Cryptosporidiosis is a major infection of humans, leading to diarrhoea and growth failure in children, diarrhoea and malnutrition in immunocompromised adults, and is associated with increased mortality in all age groups. Using the country of Zambia as an example, I review the possible approaches to treatment and prevention in a tropical setting. The current optimal therapy for cryptosporidiosis is nitazoxanide which works well in HIV uninfected children, but treatment in patients with HIV infection remains remarkably difficult. No single drug has demonstrated efficacy in a randomised trial. No vaccine is available, so the best option for prevention for the moment is filtration and clean storage of drinking water. This would be expected to reduce cryptosporidiosis dramatically, but this needs to be demonstrated directly. Water filtration would have the added benefit of protection against many other pathogens, but the paucity of alternative approaches highlights the need for a better understanding of this important human pathogen.

Three expression cDNA libraries from Trichinella spiralis worms 14 h, 20 h and 48 h post-infection (p.i.) were screened with serum from pigs experimentally infected with 20 000 T. spiralis muscle larvae. Twenty-nine positive clones were isolated from the 14 h p.i. cDNA library, corresponding to 8 different genes. A putative excretory-secretory protein similar to that of T. pseudospiralis was identified. Three clones corresponded to a T. spiralis serine proteinase inhibitor known to be involved in diverse functions such as blood coagulation and modulation of inflammation. Screening of the 20 h p.i. cDNA library selected 167 positive clones representing 12 different sequences. The clone with the highest redundancy encoded a small polypeptide having no sequence identity with any known proteins from Trichinella or other organisms. Fourteen clones displayed sequence identity with the heat shock protein (HSP) 70. HSPs are produced as an adaptive response of the parasite to the hostile environment encountered in the host intestine but their mechanism of action is not yet well defined. From the 48 h p.i. T. spiralis cDNA library, 91 positive clones were identified representing 7 distinct sequences. Most of the positive clones showed high similarity with a member of a putative T. spiralis serine protease family. This result is consistent with a possible major role for serine proteases during invasive stages of Trichinella infection and host-parasite interactions.

Life-cycle stages of the microsporidia Nosema bombycis, the pathogen causing silkworm pebrine, were separated and purified by an improved method of Percoll-gradient centrifugation. Soluble protein fractions of late sporoblasts (spore precursor cells) and mature spores were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Protein spots were recovered from gels and analysed by mass spectrometry. The most abundant differential protein spot was identified by database search to be a hypothetical spore wall protein. Using immunoelectron microscopy, we demonstrated that HSWP5 is localized to the exospore of mature spores and renamed it as spore wall protein 5 (NbSWP5). Further spore phagocytosis assays indicated that NbSWP5 can protect spores from phagocytic uptake by cultured insect cells. This spore wall protein may function both for structural integrity and in modulating host cell invasion.

Objective. Unravelling the determinants of parasite life-history traits in natural settings is complex. Here, we deciphered the relationships between biotic, abiotic factors and the variation in 4 life-history traits (body size, egg presence, egg number and egg size) in the fish ectoparasite Tracheliastes polycolpus. We then determined the factors affecting the strength of the trade-off between egg number and egg size. Methods. To do so, we used 4-level (parasite, microhabitat, host and environment) hierarchical models coupled to a field database. Results. Variation in life-history traits was mostly due to individual characteristics measured at the parasite level. At the microhabitat level (fins of fish hosts), parasite number was positively related to body size, egg presence and egg number. Higher parasite number on fins was positively associated with individual parasite fitness. At the host level, host body size was positively related to the individual fitness of the parasite; parasites were bigger and more fecund on bigger hosts. In contrast, factors measured at the environmental level had a weak influence on life-history traits. Finally, a site-dependent trade-off between egg number and egg size existed in this population. Conclusion. Our study illustrates the importance of considering parasite life-history traits in a hierarchical framework to decipher complex links between biotic, abiotic factors and parasite life-history traits.

