infected and control mice were a result of impaired ability to recognize novel stimuli rather than of impaired learning capacity in animals with latent toxoplasmosis.

Neospora caninum causes economical impact in cattle-raising farms since it is implicated as the major cause of bovine abortions. Although infection by the parasite has been widely described in mammals, the role of birds in its life-cycle is still obscure. Therefore, this work aimed to evaluate the infection by N. caninum in different chicken models. Experimental infections were conducted in 7-day-old chicks, laying hens and embryonated eggs, where samples were analysed for parasite burden, IgG antibodies and lesions promoted. Chickens demonstrated an asymptomatic infection, although with seroconversion and systemic replication of the parasite. In laying hens, no signs of vertical transmission were observed. However, embryonated eggs inoculated by the allantoic cavity route demonstrated susceptibility to infection, with mortality rates around 50% independent of the inoculum dose. Additionally, dogs became infected after ingestion of different amounts of inoculated eggs, producing either oocysts or specific IgG antibodies. The results herein presented demonstrate that chickens may be intermediate hosts of N. caninum and that embryonated eggs could be a useful model to study the parasite's biology.

The feral Soay sheep (Ovis aries L.) population on Hirta, St Kilda, is host to a diverse component parasite community, but previous parasitological studies of the population have only focussed on the metazoan species. This paper reports the first epidemiological study of the protozoan species comprising Cryptosporidium parvum, Giardia duodenalis and 11 species of Eimeria in Soay sheep across 3 years of varying host population density. Prevalence and intensity of almost all species of protozoa significantly decreased with host age, with the exception of E. granulosa, which increased in prevalence with host age. The prevalence of C. parvum appeared to vary positively with host population density but that of G. duodenalis did not vary significantly with density. Most species of Eimeria showed a distinct lag in infection level following the host population crash of 2002, taking up to 2 years to decrease. Mixed Eimeria species intensity and diversity were highest in 2002, a year of low host density. Parasite diversity decreased with host age and was higher in males. There were 5 positive pair-wise associations between protozoa species in terms of prevalence. The results of this study highlight the potential for protozoal infection to shape the evolution of parasite resistance in wild host populations harbouring diverse parasite species.

Ascaris is a large parasitic roundworm (nematode) of the small intestine of humans and pigs, which causes the socio-economically important disease, ascariasis. To better understand the relationship of Ascaris between the 2 host species, recent studies in China have focused on investigating the genetics and epidemiology of Ascaris from humans and pigs using a mutation scanning-based approach. Findings provided support for a low level of gene flow between the human and porcine Ascaris populations. Extending the studies of genotypic variability within Ascaris from humans and pigs, experimental infections of mice and pigs with selected genotypes of Ascaris were carried out. Initial results indicate that there is a significant difference in the ability of Ascaris eggs of genotype G1 (derived from human) and G3 (derived from pig) to infect and establish as adults in pigs, supporting the difference in the frequencies of these genotypes in natural Ascaris populations between pigs and humans in China. Taken together, current information supports that there is limited cross-infection of Ascaris between humans and pigs in endemic regions and that pigs are not a significant reservoir of human infection with the adult nematode in such areas.

Serum-derived fatty acids are essential for the intraerythrocytic proliferation of Plasmodium falciparum in humans. We previously reported that only limited combinations of fatty acids can support long-term parasite culture, and palmitic acid (C16:0)/oleic acid (C18:1, n-9), palmitic acid (C16:0)/vaccenic acid (C18:1, n-7), or stearic acid (C18:0) are required in these combinations, implying that these fatty acids are key molecules for intraerythrocytic parasite growth (Mi-Ichi et al.2006). Here, we analysed profiles of parasitaemia changes as well as morphologies during the erythrocytic cycle and confirmed the importance of C16:0 and C18:1, n-9. We also provide evidence that C18:1, n-9 but not other C18 monoenoic or dienoic acids maintain the synchronicity of parasite development in serum-free medium when paired with C16:0, resulting in maintained exponential growth. Thus, C18:1, n-9 is indispensable for the intraerythrocytic proliferation of P. falciparum.

