To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

Cystic echinococcosis, caused by different species of the Echinococcus granulosus complex, is an important zoonotic disease with a particular impact on pastoralist societies. In addition to the widespread taxa with synanthropic transmission, a number of Echinococcus species were described from African wild carnivores early in the 20th century. For lack of study material, most of these were later tentatively synonymized with E. granulosus. Early infection experiments with wildlife isolates gave ambiguous results due to the use of unspecified parasite material, and only recently molecular methods provided the opportunity to shed light on the confusing scenery e.g. by characterizing E. felidis from the African lion. Here we will summarize the convoluted history of Echinococcus research in sub-Saharan Africa and highlight the necessity of molecular surveys to establish the life cycles and estimate the zoonotic potential of these parasites.

Human helminthiases are common in China, especially in rural areas where sanitation conditions are poor. Soil-transmitted helminths (STHs) are predominantly found in the southern provinces. Schistosoma japonicum is also endemic to southern China. Here we review the prevalence of helminth infections and polyparasitism in China, and discuss the interactions between helminth parasites in the co-infected host. It is clear that STHs are more prevalent in rural China than previously suggested emphasizing the need for systematic control of STHs. Further, the need for improved sanitation and hygiene conditions to prevent parasite transmission is highlighted. We provide supporting evidence for human genetic susceptibility to both single helminth infection and polyparasitism, and suggest that susceptibility to helminths infections may not be independent of one or the other. We demonstrate an association between single nucleotide polymorphism (SNP) variants in IL-5 and symptomatic S. japonicum infection and discuss the potential role of IL-5 in other helminth infections. Fundamental to disease and morbidity control is adequate and effective diagnosis and surveillance of disease. We discuss the role of sICAM-1 and TNFR-I and -II as candidate markers for schistosome-induced hepatomegaly and fibrosis, and their potential for assessing disease stage and progression in schistosomiasis.

The host recognition and invasion process of Myxobolus cerebralis actinospores (triactinomyxon, TAM) was studied on a genetic level. A small-scale in vitro assay was developed to activate a large number of TAMs simultaneously, and to monitor the host invasion in the absence of live fish. The transcriptomes of non-activated and in vitro-activated TAMs were compared by suppressive subtractive hybridization (SSH) to identify parasite genes involved in the host invasion process. Differential screening and a subsequent BLAST search revealed 15 of 452 SSH-library clones expressed differently in activated TAMs. None of the 15 transcripts obtained has previously been identified from M. cerebralis. Quantitative real-time PCR was used to examine the relative expression profile of 8 selected transcripts upon TAM activation and after penetration of the host. Four of these were found to be up-regulated in activated TAMs, while expression was relatively low in non-activated TAMs and in infected fish tissue, indicating that they are relevant genes during host recognition or subsequent host invasion of M. cerebralis TAMs.

Pooled sample testing (PST) as a strategy for avoiding testing the majority of individual negative samples has been proposed for screening of diseases in low prevalence areas. There has been no standard guideline for PST in screening of Schistosoma japonicum infection of Yunnan, China. To document the optimum pool size with acceptable sensitivity of PST for screening of Schistosoma japonicum infection in this setting, an experimental pooling of each of 31 positive sera by IHA with various numbers of 24 negative sera was done. The results were used to create a statistical model which was subsequently used for simulation to predict sensitivity of the pooled serum tests in the population with varying prevalence and pool size. We found that to keep the sensitivity of PST above 90%, 1:05 should be the maximum dilution, that is, the optimum pool size should not be greater than 6. Antigen will have rather little interference if the prevalence of infection is low e.g. 1% or the antigen:antibody ratio is 1:100 or below. Pooled serum testing by IHA is an acceptable sensitive method for detecting antibody for Schistosoma japonicum infection in this area.

Infection of the snail, Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke, Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of the S. mansoni miracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia and in vitro cultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of the B. glabrata embryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to the S. mansoni gene predictions (v4.0e) either by estimating theoretical 3′ UTR lengths or using existing 3′ EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

To date, little is known about the dynamics of vertical transmission of Toxoplasma gondii in Australian marsupials. Studies in mice demonstrate that vertical transmission of T. gondii is common and that chronically infected mice can transmit T. gondii to successive generations. In this study, PCR and immunohistochemistry were used to detect T. gondii in chronically infected marsupial dams and their offspring. T. gondii was detected in the unfurred pouch young of 2 out of 10 chronically infected western grey kangaroos (Macropus fuliginosus) and in the unfurred pouch young of a brush-tailed bettong (Bettongia penicillata). Results of the study suggest that vertical transmission of T. gondii can occur in chronically infected Australian marsupials.