The present study aimed to determine the in vitro biological efficacy and selectivity of 7 novel AIAs upon bloodstream trypomastigotes and intracellular amastigotes of Trypanosoma cruzi. The biological activity of these aromatic compounds was assayed for 48 and 24 h against intracellular parasites and bloodstream forms of T. cruzi (Y strain), respectively. Additional assays were also performed to determine their potential use in blood banks by treating the bloodstream parasites with the compounds diluted in mouse blood for 24 h at 4°C. Toxicity against mammalian cells was evaluated using primary cultures of cardiac cells incubated for 24 and 48 h with the AIAs and then cellular death rates were determined by MTT colorimetric assays. Our data demonstrated the outstanding trypanocidal effect of AIAs against T. cruzi, especially DB1853, DB1862, DB1867 and DB1868, giving IC50 values ranging between 16 and 70 nanomolar against both parasite forms. All AIAs presented superior efficacy to benznidazole and some, such as DB1868, also demonstrated promising activity as a candidate agent for blood prophylaxis. The excellent anti-trypanosomal efficacy of these novel AIAs against T. cruzi stimulates further in vivo studies and justifies the screening of new analogues with the goal of establishing a useful alternative therapy for Chagas disease.

Herein we review theoretical and methodological considerations important for finding and delimiting cryptic species of parasites (species that are difficult to recognize using traditional systematic methods). Applications of molecular data in empirical investigations of cryptic species are discussed from an historical perspective, and we evaluate advantages and disadvantages of approaches that have been used to date. Developments concerning the theory and practice of species delimitation are emphasized because theory is critical to interpretation of data. The advantages and disadvantages of different molecular methodologies, including the number and kind of loci, are discussed relative to tree-based approaches for detecting and delimiting cryptic species. We conclude by discussing some implications that cryptic species have for research programmes in parasitology, emphasizing that careful attention to the theory and operational practices involved in finding, delimiting, and describing new species (including cryptic species) is essential, not only for fully characterizing parasite biodiversity and broader aspects of comparative biology such as systematics, evolution, ecology and biogeography, but to applied research efforts that strive to improve development and understanding of epidemiology, diagnostics, control and potential eradication of parasitic diseases.

The rTSSA-II (recombinant Trypomastigote Small Surface II) antigen was evaluated by ELISA to detect anti-Trypanosoma cruzi antibodies in sera from naturally infected dogs and humans. For this evaluation ELISA-rTSSA-II was standardized and groups were classified according to the results obtained through xenodiagnosis, ELISA and PCR. Sensitivity (Se), Specificity (Sp), Kappa index (KI) and area under curve (AUC) were determined. The Se was determined by using 14 sera from dogs infected with T. cruzi VI (TcVI) whereas Sp was determined by using 95 non-chagasic sera by xenodiagnosis, ELISA-Homogenate and PCR. The performance of ELISA-rTSSA-II in dog sera was high (AUC=0·93 and KI=0·91). The Se was 92·85% (1 false negative) and Sp was 100%. Two sera from dogs infected with TcI and 1 with TcIII were negative. For patients infected with T. cruzi, reactivity was 87·8% (36/41), there was only 1 indeterminate, and Sp was 100%. Fifty-four sera from non-chagasic and 68 sera from patients with cutaneous leishmaniasis did not react with rTSS-II. ELISA-rTSSA-II showed a high performance when studying sera from naturally infected dogs and it also presented 100% Sp. This assay could be an important tool to carry out sero-epidemiological surveys on the prevalence of T. cruzi circulating lineages in the region.

In the Czech Republic, sera from 551 clinically healthy adult slaughtered pigs (females, 6–8 months old) were collected during the first half of June in 2010. Sera were tested for Toxoplasma gondii-specific IgG antibodies by an enzyme-linked immunosorbent assay; samples with more than 50% S/P were considered as positive. The same samples were also analysed for Neospora caninum antibodies using a commercial competitive-inhibition enzyme-linked immunosorbent assay; samples with more than 30% inhibition were considered as positive. Antibodies against T. gondii were found in 198 pigs (36%) in all districts with prevalences ranging from 18% to 75%. Antibodies against N. caninum were found in 16 pigs (3%); positive animals were found in 4 districts with prevalences ranging from 1% to 20%. Indication of mixed infections (concurrent presence of both N. caninum and T. gondii antibodies) was found in 8 (1·5%) pigs. The results of our study indicate that pigs in the Czech Republic have a relatively high seroprevalence for T. gondii, while they have only a low seroprevalence for N. caninum. Therefore, natural infection with T. gondii seems to be very common in Czech pigs. It is the first evidence of N. caninum antibodies in pigs in the Czech Republic. These results complete data about N. caninum infection in pigs in Europe.