The present report deals with the status of hepatic and splenic glutathione-S-transferase (GST) activities in mice during experimental infection with Plasmodium yoelii nigeriensis and subsequent treatment of infected mice with mefloquine (Mf) and menadione (Md). The infection caused significant decline in the hepatic and splenic glutathione-S-transferase (GST) activities of albino and db/+ mice. The decline was observed in the levels of both cytosolic and microsomal GST(s) of liver and spleen in both types of mice. Intraperitoneal administration of mefloquine at a dose of 5 mg/kg and menadione at a dose of 100 mg/kg, twice daily from day 1 p.i. (day 0) until day 10, caused restoration in the levels of hepatic as well as splenic GST(s), albeit to varying degrees. Mf was able to suppress parasitaemia by day 5 in the case of albino mice and by day 3 in the case of db/+ mice but was unable to cure both types of mice completely. On the other hand, Md caused a delay in maturation of infection in both cases, but could not cure the mice.

Bonamia ostreae is a protozoan parasite that infects the European flat oyster Ostrea edulis, causing systemic infections and resulting in massive mortalities in populations of this valuable bivalve species. In this work, we have characterized B. ostreae actin genes and used their sequences for a phylogenetic analysis. Design of different primer sets was necessary to amplify the central coding region of actin genes of B. ostreae. Characterization of the sequences and their amplification in different samples demonstrated the presence of 2 intragenomic actin genes in B. ostreae, without any intron. The phylogenetic analysis placed B. ostreae in a clade with Minchinia tapetis, Minchinia teredinis and Haplosporidium costale as its closest relatives, and demonstrated that the paralogous actin genes found in Bonamia resulted from a duplication of the original actin gene after the Bonamia origin.

The mechanisms involved in the pathogenesis of chagasic megacolon are not completely characterized. Although autoimmunity may play a role in the pathogenesis of Chagas' disease, recent studies suggest a positive association of tissue parasitism, inflammation, and severity of lesions. The aim of this study was to evaluate the role of inflammatory cells and the occurrence of fibrosis in the colon of chagasic patients with and without megacolon. Samples from 26 patients were randomly selected and paraffin-embedded tissue blocks were sectioned and evaluated by histology and immunohistochemistry to analyse the occurrence and relation among eosinophils, mast cells, macrophages and fibrosis. Section analyses showed that the presence of eosinophils and mast cells in the analysed inflammatory cells has a direct correlation with fibrosis density in the chagasic megacolon. These data suggest that the megacolon's pathogenesis is based on a continuous process of cell damage. Our data propose that eosinophils, mast cells and macrophages may have a direct connection with the occurrence of fibrosis in the colon of chagasic patients. We believe that potential therapeutic agents against these cells could avoid the fibrosis process and contribute to prevent the development of chagasic megacolon.

P. falciparum malaria severely affects pregnant women and children. Despite immunity through lifelong exposure to malaria, pregnant women become susceptible to infections causing anaemia, abortions and low birth weight. They experience massive accumulation of infected erythrocytes (IEs) in the placenta. Adhesion of IEs to host endothelial receptors is mediated by members of a large diverse protein family called P. falciparum erythrocyte membrane protein 1 (PfEMP1). Pregnancy malaria is generally associated with the emergence of a distinct subset of parasites expressing a unique PfEMP1 that binds to the host-receptor chondroitin sulfate A (CSA). Resistance to pregnancy malaria is associated with the acquisition of antibodies that block IEs binding to placental CSA. The absence (or rare occurrence) of CSA-binding parasites in malaria patients (children, men and non-pregnant women) suggests that these parasites become virulent only during pregnancy. The molecular mechanisms used by P. falciparum to achieve the timely expression of the Pf-CSA ligand in pregnant women remain puzzling. In this review we will discuss two hypothetical mechanisms by which CSA-binding parasites may arise during pregnancy. The first, a selection process by the placenta of a distinct sub-population of P. falciparum expressing a particular PfEMP1. The second, an induction mechanism that facilitates the expression of a particular PfEMP1 protein by specific host factor(s) present only during pregnancy.

