Introduction. Species typing in leishmaniasis gains importance in diagnostics, epidemiology, and clinical studies. A restriction fragment length polymorphism (RFLP) assay of PCR amplicons from a partial heat-shock protein 70 gene (hsp70) had been described for the New World, allowing identification of some species. Methods. Based on an initial in silico analysis of 51 hsp70 sequences, most of which we recently determined in the frame of a phylogenetic study, species-specific restriction sites were identified. These were tested by PCR-RFLP on 139 strains from 14 species, thereby documenting both inter- and intra-species variability. Results. Our assay could identify Leishmania infantum, L. donovani, L. tropica, L. aethiopica, L. major, L. lainsoni, L. naiffi, L. braziliensis, L. peruviana, L. guyanensis, and L. panamensis by applying 2 subsequent digests. L. mexicana, L. amazonensis, and L. garnhami did not generate species-specific restriction fragment patterns. Conclusion. Currently no assay is available for global Leishmania species discrimination. We present a universal PCR-RFLP method allowing identification of most medically relevant Old and New World Leishmania species on the basis of a single PCR, obviating the need to perform separate PCRs. The technique is simple to perform and can be implemented in all settings where PCR is available.

This paper presents a perspective on the ecology of trematodes in snail hosts based on recent evidence. Because flukes use snails almost obligatorily as first intermediate hosts, we highlight the role of gastropods as keystone species for trematodes and their communities. After reviewing recent developments in the transmission of trematodes to and from snails, we discuss trematode communities within individual snails (infracommunities) and in snail populations (component communities). Results garnered using various protocols at the infracommunity level are reviewed. The few data available, all from marine systems, indicate that low colonization rates characterize infracommunities, suggesting that trematode infracommunities tend to be isolationist in character rather than interactive. The variety of trematode species present in a component community seems to be determined by spatial overlap of definitive hosts. Relative abundance of species in a component community shows little dependence on negative interspecific interactions at the level of the infracommunity. Temporal aspects of trematode communities are related to the life history of the host snail. The component communities of long-lived snails (mostly marine) integrate many infection episodes whereas shorter-lived snails (mostly freshwater) acquire new component communities each time host cohorts turnover.

Two lines of Plasmodium chabaudi differing in three characters have been crossed, using a technique previously described for P. yoelii. One line, termed 47AS, was characterized by an electrophoretic form of 6-phosphogluconate dehydrogenase, denoted 6PGD-2, a form of lactate dehydrogenase, denoted LDH-3, and was pyrimethamine-resistant. The second line, termed 10AJ, possessed enzyme forms 6PGD-3 and LDH-2 and was pyrimethamine-sensitive. The cross was made by permitting mosquitoes to feed on a mixture of the two lines and infecting rodents with the resulting sporozoites. The products of the cross were cloned by dilution and examined for enzyme-type and drug-response. Results showed that recombination had occurred between each of the three characters. Clones characterized by 6PGD-2/LDH-2 and 6PGD-3/LDH-3 demonstrated recombination between the enzyme markers. The drug-resistance character segregated independently of either enzyme marker.

Neospora caninum is an apicomplexan, protozoan parasite, which causes severe disease in dogs and cattle. It has previously been isolated only in the United States. A 5-week-old Boxer pup with a progressive hindlimb paresis was diagnosed as suffering from neosporosis on the basis of clinical signs and the presence of anti-Neospora antibodies in it, 2 litter-mates and its darn. Despite treatment with sulphonamides, the pup was euthanased 3 days later. The diagnosis of neosporosis was confirmed by immunohistochemical examination of muscle and CNS tissue sections from the pup. Parasites were isolated into Vero cell culture from the cerebrum, and confirmed as Neospora caninum by immunofluorescence with specific antibody, tachyzoite ultrastructure and 16S-like ribosomal RNA sequences. This isolate (designated NC-Liverpool) has been continuously passaged every 7–10 days. Its growth characteristics, ultrastructure and antigenic profile, as revealed by immunoblotting, have revealed no major differences from the American NC-1 isolate. Furthermore, no difference was seen when comparing the sequences of 16S-like ribosomal RNA and the ITS1 region of the two isolates.