Giardia lamblia is one of the most prevalent parasites of mankind and is estimated to cause over 200 million infections per year. To screen chemical libraries for compounds that perturb trophozoite proliferation we adapted a conventional culture method to 384-well plates and identified numerous inhibitors. Here we used a modified assay to screen for compounds that promote trophozoite multiplication. Trophozoite growth was reduced by dilution of the culture medium and the growth period was extended to screen 2 compound libraries comprising 1500 compounds. A total of 4 agonists of trophozoite multiplication were identified. In the presence of one of these compounds, strychnine, enhanced growth was accompanied by unusual trophozoite morphology characterized by dividing trophozoites displaying more than the 2 nuclei per cell which are normally observed. The other agonists, although belonging to 2 distinct chemical groups, are known to affect isoprenylation, indicating a link between protein or lipid isoprenylation and growth in culture. Although inhibitors of isoprenylation are known to antagonize proliferation of mammalian cells, an agonistic effect of isoprenylation modulators has to our knowledge not been described previously. These observations illustrate the power of chemical genetics for identifying pathways controlling specific traits in G. lamblia.

The aim of this study was to construct risk maps for the presence of the dominant Leishmania infantum vector, P. perniciosus, and check its usefulness (a) to predict the risk of canine leishmaniasis and (b) to define effective leishmaniasis control measures. We obtained data for the presence/absence of P. perniciosus at 167 sampling sites in southern Spain, from which we also took a series of ecological and climate-related data. The probability of P. perniciosus presence was estimated as a function of these environmental variables and generated spatial risk maps. Altitude, land use and drainage hole features (with or without PVC piping) were retained as the only predictors for the distribution of this vector species. Drainage hole features in retaining walls, with or without PVC piping, produce significant variations in the probability of P. perniciosus presence, varying from 2·3 to 91·8% if PVC piping is absent and from 0·4 to 66·5% if all holes have PVC piping. It was concluded that the use of PVC piping in drainage holes could help to reduce leishmaniasis transmission.

In the present study, we investigated the involvement of sulfated glycosaminoglycans in both the in vivo development and adhesion of T. cruzi epimastigotes to the luminal surface of the digestive tract of the insect vector, Rhodnius prolixus. Pre-incubation of T. cruzi, Dm 28c epimastigotes with heparin, chondroitin 4-sulfate, chondroitin 6-sulfate or protamine chloridrate inhibited in vitro attachment of parasites to the insect midgut. Enzymatic removal of heparan sulfate moieties by heparinase I or of chondroitin sulfate moieties by chondroitinase AC from the insect posterior midgut abolished epimastigote attachment in vitro. These treatments also reduced the labelling of anionic sites exposed at the luminal surface of the perimicrovillar membranes in the triatomine midgut epithelial cells. Inclusion of chondroitin 4-sulfate or chondroitin 6-sulfate and to a lesser extent, heparin, in the T. cruzi-infected bloodmeal inhibited the establishment of parasites in R. prolixus. These observations indicate that sulfated glycosaminoglycans are one of the determinants for both adhesion of the T. cruzi epimastigotes to the posterior midgut epithelial cells of the triatomine and the parasite infection in the insect vector, R. prolixus.

