To determine whether prolonged in vitro passage would result in attenuation of virulence in vivo, Neospora caninum tachyzoites were passaged for different lengths of time in vitro and compared for their ability to cause disease in mice. Groups of Balb/c mice were inoculated intraperitoneally with 5×106 or 1×107 of low-passage or high-passage N. caninum tachyzoites. The mice were monitored for changes in their demeanour and body weight, and were culled when severe clinical symptoms of murine neosporosis were observed. Mice inoculated with the high-passage parasites survived longer (P<0·05), and showed fewer clinical symptoms of murine neosporosis, compared to the mice receiving the low-passage parasites. The parasite was detected in the brains of inoculated mice using immunohistochemistry and ITS1 PCR. Tissue cysts containing parasites were seen in mice inoculated with both low-passage and high-passage parasites. When the in vitro growth rates of the parasites were compared, the high-passage parasites initially multiplied more rapidly (P<0·001) than the low-passage parasites, suggesting that the high-passage parasites had become more adapted to tissue culture. These results would suggest that it is possible to attenuate the virulence of N. caninum tachyzoites in mice through prolonged in vitro passage.

In spite of its limitations, the culture of Cryptosporidium parvum in monolayers of epithelial cells is a suitable model to study the interaction of this protozoan parasite with the host cell, to assay oocyst infectivity, and to screen drugs for anti-cryptosporidial activity. For unknown reasons, growth of Cryptosporidium in culture is limited in time and generally does not lead to the production of significant numbers of oocysts. In monolayers infected with high doses of oocysts, we observed that many cells remain uninfected, suggesting that some cells are less susceptible to the infection. Since C. parvum and the related species C. hominis lack many essential biosynthetic pathways, we tested whether the dependence of the parasite on host cell metabolites may favour the infection of cells in mitosis. The proportion of monolayer cells in stationary (G0/G1) phase and in mitosis (S/G2/M) was determined and the prevalence of infected cells in each subpopulation was quantified. Although C. parvum infects and develops in dividing and stationary cells, a significant preference for cells in S/G2/M phase was observed. Consistent with previous observations showing that C. parvum induces apoptosis in cell monolayers, infection was accompanied by a significant increase in the proportion of mitotic cells.

A key issue relating to developing multi-component anti-malarial vaccines, lies in studying Plasmodium vivax surface proteins' genetic variation. The present work was aimed at amplifying, cloning and sequencing the gene encoding P. vivax merozoite surface protein 5 (PvMSP5) in samples obtained from infected patients from Colombian areas having varying malaria transmission rates.Nucleotide sequence data reported in this paper are available in the GenBank, EMBL and DDBJ databases under Accessions numbers DQ341586 to DQ341601. Our results have revealed that PvMSP5 is one of the P. vivax surface proteins having greater polymorphism, this being restricted to specific protein regions. The intron and exon II (which includes the GPI anchor and EGF-like domain) were both highly conserved when compared to exon I; exon I displayed the greatest variation and most of the recombination events occurred within it. No geographical grouping was observed. The Nei-Gojobori test revealed significant positive selection in the samples analysed here, whereas Tajima and Fu and Li tests presented a neutral selection pattern. The results reflected a localized variation pattern, recombination between PvMSP5 alleles and also functional and immune pressures, where stronger selective forces might be acting on exon I than on exon II, suggesting that the latter could be an important region to be included in an anti-malarial vaccine.