The effect of Toxoplasma gondii on rat behaviour was assessed in a colony of 36 free-ranging wild/laboratory hybrid rats allowed to compete freely for food and mates in a (100 m2) outdoor naturalistic enclosure. T. gondii infection had no effect on social status or mating success, both the product of costly and competitive activities. However, the propensity to explore novel stimuli in their environment was higher in infected than uninfected individuals. These results are consistent with the hypothesis that T. gondii only affects the behavioural traits which selectively benefit the parasite, rather than causing a general alteration of rat behaviour.

NcSAG1 and NcSRS2, the two major immunodominant tachyzoite surface antigens of the apicomplexan parasite Neospora caninum, were investigated for their potential as vaccine candidates in mice. Recombinant recNcSRS2 and recNcSAG1 were expressed in Escherichia coli as poly-histidine-tagged fusion proteins. Separate groups of mice were immunized with purified recNcSAG1, recNcSRS2, or a combination of both, and were then challenged with N. caninum tachyzoites. Subsequent experiments included intramuscular vaccination of mice with the eukaryotic expression plasmid pcDNA3 containing either NcSRS2 or NcSAG1 cDNA inserts, followed by a single booster with the corresponding recombinant antigens. Immunization with a crude somatic antigen (NC1-extract) was included in the experiments. Following challenge, the presence of the parasite in the different organs was assessed by a N. caninum-specific PCR, while the parasite burden in infected brain tissue was assessed by quantitative real-time PCR. Immunization of mice employing individual recombinant antigens, or combined recNcSAG1/recNcSRS2, resulted in a lower degree of protection against cerebral infection, when compared to combined DNA/recombinant antigen vaccination. Serological analysis showed that this protective effect was associated with the occurrence of antibodies directed against native parasite antigens in those animals receiving combined DNA/recombinant antigen vaccination. Conversely, mice immunized with recombinant antigens alone generated antibodies recognizing only the recombinant antigens. Mice experiencing clinical signs such as walking disorders, rounded back, apathy and paralysis were observed only in the untreated positive control groups, but never in the vaccinated groups. Our results suggest that a combined DNA/recombinant antigen-vaccine, based on NcSAG1 and NcSRS2, respectively, exhibited a highly significant protective effect against experimentally induced cerebral neosporosis in mice.

Significant mortalities of the Pacific oyster, Crassostrea gigas, have been reported worldwide since the 1950s. The impact these re-occurring mortality events have had on the C. gigas industry has highlighted the necessity to determine the factors that may be causing these mortalities. This study investigated the possible role of ostreid herpes virus (OsHV-1) in C. gigas mortalities over 2 successive summers at 2 study areas in Ireland. A single sample of adult C. gigas, which had been experiencing mortalities at one of the sites was screened. Successive cohorts of C. gigas spat obtained from a hatchery outside Ireland was relayed to both sites in 2003 and in 2004. Spat were screened each year prior to relaying. Samples were collected every 2 weeks and mortality counts were recorded and observed at both sites. Polymerase chain reaction (PCR) analysis and subsequent sequencing indicated that a previously undocumented variant genotype of OsHV-1 was present in the single cohort of adult C. gigas and in seed and juveniles at both sites, in both years. Analysis suggests that the Irish OsHV-1 μvar variant genotype is closely related to OsHV-1 μvar, first described in France in 2008.