Multiplication of Plasmodium parasites within human erythrocytes is essential to malarial disease. The cell-division cycle of this organism, however, is still poorly understood. In other eukaryotes, various techniques for (apparent) cell-cycle synchronization have been used to shed light on the mechanisms involved in cell division and its control. Thus far there is no technique for cell-cycle synchronization (as opposed to selection of parasites of a limited age-range) in Plasmodium. We therefore investigated the possibility that inhibitors of DNA synthesis, the mitotic spindle, or cell-cycle control elements (such as cyclin-dependent kinases) could be used to synchronize P. falciparum cultures to a particular cell-cycle phase. Surprisingly, most of these compounds did not cause a block at a specific phase. Three compounds, Hoechst 33342, roscovitine and L-mimosine, did block development at the trophozoite–schizont transition (S or G2 phase). The block caused by the latter 2 inhibitors was reversible, suggesting that they might be used as synchronizing agents. However, a consideration of the perturbing effects of inhibitors and problems with ‘batch’ synchronization techniques in general lead us to believe that any results obtained using roscovitine- or L-mimosine-treated parasites may not be reflective of the normal cell cycle.

The ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes, was found to express an antigen that elicits antibodies in rabbits and tiger puffer (Takifugu ruburipes). Serum from rabbits and fish immunized with theronts had agglutination/immobilization activity against theronts in vitro; fish serum antibody levels (measured by enzyme-linked immunosorbent assays: ELISA) correlated with this activity. Anti-theront antibody levels in fish were significantly higher in the immunized group as compared with control fish at 2 weeks after booster immunization (injection of bovine serum albumin; Student's t-test, P<0·01). Biochemical analyses indicated that a Triton X-114-soluble 32 kDa theront integral membrane protein may be the agglutination/immobilization antigen. Indirect immunofluorescence staining of theronts suggested that this 32 kDa antigen was expressed on the surface of cilia. The full-length 32 kDa antigen cDNA contained 1147 basepairs, encoding a 328-amino acid protein including hydrophobic N- and C-termini. As with Tetrahymena and Paramecium spp., TAA and TAG appear to be used as glutamine codons in the 32 kDa antigen gene.

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.

Benzimidazole (BZ) resistance is widespread and appears to be readily selected in a variety of nematode parasites of animals. There have been reports of a lack of efficacy of BZ anthelmintics against soil transmitted nematode parasites of humans. However, resistance to BZs in nematodes of humans has not been confirmed. It is difficult to perform tests to confirm anthelmintic resistance in humans for a variety of technical and ethical reasons. The use of anthelmintic drugs for the control of helminth parasites in people is increasing massively as a result of numerous programmes to control gastrointestinal nematode parasites in children, the Global Program for the Elimination of Lymphatic Filariasis and other programmes. Many of these programmes are dependent on BZ anthelmintics and this will increase the pressure for resistance development to BZ anthelmintics in nematode parasites of people. We need to perform monitoring for anthelmintic resistance in these programmes and we need new tools to make that monitoring sensitive, inexpensive and practical. There is a real need for DNA-based markers for BZ resistance in nematode parasites of humans. We have a reasonable understanding of the molecular mechanisms and genetics of BZ resistance in some nematode parasites of animals and similar mechanisms are likely to prevail in nematodes of humans. Based on the likelihood that similar single nucleotide polymorphisms (SNPs) will be involved in BZ resistance in human, as in animal nematode parasites, rapid SNP assays have been developed for possible BZ resistance development in Wuchereria bancrofti.

Malaria parasites in the genus Plasmodium are now placed within 11 subgenera based on morphology under the light microscope, life-history traits, and host taxon. The phylogenetic significance of these characters, however, is problematic because the observed variation could be homoplasious. Using Plasmodium infections found in 2632 birds of many avian families collected in the USA, and several samples from other locations, we compared identifications to subgenus based on morphology in blood smears with a 2-gene molecular phylogeny (the first for avian Plasmodium) to determine if the 5 avian Plasmodium subgenera represent monophyletic groups. Phylogenetic trees recovered by parsimony, likelihood, and Bayesian methods presented nearly identical topologies. The analysis allowed testing the hypothesis of monophyly for the subgenera. Monophyly of the subgenera Haemamoeba, Huffia, and Bennettinia was supported by the analysis. The distinctive morphology of Haemamoeba species appears to have evolved once. Most samples identified to Novyella also fell within a monophyletic clade with the exception of 2 samples that fell basal to all other avian Plasmodium. Samples of the subgenus Giovannolaia did not form a monophyletic group. Thus, the characters used by parasitologists for over a century to define subgenera of Plasmodium vary in their phylogenetic significance.