Neospora caninum is an apicomplexan parasite and the aetiological agent of bovine neosporosis, one of the main causes of reproductive failure worldwide. We have generated 2 independent transgenic knock-in clones, Nc-1SAG4c1.1 and Nc-1SAG4c2.1, that express the bradyzoite stage-specific protein NcSAG4 in the tachyzoite stage. These clones have similar growth rates in vitro as the wild-type (WT) strain Nc-1. Studies in a cerebral mouse model of infection revealed a slightly lower rate of detection of the transgenic strains in brains during the chronic phase of infection. However, a pregnant mouse model of infection revealed a reduction in the virulence of the Nc-1SAG4c1.1 strain despite the same tachyzoite expression of NcSAG4 and a similar anti-NcSAG4 response displayed by mice inoculated with Nc-1 SAG4c1.1 or Nc-1 SAG4c2.1 parasites. This behaviour may be related to the reduced ability of the Nc-1SAG4c1.1 parasites to invade host cells, which was observed in in vitro assays. The apparent reduction in persistence and the high growth rate of the transgenic strains, together with their constitutive expression of the protein NcSAG4, may be useful features for future immunoprophylaxis trials based on a safe live attenuated vaccine.

Objective. Glycogen synthase kinase 3 (GSK-3) is a promising target for the treatment of various human diseases such as type 2 diabetes, Alzheimer's disease and inflammation. Successful inhibition of the homologues of this kinase in Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani makes the kinase an attractive target for the treatment of malaria, trypanosomiasis and leishmaniasis, respectively. The aim of this work was to compare the binding sites of the GSK-3 kinases of different parasites and to analyse them as possible targets for therapeutic compounds. Methods. Both a sequence alignment and homology models of the structure of 21 different GSK-3 homologues belonging to mammals, insects, pathogenic fungi, nematodes, trematodes and protozoa have been analysed, 17 of them being studied for the first time. Results. The structure of the kinases and, in particular, their binding sites, were found to be rather conserved, possessing small insertions or deletions and conserved amino acid substitutions. Nevertheless, the kinases of most species of parasite did have some amino acid differences from the human kinase, which could be exploited for the design of selective drugs. Conclusion. Comparison of the human and parasite GSK-3 ATP binding site models has shown that the development of selective drugs affecting parasite GSK-3 is possible. Known inhibitors of human GSK-3 can also be used as starting scaffolds for the search for drugs acting against parasitic diseases.

The worldwide economic impact of Neospora caninum infection has caused the development of effective vaccines to become one of the main goals in the field of neosporosis research. In this study, the protection conferred by antigens from inactivated whole tachyzoites (TZ) and a tachyzoite-bradyzoite mixture (TZ-BZ) of N. caninum (Nc-Spain7 isolate) incorporated into a water-in-oil emulsion (W/O) and aluminium hydroxide-ginseng extract (Al/G) was evaluated in mouse models of congenital and cerebral N. caninum infection. Immunization with TZ-BZ induced congenital and cerebral neosporosis exacerbation that was mainly characterized by reduced neonatal median survival time and increased parasite presence in adult mouse brains. The immune response of mice immunized with TZ-BZ was characterized by an increase in IFN-γ expression prior to challenge and an increase in IL-4 expression accompanied with significantly higher levels of antibodies against 2 recombinant bradyzoite-specific proteins (rNcSAG4 and rNcBSR4) after challenge. Immunization with TZ in W/O significantly reduced neonatal mortality, vertical transmission as well as parasite presence in adult mouse brains and induced a strong humoral immune response. The current study demonstrates the critical role of stage-specific antigens and adjuvants on the development of effective inactivated vaccines for the prevention of N. caninum infection.

Sirtuin proteins form a family of NAD+-dependent protein deacetylases that are considered potential drug targets against parasites. Here, we present the first characterization of a sirtuin orthologue from Leishmania amazonensis, an aetiological agent of American tegumentary leishmaniasis that has been the subject of many studies focused in the development of therapeutic approaches. The protein has high sequence identity with other Kinetoplastid Silent information regulator 2 Related Protein 1 (Sir2RP1) and was named LaSir2RP1. The gene exists as a single copy, encoding a monomeric protein (LaSir2RP1) of approximately 41 kDa that has NAD+-dependent deacetylase activity. LaSir2RP1 was immunodetected in total protein extracts, in cytoplasmic granules, and in the secreted material of both promastigotes and lesion-derived amastigotes. Analysis of both lectin‑affinity purified promastigote and amastigote extracts revealed the presence of a major enriched protein of approximately 66 kDa that was recognized by an anti-LaSir2RP1 serum, suggesting that a parasite sirtuin could be glycosylated in vivo.

