An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.

Metabolomics analysis, which aims at the systematic identification and quantification of all metabolites in biological systems, is emerging as a powerful new tool to identify biomarkers of disease, report on cellular responses to environmental perturbation, and to identify the targets of drugs. Here we discuss recent developments in metabolomic analysis, from the perspective of trypanosome research, highlighting remaining challenges and the most promising areas for future research.

Previously, we described the protective action of the immunomodulatory extract of Kalanchoe pinnata (Kp) in murine and human cutaneous leishmaniasis. In the present study, we investigated the effectiveness of Kp against visceral leishmaniasis, using the BALB/c mouse model of infection with Leishmania chagasi. Mice receiving oral daily doses of Kp (400 mg/kg) for 30 days displayed significantly reduced hepatic and splenic parasite burden, when compared with untreated animals. Protectiveness was accompanied by a reduction in parasite-specific IgG serum levels, and impaired capacity of spleen cells to produce IL-4, but not IFN-γ and nitric oxide upon antigen recall in vitro. The reference drug Pentostam (72 mg/kg) given by the intra-peritoneal route on alternate days produced an anti-leishmanial effect similar to oral Kp. Our findings show that the oral efficacy of Kp, seen previously in murine cutaneous leishmaniasis, extends also to visceral leishmaniasis caused by L. chagasi, a difficult to treat and lethal disease of man.

Taenia crassiceps is a cestode parasite of wild and domestic animals that rarely affects humans; it has been widely used as an experimental model. The asexual proliferation by budding is a useful attribute of T. crassiceps cysticerci, which allows the various strains to be maintained indefinitely in the peritoneal cavity of inbred mice. Over the last 50 years, experimental results using larval and adult stages of T. crassiceps have yielded much information on the morphology, infectivity, proliferation dynamics, host immune response, endrocrinological responses and vaccine research, all of which have contributed to our knowledge of cestode biology.

Human African trypanosomiasis (HAT) or sleeping sickness is caused by protozoan parasites Trypanosoma brucei gambiense and T. b. rhodesiense. Despite the enormous technological progress in molecular parasitology in recent years, the diagnosis of HAT is still problematic due to the lack of specific tools. To date, there are two realities when it comes to HAT; the first one being the world of modern experimental laboratories, equipped with the latest state-of-the-art technology, and the second being the world of HAT diagnosis, where the latest semi-commercial test was introduced 30 years ago (Magnus et al.1978). Hence, it appears that the lack of progress in HAT diagnosis is not primarily due to a lack of scientific interest or a lack of research funds, but mainly results from the many obstacles encountered in the translation of basic research into field-applicable diagnostics. This review will provide an overview of current diagnostic methods and highlight specific difficulties in solving the shortcomings of these methods. Future perspectives for accurate, robust, affordable diagnostics will be discussed as well.

Certain species of the protozoan genus Besnoitia cause clinical disease in livestock and wildlife. In the present paper a new species, Besnoitia neotomofelis is described from the southern planes woodrat (Neotoma micropus). The parasite was detected by bioassay of woodrat tissues in outbred Swiss Webster mice in an attempt to isolate Toxoplasma gondii. Initially, the organism was misdiagnosed as T. gondii because it was highly pathogenic for mice and its tachyzoites resembled T. gondii tachyzoites. Further studies revealed that it differed structurally and biologically from T. gondii. Tachyzoites were successfully cultivated and maintained in vitro in bovine monocytes and African green monkey kidney cells, and in vivo in mice. Non-dividing, uninucleate tachyzoites were approximately 1×5 μm in size. Longitudinally-cut bradyzoites in tissue sections measured 1·5–1·6×7·7–9·3 μm. Tissue cysts were microscopic, up to 210 μm long, and were infective orally to mice. Cats fed tissue cysts shed unsporulated 13×14 μm sized oocysts. All mice inoculated with B. neotomofelis died of acute besnoitiosis, irrespective of the dose, and Norwegian rats became infected but remained asymptomatic. Entero-epithelial stages (schizonts, gamonts) were found in cats fed tissue cysts. Large (up to 40×50 μm) first-generation schizonts developed in the lamina propria of the small intestine of cats. A second generation of small sized (8 μm) schizonts containing 4–8 merozoites was detected in enterocytes of the small intestine. Gamonts and oocysts were seen in goblet cells of the small intestinal epithelium. Tachyzoites were present in mesenteric lymph nodes of cats. Phylogenetic analysis indicated that B. neotomofelis was related to other Besnoitia species from rodents, rabbits, and opossums. Besnoitia neotomofelis is distinct from the 3 other species of Besnoitia, B. wallacei, B. darlingi and B. oryctofelisi that utilize cats as a definitive host.