We compared the distribution of the digenean Pholeter gastrophilus in the stomach of 27 harbour porpoises, Phocoena phocoena, 27 striped dolphins, Stenella coeruleoalba, 18 bottlenose dolphins, Tursiops truncatus, and 100 long-finned pilot whales, Globicephala melas. The stomach of these species is composed of 4 chambers of different size, structure and function. In all species, P. gastrophilus was largely restricted to the glandular region of the stomach, but the parasite tended to favour the fundic chamber in bottlenose dolphins and harbour porpoises, the pyloric chamber in pilot whales, and none in striped dolphins. However, predictability at infrapopulation level was generally low, suggesting a weak preference of P. gastrophilus for any of the chambers. Three hypotheses were tested to investigate a common cause for the distribution of P. gastrophilus in all host species, namely, colonization of chambers was (1) sequential, (2) dependent on chamber size, or (3) dependent on the passage time of food through the whole stomach. The latter hypothesis was indirectly tested by assuming, based on previous evidence from other vertebrates, that the greater the size of the stomach and/or the energy content of prey, the greater the delay of food passage. We found no compelling evidence that chamber colonization was sequential, or related to chamber size in any species. However, the distribution of P. gastrophilus was significantly more anteriad when the host species had larger stomachs and, particularly, when hosts fed on prey with higher caloric content. Accordingly, the stomach distribution of P. gastrophilus at this scale seems to be passively driven by features of the diet and digestive physiology of each host species. This study provides a general framework to formulate null hypotheses in future studies on microhabitat choice by parasites.

A flagellate isolated from the intestinal tract of a reduviid bug Ricolla simillima (Heteroptera) in Costa Rica was found to represent a new trypanosomatid species by the phylogenetic analysis of small subunit ribosomal RNA (SSU rRNA), glyceraldehyde phosphate dehydrogenase (GAPDH) and large subunit of RNA polymerase II (RPOIILS) genes. The phylogenetic position of this trypanosomatid, together with its typical promastigote morphology and the host identity, allowed its classification as a species that belongs to the polyphyletic genus Leptomonas. Interestingly, the new species was revealed as a member of the novel phylogenetic clade representing the closest known relative of Leishmania. With the new species used as an outgroup to root the Leishmania RPOIILS phylogenetic tree, the lineage of the Neotropical species L. enriettii was found to branch off early, and was followed by a deep split between the Old World and the remaining New World species. This tree topology supports the hypothesis that the initial transition to dixenous parasitism in this group pre-dated the continental split and that afterwards the Neotropical and the Old World groups evolved largely independently.

The treatment and prevention of parasitism in both humans and livestock continues to rely almost exclusively on the use of antiparasitic drugs – an approach which has limitations, particularly as reinfection, which occurs rapidly in endemic regions, is not prevented. In addition, the widespread appearance of drug-resistant parasites of animals (Kaplan, 2004;) together with emerging evidence of resistance problems in human parasites (Fallon et al. 1995; Ismail et al. 1996; De Clerq et al. 1997; East African Network for Monitoring Antimalarial Treatment, 2003), emphasise the importance of developing alternative methods of control, with anti-parasite vaccines a prime target.

Blood schizontocidal activity of 10 selected cis-fused cyclopenteno-1,2,4-trioxanes (namely Fenozan compound nos 6, 7, 11, 27, 32, 39, 44, 45, 48 and 51) have been re-investigated to establish their curative doses against the multidrug-resistant Plasmodium yoelii nigeriensis strain, which is lethal in Swiss mice. Freshly prepared formulations of these compounds prepared either in neutral groundnut (peanut) oil or in dimethyl sulfoxide (DMSO)-Tween-water, were compared for their antimalarial activity. Only 2 compounds, namely Fenozan derivatives 11 and 45, formulated in neutral groundnut oil for oral administration, showed highest activity with 100% cure rate in MDR P. yoelii nigeriensis-infected mice, while the DMSO-Tween-water formulations were inactive. Fenozan-48 produced 72·2% cure, when administered orally in groundnut oil (formulation) while its DMSO-Tween formulation was inactive. In the case of Fenozan 7, the oil and DMSO-Tween formulations produced 92·3 and 76·0% cures respectively. Fenozan derivatives nos 6, 27, 32, 39, 44 and 51 were not protective either in groundnut oil or DMSO-Tween oral formulations. The present study has applied more rigorous criteria for selection of active compounds, and has identified the 3,3-spirocyclopentane derivative Fenozan 11, and the 3,3-spirohydropyran derivative Fenozan 45, as potential blood schizontocides which can completely eliminate multidrug-resistant malaria infection in mice. Both these compounds are candidates for pre-clinical development. The present study advocates the preferred use of an oil vehicle for oral evaluation of potential antimalarial trioxanes/fenozans instead of the DMSO formulation, which gives inferior curative efficacy.