The vectorial competence of Phlebotomus sergenti for 3 Old World species of Leishmania, L. tropica, L. major and L. donovani, was investigated in vivo and by in vitro midgut binding assays using living promastigotes and purified lipophosphoglycan (LPG). P. sergenti consistently showed a high specificity for L. tropica strains, which were able to develop mature, potentially transmissible infections. The loss of infection with L. major and L. donovani correlated with the excretion of the digested bloodmeal. These strains were able to produce sustained infections in the midguts of their appropriate vectors, P. papatasi and P. argentipes, respectively. In in vitro binding assays, a significantly higher number of L. tropica procyclic promastigotes attached to the midgut lining of P. sergenti, compared to those of L. major and L. donovani (P < 0·05). The prediction that the species specificity of midgut attachment is controlled by polymorphic structures on the parasite LPG was supported by the finding that P. sergenti midguts were intensely stained following incubation with purified phosphoglycan (PG) from L. tropica compared with PGs from L. major or L. donovani. The results provide further evidence that LPG structural polymorphisms are driven by the species diversity of molecules present on the sandfly midgut that function as parasite attachment sites.

The responses of susceptible Ndama and Zebu cattle to needle challenge with Trypanosoma congolense were followed using parasitological, haematological and radio-isotopic methods and compared with those of corresponding uninfected animals. In both breeds, infection became patent at the same time but peak parasitaemias were significantly lower, were attained later and were of short duration in the Ndama. All infected animals became anaemic, the severity of which correlated with the level and duration of parasitaemia. However, even when parasites could no longer be detected in the blood, packed cell volumes showed little tendency to recover. The anaemia was due to increased intravascular red cell destruction and was more pronounced in the Zebu. Haemodilution was not a feature. Increased red cell synthesis occurred in infected animals of both breeds but particularly in the Zebu; this accounted for the capacity to maintain packed cell volume levels following the initial drop associated with parasitaemia. However, in most cases red cell synthesis was less than expected from the degree of anaemia, suggesting impairment of bone marrow function. Measurement of red cell iron utilization indicated that this was due to defective iron re-utilization from degraded red cells arising from reticulo-endothelial blockade. It is concluded that the anaemia in this disease and its underlying processes are broadly in line with the number of parasites in the blood and that the superior resistance of the Ndama cattle lies in their ability to control parasitaemia rather than their capacity to mount a more efficient erythropoietic response.

The phylogenetic affinities of the oyster pathogen Perkinsus marinus were investigated with morphology, 18S-like rDNA data and actin sequence data. Morphological investigations revealed that Perkinsus species do not have a conoid and that other criteria which have been used to place them in the Apicomplexa are general to alveolates. When considered separately, 18S-like rDNA and actin data sets each support a closer affinity for Perkinsus marinus with the dinoflagellates. However, each of these separate analyses possess their own biases and weaknesses. Use of the phylogenetic principle of ‘total evidence’ in which data sets are combined in simultaneous analysis yielded a more robust hypothesis that is stable both to character and taxonomic sampling. The resulting cladogram strongly corroborates the placement of Perkinsus species with the Dinoflagellida and not with the Apicomplexa.

A new mathematical model of Plasmodium falciparum asexual parasitaemia is formulated and fitted to 35 malaria therapy cases making a spontaneous recovery after primary inoculation. Observed and simulated case-histories are compared with respect to 9 descriptive statistics. The simulated courses of parasitaemia are more realistic than any previously published. The model uses a discrete time-step of 2 days. Its realistic behaviour was achieved by the following combination of features (i) intra-clonal antigenic variation, (ii) large variations of the variants' baseline growth rate, depending on both variant and case, (iii) innate autoregulation of the asexual parasite density, variable among cases, (iv) acquired variant-specific immunity and (v) acquired variant-transcending immunity, variable among cases. Aspects of the model's internal behaviour, concerning variant dynamics, as well as the respective contributions of the three control mechanisms (iii) – (v), are displayed. Some implications for pathogenesis and control are discussed.