The dense granule antigen 4 (GRA4) is known as an immundominant antigen of Toxoplasma gondii and, therefore, is considered as a vaccine candidate. For further evaluation of its vaccine effect, a recombinant plasmid and vaccinia virus, both expressing GRA4, were constructed, and a heterologous prime-boost vaccination regime was performed in a mouse model. The mice immunized with the heterologous prime-boost vaccination regime showed a high level of specific antibody response against GRA4 and a significantly high level of gamma interferon (IFN-γ) production and survived completely against a subsequent challenge infection with a lethal dose of T. gondii. In addition, the formation of cysts was inhibited in the mice vaccinated with the heterologous prime-boost vaccination regime. These results demonstrate that the heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, could induce both humoral and cellular immune responses and provide effective protection against lethal acute and chronic T. gondii infections in mice.

This study, based on correlative data, tests the hypothesis that infections with Eimeria spp. parasites exert a significant loss of fitness of bank voles (Clethrionomys glareolus) reflected in lower reproductive success and survival, declining host population densities and are associated positively with population size. The study was conducted in 20 mainland and 27 island populations in central Finland during May–September in 1999. Faecal samples showed that 28% of 767 individuals were infected with Eimeria spp. The presence of Eimeria parasites was higher in dense mainland populations than in sparsely populated islands. Eimerian infections increased during the course of the breeding season, probably as a result of the high infection rate of young individuals. Accordingly, the body masses of bank voles were negatively related to the presence of Eimeria spp. Reproductive output, as measured by the breeding probability of females and litter size, was not associated with the presence of eimerian infection. Interestingly, the body condition of the infected mothers appeared to be low. Moreover, mother's body condition was the single most important variable studied that showed a positive correlation to pup's body condition at birth. On small islands ([les ]3·2 ha) that were comprehensively trapped, the mean number of Eimeria spp. in the bank vole population was negatively related to density changes of the bank vole population during the study. Our data are consistent with the idea that infection with coccidian parasites may be one of the factors responsible for declining host populations in small, isolated populations.

Since the first description of Marteilia refringens (Paramyxea) in flat oysters Ostrea edulis in 1968 in the Aber Wrach, Brittany (France), the life-cycle of this parasite has remained unknown. However, recent studies, conducted in the ‘claire’ system, have proposed the planktonic copepod Acartia grani as a potential intermediate host for the parasite. Nevertheless, experimental transmission of the parasite through the copepod has failed. Recent studies in this field have reported the presence of the parasite in zooplankton from the bays of the Delta de l'Ebre, a more complex and natural estuarine environment than that of the claire. As a result, 2 new Marteilia host species were proposed: the copepods Oithona sp. (Cyclopoida) and an indeterminate Harpaticoida. Consequently, the objective of the present work was to study the dynamics of Marteilia in the zooplankton community from one of the bays, Alfacs Bay, as well as the dynamics of the parasite in cultivated mussels during 1 complete year. Six different zooplankton taxa appeared to be parasitized by M. refringens, including copepods (3 Calanoida, Acartia discaudata, A. clausi and A. italica; 1 Cyclopoida, Oithona sp.; and 1 Harpacticoida, Euterpina acutifrons), and larval stages of decapod crustaceans (zoea larvae of Brachyura, probably Portumnus sp.). These taxa are thus proposed as new subjects for study, since they could be intermediate hosts in the infection process of mussels by Marteilia.

Epidemiological factors of tick-borne equine piroplasmoses, caused by Theileria equi and Babesia caballi, were investigated using logistic regression (GLM) and general additive models (GAM) based on the prevalences determined in 510 domestic horses and in ticks in S.W. Mongolia by indirect immunofluorescence antibody test (IFAT) and/or multiplex PCR. Prevalences of T. equi and B. caballi in horses were 66·5% (95% CI: 62·1–70·7) and 19·1% (15·6–22·9), respectively by PCR and 78·8% (74·9–82·3) and 65·7% (61·3–69·9) by IFAT. Of 166 ticks analysed from PCR- and IFAT-negative horses 1 was PCR positive for B. caballi and none for T. equi. GAM demonstrated non-linear increasing proportions of T. equi-PCR and -IFAT positive horses with age suggesting persistent infection. In contrast, the B. caballi-PCR prevalence decreased with age despite a concurrent increase in the proportion of IFAT-positive animals suggesting parasite elimination. The tick (Dermacentor nuttalli) burden of the horses increased with age and decreased with advancing season. Geldings were more likely to be infected with, and seroconvert to, T. equi. Neither herd affiliation, date of sample collection nor abundance of tick infestation had a significant influence on parasite prevalence.