Reliable detection, discrimination and quantification of parasites are important for host-parasite studies and diagnostics. Microsporidial infections are problematic in this respect. Their discrimination and quantification using light microscopy is difficult because spores are the only light microscopically visible form of the parasite and they offer few distinct characters. We developed a quantitative PCR (qPCR) assay based on SYBR Green chemistry to quantify the microsporidia Glugoides intestinalis, Octosporea bayeri and Ordospora colligata in their host, the freshwater crustacean Daphnia magna. The assay allows the quantification of infection intensities in whole animals and is more than an order of magnitude more sensitive than light microscopy. Sampling and DNA extraction account for more than 90% of the residual variance in infection intensity data and this variance considerably impairs the resolution of qPCR. Where higher resolution is required, we propose using the ratio of parasite to host DNA as the measure of infection intensity. We show that this measure is robust and greatly improves resolution of qPCR. Additionally, this method can be applied to compare samples of unequal volume.

Toxoplasma gondii, a cosmopolitan protozoan parasite, is known to induce behavioural alterations in rodents and may exert an effect on human personality and behaviour. The mechanism of parasite-induced alterations in host behaviour has not been described, but it was hypothesized that development of Toxoplasma tissue cysts in the brain could affect the dopaminergic neuromodulatory system. In this study, we tested the effect of latent Toxoplasma infection on mouse behaviour associated with activity of the dopaminergic system, i.e. locomotion in a novel environment and exploration test. Additionally, we examined the behavioural response of Toxoplasma-infected mice to a selective dopamine uptake inhibitor, GBR 12909. In both genders, Toxoplasma infection decreased locomotion in the open field. Infected females displayed an increased level of exploration in the holeboard test. GBR 12909 induced suppression in holeboard-exploration in the infected males, but had an opposite effect on the controls. These results suggest an association between Toxoplasma gondii infection and changes in the dopaminergic neuromodulatory system.

Plasmodium falciparum has developed resistance to most anti-malarials; therefore, an investigation of potential targets should be performed. DNA helicases are enzymes that catalyse the unwinding of double-stranded DNA to provide single-stranded templates for DNA replication, repair and recombination. In this study, a DNA helicase (PfDH A) was purified from a crude extract of Plasmodium falciparum. DNA helicase activity was measured by assaying unwinding activity. The apparent molecular weight of PfDH A as determined by SDS-PAGE was 90 kDa. PfDH A moved unidirectionally in the 3′ -to- 5′ direction along the bound strand and preferred a fork-like substrate structure and could not unwind blunt-ended duplex DNA. Unwinding activity required Mg2+ and could be inhibited by 200 mM NaCl or KCl and was dependent on hydrolysis of ATP or dATP. Anthracyclines, including daunorubicin, nogalamycin, doxorubicin, and aclarubicin, inhibited PfDH A activity with IC50 values of 2, 5, 8 and 9 μM, respectively. Based on the results, PfDH A differs from all known human DNA helicases. However, its function and roles in parasite DNA replication need to be elucidated in the future.