Reproducible growth of gametocytes of Plasmodium falciparum in vitro was obtained from ring-stages taken directly from naturally infected patients and from the same material following storage in liquid nitrogen. Progressive sexual differentiation in vitro was examined for a finite period of 9 days in microcultures and was, for convenience, divided into 5 stages using established morphological criteria (Hawking, Wilson & Gammage, 1971). This microculture system was adapted as a bioassay for various anti-metabolites. Drug activity was measured by observing the inhibition of the established pattern of sequential development in experimental as compared to control cultures. Inhibitors used were directed against DNA, RNA and protein metabolism and microtubule assembly. As a result of these studies it is proposed that the sexual cell-cycle of P. falciparum is characterized by 4 phases. (1) A G1 period which lasts only a few hours. (2) The S phase, where DNA synthesis occurs, occupies the remainder of the first 2 days of development – both G1 and S are confined to stage I and II gametocytes. (3) G2, which is subdivided into 2 sections: G2A, characterized by stage II and III gametocytes, in which significant RNA and protein synthesis continue to occur; and G2B, where there is a progressive increase in transcription control resulting in the depression of both RNA and protein synthesis. Nonetheless, continued morphological differentiation occurs in the latter section transforming the parasites to stage IV and the morphologically and functionally mature stage V. The final M phase is marked by the brief and explosive events of gametogenesis, during which further protein synthesis occurs de novo. The proposed cell-cycle is examined as a model for studies on the activity of gametocytocidal compounds.

Schistosomes are parasitic flatworms that cause schistosomiasis, a neglected tropical disease that affects hundreds of millions worldwide. Treatment and control of schistosomiasis relies almost entirely on the single drug praziquantel (PZQ), making the prospect of emerging drug resistance particularly worrisome. This review will survey reports of PZQ (and other drug) resistance in schistosomes and other platyhelminths, and explore mechanisms by which drug resistance might develop. Newer genomic and post-genomic strategies that offer the promise of better understanding of how drug resistance might arise in these organisms will be discussed. These approaches could also lead to insights into the mode of action of these drugs and potentially provide markers for monitoring the emergence of resistance.

A pure strain of Entamoeba histolytica has been isolated and cultivated, and an attempt has been made to study and describe its whole life-history in detail.

This strain (K. 28 c) was derived from the dysenteric dejecta of a kitten experimentally infected per os by means of typical cysts from the faeces of a monkey (Macacus sinicus). It has now been under continuous cultivation for about 20 months (220 serial subcultures), and its development in vitro has been uniform throughout.

Methods have been devised, and are here described, whereby any desired stage in the life-history of this strain—amoebae, cysts, and all intermediate stages (including encystation and excystation)—can be readily procured in vitro at will.

Detailed study has shown that the trophic amoebae multiply in cultures by simple binary fission only, as they do in their natural hosts. Their mode of division is briefly described.

Encystation also occurs in vitro just as it does in the bowel, with formation of characteristic precystic amoebae and the final production of typical quadrinucleate cysts.

Excystation has been carefully studied, and it has been found that a single quadrinucleate, amoeba escapes from each cyst through a minute perforation in its wall. An account is given of this remarkable process, which has not been described previously.

The 4-nucleate excysted (metacystic) amoeba has been found to produce a new generation of trophic forms by a complicated series of nuclear and cytoplasmic divisions, which are described in detail for the first time. The final result of this subdivision is the production of eight uninucleate amoebulae by each quadrinucleate amoeba hatched from a cyst.

These amoebulae are young trophic amoebae, and not gametes or conjugants. No sexual phenomena of any sort have been observed during the metacystic itages: and the life-history of E. histolytica, as visible in vitro, is thus wholly sexual.

A development similar to that here described in the case of Strain K. 28 c has been found to occur in many other cultivated strains of E. histolytica— including a strain isolated directly from man, and a human strain experiaentally implanted in a monkey (M. sinicus) and recovered therefrom in pure culture. There are therefore good reasons for concluding that the development were described is not abnormal, and that it is probably closely parallel to that which occurs naturally inside man.