Infection with parasitic helminths takes a heavy toll on the health and well-being of humans and their domestic livestock, concomitantly resulting in major economic losses. Analyses have consistently revealed bioactive molecules in extracts of helminths or in their excretory/secretory products that modulate the immune response of the host. It is our view that parasitic helminths are an untapped source of immunomodulatory substances that, in pure form, could become new drugs (or models for drug design) to treat disease. Here, we illustrate the range of immunomodulatory molecules in selected parasitic trematodes, cestodes and nematodes, their impact on the immune cells in the host and how the host may recognize these molecules. There are many examples of the partial characterization of helminth-derived immunomodulatory molecules, but these have not yet translated into new drugs, reflecting the difficulty of isolating and fully characterizing proteins, glycoproteins and lipid-based molecules from small amounts of parasite material. However, this should not deter the investigator, since analytical techniques are now being used to accrue considerable structural information on parasite-derived molecules, even when only minute quantities of tissue are available. With the introduction of methodologies to purify and structurally-characterize molecules from small amounts of tissue and the application of high throughput immunological assays, one would predict that an assessment of parasitic helminths will yield a variety of novel drug candidates in the coming years.

Plasmodium falciparum parasites use multiple ligand-receptor interactions to invade human erythrocytes. Variant expression levels of members of the PfRh and PfEBA ligand families are associated with the use of different erythrocyte receptors, defining invasion pathways. Here we analyse a major polymorphism, a large sequence deletion in the PfRh2b ligand, and erythrocyte invasion profiles in uncultured Senegalese isolates. Parasites vary considerably in their use of sialic acid-containing and protease-sensitive erythrocyte receptors for invasion. The erythrocyte selectivity index was not related to invasion pathway usage, while parasite multiplication rate was associated with enhanced use of a trypsin-resistant invasion pathway. PfRh2b protein was expressed in all parasite isolates, although the PfRh2b deletion was present in a subset (~68%). Parasites with the PfRh2b deletion were found to preferentially utilize protease-resistant pathways for erythrocyte invasion. Sialic acid-independent invasion is reduced in parasites with the PfRh2b deletion, but only in isolates derived from blood group O patients. Our results suggest a significant role for PfRh2b sequence polymorphism in discriminating between alternative erythrocyte receptors for invasion and as a possible determinant of virulence.

Several other journal supplements have documented progress made in the control of schistosomiasis in Egypt, China and Brazil, however, with more than 97% of the schistosome infections now estimated to occur in Africa, the relevance of this special issue in Parasitology cannot be overemphasized. In total, 18 articles are presented, inclusive of a lead-editorial from the WHO highlighting a seminal resolution at the 54th World Health Assembly in 2001 that advocated de-worming. Facilitated by a US$ 30 million grant from the Bill and Melinda Gates Foundation in 2002, the Schistosomiasis Control Initiative subsequently fostered implementation of large-scale schistosomiasis (and soil-transmitted helminthiasis) control programmes in six selected African countries. From 2005, CONTRAST, a European union-funded consortium, was formed to conduct multi-disciplinary research pertaining to optimisation of schistosomiasis control. Progress made in schistosomiasis control across sub-Saharan Africa since the turn of the new millennium is reviewed, shedding light on the latest findings stemming from clinical, epidemiological, molecular and social sciences research, inclusive of public health interventions with monitoring and evaluation activities. New opportunities for integrating the control of schistosomiasis and other so-called neglected tropical diseases are highlighted, but more importantly, several opportune questions that arise from it frame the remaining challenges ahead for an enduring solution.

This study was conducted to determine the genotypes of Giardia duodenalis isolated from human faecal samples at Pos Betau, Pahang, Malaysia. Faecal specimens were collected and examined for G. duodenalis cysts using Trichrome staining techniques. Molecular identification was carried out by the amplification of a region of the small subunit of the nuclear ribosomal RNA (SSU rRNA) gene using nested PCR and subsequent sequencing. The sequences from 15 isolates from G. duodenalis were subjected to phylogenetic analysis (including appropriate outgroups) using the neighbor-joining and maximum parsimony methods. The trees identified G. duodenalis assemblages A and B, with a predominance of assemblage B. The predominance of anthroponotic genotypes indicates the possibility of anthroponotic transmission of these protozoa in this Semai Pahang Orang Asli community.