Cryptosporidium parvum is a zoonotic protozoan parasite that mainly affects the ileum of humans and livestock, with the potential to cause severe enteric disease. We describe the complete life cycle of C. parvum in an in vitro system. Infected cultures of the human ileocecal epithelial cell line (HCT-8) were observed over time using electron microscopy. Additional data are presented on the morphology, development and behavioural characteristics of the different life-cycle stages as well as determining their time of occurrence after inoculation. Numerous stages of C. parvum and their behaviour have been visualized and morphologically characterized for the first time using scanning electron microscopy. Further, parasite-host interactions and the effect of C. parvum on host cells were also visualized. An improved understanding of the parasite's biology, proliferation and interactions with host cells will aid in the development of treatments for the disease.

Infection by a parasite often induces behavioural changes in the host and these changes may benefit either the host or the parasite. However, whether these changes are active host defence mechanisms or parasitic manipulations or simply incidental byproducts of the infection is not always clear. It has been suggested that understanding the proximate mechanisms of these changes as well as comparative studies could help distinguish these alternatives better. Behavioural fever is a common response to an infection in many animals and we investigated the phenomenon in the novel host-parasite relationship between the honeybee and the temperature-sensitive microsporidian Nosema ceranae. Our results show that infected bees prefer higher temperatures and even though this seems to benefit the pathogen, the proximate mechanism underlying this change is the pathological stress underlying the infection. Especially because it is a new host-parasite relationship, it is best to label the observed behavioural change as a case of incidental benefit although this does not rule out selection acting on it. We discuss the importance of looking at the behavioural outcomes of host-parasite relationships and the importance of studying them at multiple levels for understanding their origin and maintenance.

The epidemiological and ecological processes which govern the success of multiple-species co-infections are as yet unresolved. Here we investigated prior versus late residency within hosts, meaning which parasite contacts the host first, to determine if the outcomes of intra-host competition are altered. We infected a single genotype of the waterflea Daphnia galeata with both the intestinal protozoan Caullerya mesnili and the haemolymph fungus Metschnikowia sp. (single genotype of each parasite species), as single infections, simultaneous co-infections and as sequential co-infections, with each parasite given 4 days prior residency. Simultaneous co-infections were significantly more virulent than both single infections and sequential co-infections, as measured by a decreased host life span and fecundity. Further, in addition to the Daphnia host, the parasites also suffered fitness decreases in simultaneous co-infections, as measured by spore production. The sequential co-infections, however, had mixed effects: C. mesnili benefited from prior residency, whereas Metschnikowia sp. experienced a decline in fitness. Our results show that multiple-species co-infections of Daphnia may be more virulent than single infections, and that prior residency does not always provide a competitive advantage.

Neurocysticercosis is a public health problem in many developing countries and is the most frequent parasitic disease of the brain. The human tapeworm carrier is the main risk factor for acquiring neurocysticercosis. Since the parasite lodges only in the human intestine, experimental models of Taenia solium taeniosis have been explored. Macaques, pigs, dogs, cats and rabbits are unsuccessful hosts even in immunodepressed status. By contrast, rodents are adequate hosts since tapeworms with mature, pregravid and, in some cases, gravid proglottids develop after infection. In this review, information that has been generated with experimental models of taeniosis due to T. solium is discussed. Initially, the use of the model for immunodiagnosis of human taeniosis and evaluation of intervention measures is summarized. Next, descriptions of tapeworms and comparison of hamsters, gerbils and other mammals as experimental models are discussed, as well as data on the humoral immune response, the inflammatory reaction and the production of cytokines associated to Th1 and Th2 responses in the intestinal mucosa. Finally, evaluation of protection induced against the development of tapeworms by recombinant T. solium calreticulin in hamsters is summarized and compared to other studies.

Prevalence and abundance of Bartonella spp. infections were studied over a 3-year period in woodland and grassland rodents in North-Eastern Poland. Prevalence of bacterial infections was similar in the two rodent communities, with one leading host species in each habitat (46·3% in Apodemus flavicollis versus 29·1% in Myodes glareolus in forest, or 36·9% in Microtus arvalis versus 13·7% in Mi. oeconomus in grassland). Prevalence/abundance of infections varied markedly across the 3 years with 2006 being the year of highest prevalence and abundance. Infections were more common during autumn months in My. glareolus and A. flavicollis, and in juvenile and young adult (age classes 1 and 2) My. glareolus and Mi. oeconomus than in adults (age class 3). Higher prevalence and abundance of Bartonella infections were found in male A. flavicollis in comparison to females. These data are discussed in relation to the parasite genotypes identified in this region and with respect to the role of various ecological factors influencing Bartonella spp. infections in naturally infected host populations.