The prevalence of Cryptosporidium in calves younger than 10 weeks was estimated in a cross-sectional epidemiological study on 100 dairy (n=499) and 50 beef (n=333) farms in East Flanders (Belgium), using a previously evaluated immunofluorescence assay (Merifluor®). The calf prevalence was 37% (95% Probability Interval (PI): 7–70%) in dairy calves and 12% (95% PI: 1–30%) in beef calves. To elucidate the genetic diversity, the Cryptosporidium 18S ribosomal DNA and the 70 kDa heat shock protein gene were targeted. In the majority of the samples C. parvum was present, although C. bovis was also identified, all but one in calves older than 1 month. The porcine-specific C. suis was identified in 1 beef calf. Subtyping of C. parvum positive isolates by sequence analysis of the 60 kDa glycoprotein gene indicated the presence of 4 allele IIa subtypes, along with 1 subtype IIdA22G1. The subtype IIaA15G2R1 was most prevalent, next to subtype IIaA13G2R1 and IIaA16G2R1, and a new subtype IIaA14G2R1. The results of the present study indicate a high prevalence of Cryptosporidium infections in calves in Belgium and confirm that these calves should be considered as a potential zoonotic reservoir for human infections.

Considering the epidemic situation of gambiense human African trypanosomiasis (HAT) at the end of the twentieth century, the World Health Organization (WHO) and partners strengthened disease control and surveillance. Over the last 15 years, the activities implemented through the National Control Programmes have brought gambiense HAT under control and now its elimination is deemed as an achievable goal. In 2012, WHO targeted gambiense HAT for elimination as a public health problem by 2020. The final goal will be the sustainable disease elimination by 2030, defined as the interruption of the transmission of gambiense HAT. The elimination is considered feasible, because of the epidemiological vulnerability of the disease, the current state of control, the availability of strategies and tools and international commitment and political will. Integration of activities in the health system is needed to ensure the sustainability of the elimination. The development of user-friendly diagnostic and treatment tools will facilitate the integration process. Adequate funding is needed to implement activities, but also to support research that will make the elimination sustainable. A long-term commitment by donors is needed and ownership of the process by endemic countries is critical.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent antimalarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

Parasitologists are fond of alluding to the possible antiquity of their chosen group. They may even place the origin in a specific period of geological time and trace its co-evolution, or lack thereof, with the hosts. These speculations usually make only passing comments on the actual fossil record, but a critical examination suggests that this area can throw much light on both the origins and evolution of parasitism. This review is concerned primarily with the fossil record and its immediate implications. It does not appraise parasite evolution in the light of evidence from sources, often of excellent quality, such as comparative anatomy and physiology or ecology. As some parasitologists may not be entirely familiar with the geological column and time scale, Table 1 depicts these and indicates some records of parasitism mentioned here, together with the major events in this history of life.

The susceptibility of Brugia malayi and Onchocerca lienalis microfilariae to H2O2 and NO either in cell-free culture or from IFNγ-activated macrophages was examined. In cell-free culture, O. lienalis microfilariae were highly susceptible to H2O2 induced toxicity, exhibiting rapid reductions in motility and viability. The addition of exogenous catalase abrogated H2O2-induced killing. In contrast, B. malayi microfilariae were relatively resistant to H2O2, with concentrations as high as 50 μM having no effect on motility or viability. On exposure to NO, both species showed reductions in motility within 5–30 min, but longer was required to see effects on the viability of microfilariae. Parasites incubated with IFNγ-activated macrophages also exhibited marked reductions in motility and viability. In cultures with B. malayi and activated macrophages, inhibition of these effects was achieved by the addition of either L-NMMA, to abolish NO production, or neutralizing anti-TNFα antibodies. Attempts to inhibit parasite killing by the addition of catalase to macrophage cultures were ineffective. The results of this study show that B. malayi and O. lienalis microfilariae have different susceptibility to H2O2, but are equally affected by exposure to NO. Moreover both species are killed by IFNγ-activated macrophages and in the case of B. malayi, killing is dependent on the generation of NO via TNFα.