The intestinal protozoan parasite Giardia lamblia causes diarrhoea in humans and animals. In the present study, we used the C57BL/6 inbred mouse model to assess the impact of a nematode (Trichinella spiralis) infection on the course of a G. lamblia (clone GS/M-83-H7) infection. Acute trichinellosis coincided with transient intestinal inflammation and generated an intestinal environment that strongly promoted growth of G. lamblia trophozoites although the local anti-Giardia immunoglobulin (Ig) A production was not affected. This increased G. lamblia infection intensity correlated with intestinal mast cell infiltration, mast cell degranulation, and total IgE production. Furthermore, a G. lamblia single-infection investigated in parallel also resulted in intestinal mast cell accumulation but severe infiltration was triggered in the absence of IgE. Recently, intestinal mast cells emerging during a G. lamblia infection were reported to be involved in those immunological mechanisms that control intestinal proliferation of the parasite in mice. This anti-giardial activity was assumed to be related to the capacity of mast cells to produce IL-6. However, this previous assumption was questioned by our present immunohistological findings indicating that murine intestinal mast cells, activated during a G. lamblia infection were IL-6-negative. In the present co-infection experiments, mast cells induced during acute trichinellosis were not able to control a concurrent G. lamblia infection. This observation makes it feasible that the T. spiralis infection created an immunological and physiological environment that superimposed the anti-giardial effect of mast cells and thus favoured intestinal growth of G. lamblia trophozoites in double-infected mice. Furthermore, our findings raise the possibility that intestinal inflammation e.g. as a consequence of a ‘pre-existing’ nematode infection is a factor which contributes to increased susceptibility of a host to a G. lamblia infection. The phenomenon of a ‘pre-existing’ nematode infection prior to a G. lamblia infection is a frequent constellation in endemic areas of giardiasis and may therefore have a direct impact on the epidemiological situation of the disease.

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii, the causative agent of toxoplasmosis in humans and domestic animals. However, in contrast to T. gondii, N. caninum represents a major cause of abortion in cattle, pointing towards distinct differences in the biology of these two species. There are 3 distinct key features that represent potential targets for prevention of infection or intervention against disease caused by N. caninum. Firstly, tachyzoites are capable of infecting a large variety of host cells in vitro and in vivo. Secondly, the parasite exploits its ability to respond to alterations in living conditions by converting into another stage (tachyzoite-to-bradyzoite or vice versa). Thirdly, by analogy with T. gondii, this parasite has evolved mechanisms that modulate its host cells according to its own requirements, and these must, especially in the case of the bradyzoite stage, involve mechanisms that ensure long-term survival of not only the parasite but also of the host cell. In order to elucidate the molecular and cellular bases of these important features of N. caninum, cell culture-based approaches and laboratory animal models are being exploited. In this review, we will summarize the current achievements related to host cell and parasite cell biology, and will discuss potential applications for prevention of infection and/or disease by reviewing corresponding work performed in murine laboratory infection models and in cattle.

Ectoparasites of livestock are of great economic and social importance but their effective control remains difficult. The feasibility of vaccination as a novel control measure was established over a decade ago with the commercial release of a recombinant vaccine against the cattle tick Boophilus microplus. Since then, research has continued on ticks and other ectoparasites. While some ectoparasite species will undoubtedly be refractory to immunological control, for others there has been a steady accumulation of knowledge of partially protective antigens, now accelerating through the application of genomic technologies. Nevertheless, progress towards usable, commercially available vaccines has been limited by a number of factors. The number of highly effective antigens is still very small. Although some classes of antigen have been investigated in more detail than others, we have no systematic knowledge of what distinguishes an effective antigen. Much hope has been placed on the potential of multi-antigen mixtures to deliver the efficacy required of a successful vaccine but with little experimental evidence. The application of current knowledge across parasite and host species needs to be explored but little has been done. In most cases, the path to commercial delivery is uncertain. Although many constraints and challenges remain, the need for vaccines and our capacity to develop them can only increase.