In order to investigate the dynamics of Septin4 (Sept4) expression and its function in the formation of fibrotic livers in mice infected with Schistosoma japonicum, we constructed the mouse model of S. japonicum egg-induced liver fibrosis for 24 weeks. Immunohistochemical staining, qRT-PCR and Western blot were used to detect the expression of Sept4 and α-smooth muscle actin (α-SMA). We found Sept4 localized in the perisinusoidal space where hepatic stellate cells (HSCs) distribute in the periphery of circumoval granulomas and the portal venule. The expression of Sept4 and α-SMA had a similar significant tendency of an up-regulation to a peak at 12 weeks post-infection (p.i.) followed by a down-regulation. At 24 weeks p.i. both were at a low level. These results suggest that Sept4 and α-SMA may interact together in HSCs. Based on this evidence, we hypothesize that Sept4 seems to be involved in the formation of inflammatory granulomata and subsequent liver fibrosis by regulating HSCs activation.

Oropsylla hirsuta is the primary flea of the black-tailed prairie dog and is a vector of the plague bacterium, Yersinia pestis. We examined the population genetic structure of O. hirsuta fleas collected from 11 prairie dog colonies, 7 of which had experienced a plague-associated die-off in 1994. In a sample of 332 O. hirsuta collected from 226 host individuals, we detected 24 unique haplotype sequences in a 480 nucleotide segment of the cytochrome oxidase II gene. We found significant overall population structure but we did not detect a signal of isolation by distance, suggesting that O. hirsuta may be able to disperse relatively quickly at the scale of this study. All 7 colonies that were recently decimated by plague showed signs of recent population expansion, whereas 3 of the 4 plague-negative colonies showed haplotype patterns consistent with stable populations. These results suggest that O. hirsuta populations are affected by plague-induced prairie dog die-offs and that flea dispersal among prairie dog colonies may not be dependent exclusively on dispersal of prairie dogs. Re-colonization following plague events from plague-free refugia may allow for rapid flea population expansion following plague epizootics.

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.

In Trypanosoma evansi infections changes in the haemogram are commonly observed, and the enzyme adenosine deaminase (ADA) plays an important role in the production and differentiation of blood cells. Thus, the aim of this study was to evaluate the activity of ADA in serum, erythrocytes and lymphocytes of rats infected with T. evansi compared to non-infected rats. Thirty adult rats were used, divided into 3 uniform groups. The animals in groups A and B were infected intraperitoneally with 2×106 trypomastigotes/rat. Rodents from group C (control group), were not-infected. Blood collection was performed on days 4 and 20 post-infection (p.i.) in order to obtain acute and chronic infection stages of disease. The blood was used to assess the activity of ADA. In the blood, reduced haematocrit and increased lymphocytes were correlated with ADA activity in erythrocytes and lymphocytes. We observed reduction of ADA activity in serum and erythrocytes in rats infected with T. evansi compared to non-infected rats (P<0·05). ADA activity in lymphocytes was decreased after 4 days, when the parasitaemia was high and increased after 20 days, when the number of circulating parasites was low. In conclusion, our results showed that the ADA activity was altered in serum, lymphocytes and erythrocytes of rats, concomitantly with haematological parameters, in experimental infection by T. evansi.

In recent years, the isolation of parasitic DNA from faecal samples and PCR techniques, have been improved and simplified. Moreover, the introduction of real-time PCR has made it possible to multiplex different targets into one reaction. These new technical possibilities make it feasible to introduce PCR with its unsurpassed sensitivity and specificity in a routine laboratory setting for the diagnosis of intestinal parasites. Detection rates of the parasitic infections included in the PCR are increased significantly compared with microscopy. Molecular diagnostics, especially in children, reveal a possible cause of the gastrointestinal complaints in many more cases compared with conventional methods. Usually in GP patients no other pathogenic parasites are detected using microscopy and in the returning travellers additional parasites are found with microscopy in a minority of cases only. Multiplex real-time PCR offers a highly sensitive and specific diagnostic alternative for labour intensive microscopy in clinical laboratory practice. Additional diagnostic methods for the detection of parasitic infections that are not included as PCR target can be limited to a selected group of patients.