The blackcap (Sylvia atricapilla) is a common Palearctic migratory warbler, and haemosporidian parasites are common in this species. However, genetic and phenotypic diversity of haemosporidians in warblers has been insufficiently investigated and poorly linked. We addressed this issue by combining molecular and microscopy data for detection of pigment-forming haemosporidians of the genera Haemoproteus and Plasmodium. Blood samples from 498 blackcaps were collected at 7 different sites in Europe and investigated for these parasites by polymerase chain reaction (PCR)-based techniques and microscopic examination. In all, 56% of the birds were infected by at least 1 out of 25 distinct mitochondrial cytochrome b (cyt b) gene lineages of these haemosporidians. It is concluded that the blackcap is infected not only with blackcap specific haemosporidians, but also with Haemoproteus majoris, which is a host generalist and common in birds belonging to the Paridae. Haemoproteus pallidulus sp. nov. is described based on morphology of its blood stages and segments of the cyt b and dihydrofolate reductase/thymidylate synthase (DHFR-TS) genes. This study provides evidence that genetic diversity of haemosporidian parasites might be positively correlated with migratory strategies of their avian hosts; it also contributes to the value of both microscopy and molecular diagnostics of avian blood parasites.

The composition and structure of a community of Eimeria was investigated in a population of Eastern cottontail (Sylvilagus floridanus) introduced into Italy. Eight Eimeria species were found, of which all but 1 had North American origins and were presumably introduced into Italy together with imported cottontails. The success of cottontails in spreading microparasites is probably related to their massive release for hunting purposes. Nearly all cottontails were infected with at least 1 Eimeria species, with bimonthly prevalence ranging from 0–6·3% (E. leporis) to 42·9–89·3% (E. environ). Bayesian model averaging and multivariate techniques were used to investigate the relationships between the occurrence of each parasite and the structure of the relative community. Among the host parameters, only sex was found to be associated with the prevalence of E. honessi, while the rest of the parameters were only weakly correlated with prevalence and species richness. This indicates that individual phenotypic host characteristics are probably less important than environmental factors in determining levels of parasite prevalence and diversity. The community of Eimeria species was probably structured by competition, with less species co-occurrence than expected under a null hypothesis. This was made evident by the low co-occurrence of E. environ and E. neoirresidua with E. poudrei, E. honessi, and E. maior.

Encephalitozoon cuniculi is an obligate intracellular pathogen that has a wide host distribution, but primarily affects rabbits. The aim of this study was to characterize both the cell-mediated and the antibody response in rabbits after experimental infection using 2 different infection routes: oral and ocular. SPF rabbits were infected with low (103 spores) and high (107 spores) infection doses. Monitored parameters included clinical signs, detection of spores in urine, antibody response detected with ELISA, and cell-mediated immunity detected by antigen-driven lymphocyte proliferation. At week 13 post-infection, half of the rabbits in each group were suppressed by intramuscular administration of dexamethasone. At week 18 post-infection, animals were euthanized. Clinical signs were mild with exacerbation after immunosuppression. Spores in urine and antigen-specific cell-mediated immunity were detected from weeks 5 and 4 post-infection, respectively. Specific IgM was detected 1 week after infection, and IgG antibodies followed 1 week later in rabbits infected with the high dose. Immunological responses were dose dependent. The authors can conclude that both oral and ocular experimental infection with E. cuniculi resulted in an immune response of the infected animals. Rabbits could be used as an experimental model for the study of ocular microsporidiosis.

Babesia sp. BQ1 (Lintan) is a Babesia isolated from sheep infested with Haemaphysalis qinghaiensis in China, and is closely related to B. motasi based on the 18S rRNA gene sequence. In the present study, an ELISA was developed with merozoite antigens of Babesia sp. BQ1 (Lintan) (BQMA) purified from in vitro culture. When the positive threshold was chosen as 30% of the antibodies rate, evaluated with 198 negative sera, the specificity was 95·5%. Except for Babesia sp. Tianzhu, there was no cross-reaction between BQMA and positive sera from Babesia sp. BQ1 (Ningxian)-, Babesia sp. Hebei-, Babesia sp. Xinjiang-, Theileria luwenshuni-, T. uilenbergi-, or Anaplasma ovis-infected sheep, which are the dominant haemoparasites of small ruminants in China. Specific antibodies against Babesia sp. BQ1 (Lintan) were produced 1 or 2 weeks post-infection and a high level of antibodies persisted for more than 8 months in experimentally infected sheep. This ELISA was tested on 974 sera collected from field-grazing sheep in 3 counties of Gansu province, northwestern China to evaluate the seroprevalence of Babesia sp. BQ1 (Lintan) infection and the average positive rate was 66·84%. The feasibility of increasing the specificity of this BQMA-based ELISA, by using some BQMA antigens for serodiagnosis is discussed.

Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (~82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

(-)-Epigallocatechin-3-gallate (EGCG) is the major tea catechin and accounts for 50–80% of the total catechin in green tea. (-)-Epigallocatechin-3-gallate has antioxidant, anti-inflammatory, anti-microbial, anti-cancer, and anti-trypanocidal activities. This report describes the inhibitory effect of (-)-Epigallocatechin-3-gallate on the in vitro growth of bovine Babesia parasites and the in vivo growth of the mouse-adapted rodent babesia B. microti. The in vitro growth of the Babesia species was significantly (P<0·05) inhibited in the presence of micromolar concentrations of EGCG (IC50 values=18 and 25 μM for B. bovis, and B. bigemina, respectively). The parasites showed no re-growth at 25 μM for B. bovis and B. bigemina in the subsequent viability test. The drug significantly (P<0·05) inhibited the growth of B. microti at doses of 5 and 10 mg/kg body weight, and the parasites completely cleared on day 14 and 16 post-inoculation in the 5 and 10 mg/kg treated groups, respectively. These findings highlight the potentiality of (-)-Epigallocatechin-3-gallate as a chemotherapeutic drug for the treatment of babesiosis.

Cryptosporidium parvum, belonging to the phylum Apicomplexa, is a major cause of waterborne gastroenteritis throughout the world. The sporozoites are thought to invade host enterocytes using an active process termed gliding motility. However, the biological and morphological changes within the sporozoites during this process are not fully understood. In the present study, excysted sporozoites of C. parvum were analysed ultrastructurally in vitro and their viability was evaluated using fluorescent dyes. The sporozoites excysted from oocysts changed morphologically from banana-shaped to rod-shaped and finally to a rounded shape, in culture media in 3 h. Transmission microscopy revealed that the distance between the apical end and the nucleus was markedly reduced, dense granules were present close to the rhoptry in the apical region, amylopectin granules were absent, and membranes of round sporozoites were less clear. A fluorescent assay showed that the rate of survival decreased from 89% to 56% at 0–3 h (84·3% for banana-shaped and 49·2% for rod-shaped sporozoites). Therefore, post-excysted sporozoites in vitro underwent morphological changes and a rapid loss of viability. This staining method is useful, inexpensive and provides an alternative to more costly and intensive flow cytometric assays or infectivity assays with host cells in vitro.

Clinical and environmental isolates of pathogens are often unique and may be unculturable, yielding a very limited amount of DNA for genetic studies. Cryptosporidium in particular are difficult to propagate. Whole genome amplification (WGA) is a valuable technique for amplifying genomic material. In this study, we tested 5 WGA commercial kits using Cryptosporidium clinical isolates. DNA of 5 C. hominis and 5 C. parvum clinical isolates and C. parvum IOWA reference strain were used. The majority of the samples were amplified by all of the kits tested. The integrity and fidelity of the amplified genomic DNA were assessed by sequence analysis of several PCR products of varying length. We found evidence that one kit in particular may be more error prone while another seemed the more suitable kit for Cryptosporidium clinical samples, generating high molecular weight DNA from all the samples with high fidelity. Thus WGA was found to be a useful technique for producing amplified DNA suitable for downstream genotyping techniques and archiving of Cryptosporidium clinical isolates.

We have previously discovered a unique mechanism of epigenetic transcriptional gene silencing in the Entamoeba histolytica trophozoites of strain HM-1:IMSS that resulted in the persistent downregulation of the amoebapore A (ap-a) gene, and that could be successfully applied to silence other virulence genes (cpA5, lgl1). In order to understand how the silencing is maintained throughout generations, we analysed whether modifications occurred at the chromatin level. Chromatin immunoprecipitation assays were done with antibodies specific to the methylated lysine 4 of E. histolytica histone H3. When the genes were in a transcriptionally silent state, the methylation levels of H3K4 in their coding region were significantly reduced. In contrast, the levels of core histone H3 were consistently higher in the silenced genes. Controlled chromatin digestion with micrococcal nuclease was used to assess changes in nucleosome compaction. We found a significant resistance to digestion in the promoter region of the silenced ap-a and cpA5 genes as compared to the parental strain that expresses those genes. Our data lend further support to the idea that histone modifications and heterochromatin formations are at the basis of the transcriptional silencing of genes in E. histolytica.