The pro-inflammatory cytokine tumour necrosis factor alpha (TNF-α) is associated with malaria virulence (disease severity) in both rodents and humans. We are interested in whether parasite genetic diversity influences TNF-mediated effects on malaria virulence. Here, primary infections with genetically distinct Plasmodium chabaudi chabaudi (P.c.c.) clones varied in the virulence and cytokine responses induced in female C57BL/6 mice. Even when parasitaemia was controlled for, a greater day 7 TNF-α response was induced by infection with more virulent P.c.c. clones. Since many functions of TNF-α are exerted through TNF receptor 1 (TNFR1), a TNFR-1 fusion protein (TNFR-Ig) was used to investigate whether TNFR1 blockade eliminated clone virulence differences. We found that TNFR-1 blockade ameliorated the weight loss but not the anaemia induced by malaria infection, regardless of P.c.c. clone. We show that distinct P.c.c. infections induced significantly different plasma interferon gamma (IFN-γ), interleukin 6 (IL-6) and interleukin 10 (IL-10) levels. Our results demonstrate that regardless of P.c.c. genotype, blocking TNFR1 signalling protected against weight loss, but had negligible effects on both anaemia and asexual parasite kinetics. Thus, during P.c.c. infection, TNF-α is a key mediator of weight loss, independent of parasite load and across parasite genotypes.

More than 200 species of avian Haemosporidia (genera Plasmodium, Haemoproteus, and Leucocytozoon) have been described based primarily on morphological characters seen in blood smears. Recent molecular studies, however, suggest that such methods may mask a substantial cryptic diversity of avian haemosporidians. We surveyed the haemosporidians of birds sampled at 1 site in Israel. Parasites were identified to species based on morphology, and a segment of the parasite's cytochrome b gene was sequenced. We compared 3 species concepts: morphological, genetic, and phylogenetic. Fifteen morphological species were present. Morphological species that occurred once within our dataset were associated with a unique gene sequence, displayed large genetic divergence from other morphological species, and were not contained within clades of morphological species that occurred more than once. With only 1 exception, morphological species that were identified from multiple bird hosts presented identical sequences for all infections, or differed by few synonymous substitutions, and were monophyletic for all phylogenetic analyses. Only the morphological species Haemoproteus belopolskyi did not follow this trend, falling instead into at least 2 genetically distant clades. Thus, except for H. belopolskyi, parasites identified to species by morphology were supported by both the genetic and phylogenetic species concepts.

Haemosporidian parasites are common in birds in which they act as an important selective pressure. While most studies so far have focused on the effect of their prevalence on host life-history traits, no study has measured the effect of parasitaemia. We developed molecular methods to detect, identify and quantify haemosporidia in 2 natural populations of the Blackbird Turdus merula. Three different parasite genotypes were found – 1 Haemoproteus and 2 Plasmodium. A PCR-RFLP screening revealed that only approximately 3% of blackbirds were free of parasites, compared to the 34% of uninfected birds estimated by blood smear screening. A quantitative PCR (q-PCR) assay revealed a weaker parasitaemia in microscopically undetected parasites compared to microscopically detected ones. Large parasitaemia differences were found between parasite species, suggesting either differing parasite life-histories or host resistance. Parasitaemias were also weaker in male hosts, and in urban habitats, suggesting that both host factors (e.g. immunity) and habitat characteristics (e.g. vector availability) may modulate parasite density. Interestingly, these differences in parasitaemia were comparable to differences in parasite prevalence estimated by smear screening. This suggests that previous results obtained by smear screening should be reinterpreted in terms of parasitaemia instead of parasite prevalence.

Plasmodium falciparum causes the most severe form of malaria. Utilization of fatty acids in serum is thought to be necessary for survival of this parasite in erythrocytes, and thus characterization of the parasite fatty acid metabolism is important in developing a new strategy for controlling malaria. Here, we examined which combinations of fatty acids present in human serum support the continuous culture of P. falciparum in serum-free medium. Metabolic labelling and gas chromatography analyses revealed that, despite the need for particular fatty acids for the growth of intraerythrocytic P. falciparum, it can metabolize a broad range of serum-derived fatty acids into the major lipid species of their membranes and lipid bodies. In addition, these analyses showed that the parasite's overall fatty acid composition reflects that of the medium, although the parasite has a limited capacity to desaturate and elongate serum-derived fatty acids. These results indicate that the Plasmodium parasite is distinct from most cells, which maintain their fatty acid composition by coordinating de novo biosynthesis, scavenging, and modification (desaturation and elongation).

Recently we showed that de novo ribosome biosynthesis is transcriptionally regulated in Coccidia, depending on their life-cycle stage. Since the expression of ribosomal protein genes is likely coordinated, the transcriptional control of all Toxoplasma gondii ribosomal protein (RP) genes was analysed. Therefore, the complete set of all cytoplasmic RPs was defined, containing 79 different RPs in T. gondii. RP genes were randomly distributed over the genome, each with a unique upstream region with the exception of 8 RP genes which were paired in a head-to-head orientation. To study if the RP genes share conserved promoter elements, a database was made containing upstream sequences of all T. gondii RP genes. Promoter activity was confirmed for the upstream sequences of 8 RP genes, some of which are comparable in strength to the alpha-tubulin promoter. In the complete set of RP upstream sequences 2 novel and highly conserved elements were identified, named Toxoplasma Ribosomal Protein (TRP)-1 (consensus: TCGGCTTATATTCGG) and TRP-2 ([T/C]GCATGC[G/A]). TRP-1 and/or TRP-2 were present in 95% of all RP upstream sequences and moreover, were specifically localized in a small region near the presumptive transcriptional start site (10–330 bp upstream). Although TRP elements were mostly absent in known T. gondii promoters, they are present elsewhere in the T. gondii genome suggesting that they operate not only in RP genes but in a larger set of genes. The identification of TRP elements creates a basis to further study the underlying mechanism by which RP transcription is controlled in T. gondii.The nucleotide sequence data reported in this paper are available in the Third Party Annotation Section of the DDBJ/EMBL/GenBank databases under the Accession numbers TPA: BK004896-BK004974.

Recombinant vaccines have been developed which are highly effective in preventing infection with Taenia ovis in sheep, Taenia saginata in cattle, Taenia solium in pigs and Echinococcus granulosus in livestock animals. T. ovis and T. saginata are economically significant parasites and the commercial success or otherwise of vaccines against them will rely on their economic value. E. granulosus and T. solium are zoonotic parasites that cause cystic hydatid disease and neurocysticercosis, respectively, in humans. Vaccines against these parasites have been developed to assist with the control of transmission of the human diseases rather than for prevention of infections in livestock per se. Regions of high prevalence for cystic hydatid disease and neurocysticercosis occur primarily in the developing world. As a consequence, vaccines against them are of little or no commercially interest – they are Orphan Vaccines. Lack of commercial interest in these vaccines has made public sector support for their development necessary well beyond the research phase trough into completion of commercial scale-up and other more commercially-related assessments. Practical use of the vaccines will require commercial-scale production according to international manufacturing standards. Identifying partners and support in this endeavour is now of prime importance in efforts to achieve the potential of these vaccines as new tools for the control of cystic hydatid disease and neurocysticercosis.

Clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum and a Blastocystis sp. were established for the first time. Single microbes were successfully isolated from a mixture of micro-organisms obtained from caecal contents of turkeys, using a micromanipulation approach. The cloned parasites were propagated in vitro and maintained through continuous passages multiplying to high numbers. Identification of the protists was done by morphological investigation identifying various forms of each parasite. PCR and partial sequencing of the small subunit rRNA were used to confirm clonality and to determine the relationship of the cloned parasites with known protozoan parasites. The clonal cultures established by this technique will be useful to gain more insight into the biological repertoire of the organisms. In addition, refined infection experiments in different poultry species can now be performed to elucidate the pathological pathways of the respective protozoa.