In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic α-helical peptides P5 and P6, corresponding to the N-terminus of the pore-forming protein from Triatoma infestans, a blood-sucking insect, and a β-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells. P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. Samples of serum, feces, and tissues from feral cats from St Kitts, West Indies were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test, and found in 71 of 96 (73·9%) of cats with titres of 1:10 in six, 1: 20 in six,1:40 in seven,1: 80 in three, 1: 160 in 10, 1:320 in 13, 1:640 in nine, and 1:1,280 or higher in 17. Tissues of 10 cats were bio-assayed in mice. Toxoplasma gondii was isolated from tissues of 7 cats; from hearts of 6, from tongue of 5, and brains of 3 cats. All 7 isolates were avirulent for mice. Toxoplasma gondii oocysts were not found in the feces of 51 cats. Genotyping of these 7 T. gondii isolates by 10 multi-locus PCR-RFLP markers, including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico, revealed 4 genotypes, including clonal Type II, Type III and 2 unique genotypes. Five of the 7 cats had infection with 2 genotypes, indicating high frequency of mixed infection in the cat population on the St Kitts island.

Mice fed 1·5 mg ochratoxin A (OTA) per kg body weight and infected with Trypanosoma brucei rhodesiense were compared with trypanosome-infected placebo-fed and uninfected OTA-fed controls. Uninfected OTA-fed mice showed fever, lethargy, facial and eyelid oedemas, mild hepatitis and nephritis, and high survival. Infected placebo-fed controls had mean pre-patent period (PPP) of 3·26 days, lethargy, dyspnoea, fever, facial and scrotal oedema, survival of 33–65 days, reduced red cell counts (RCC: 10·96–6·87×106 cells/μl of blood), packed cell volume (PCV: 43·19–26·36%), haemoglobin levels (Hb: 13·37–7·92 g/dL) and mean corpuscular volume (MCV) of 37·96–41·31 fL, hepatosplenomegaly, generalized oedemas, heart congestion, hepatitis and nephritis. Compared to infected placebo-fed controls, infected OTA-fed mice had significantly (P<0·05) shorter mean PPP (2·58 days), reduced survival (6–47 days), more pronounced fever and dyspnoea. The latter had significantly (P<0·05) reduced RCC (10·74–4·56×106 cells/μl of blood), PCV (43·90–20·78%), Hb (13·06–5·74 g/dL), increased MCV (39·10–43·97 fL), severe generalized oedemas, haemorrhages, congestion, hepatic haemosiderosis, hepatitis, nephritis, endocarditis, pericarditis and exclusively, splenic macrophage and giant cell hyperplasia, expanded red pulp and splenic erythrophagocytosis. It was concluded that OTA aggravated the pathogenesis of T. b. rhodesiense infection in mice, and should therefore be taken into consideration during trypanosomosis control programmes.

Histomonas meleagridis is a protozoan parasite of various galliform birds causing a type of enterohepatitis termed histomonosis or ‘blackhead disease’. Due to the ban of chemotherapeutic substances and an increase in free-range poultry production, histomonosis is currently a re-emerging disease. So far limited molecular knowledge is available. In the present work, mRNAs coding for antigenic proteins of H. meleagridis were identified. For this purpose, a cDNA expression library was constructed from a mono-eukaryotic culture of H. meleagridis. The library was screened with polyclonal rabbit serum raised against purified H. meleagridis trophozoites. Polyclonal rabbit serum specifically recognized the same major H. meleagridis antigens as chicken and turkey sera originating from animal trials, but displayed a significantly lower bacteria-dependent background signal. After 2 rounds of screening, a total of 95 positive clones were sequenced. Bioinformatics analyses were performed on nucleotide and deduced amino acid sequences, identifying 37 unique clones. Based on the homology to other protozoan parasites, mostly Trichomonas vaginalis, the clones were grouped according to functional aspects: structural proteins, possible surface proteins, oxygen reducing proteins, ribosomal proteins, protein kinases and various other intracellular proteins.

Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms at the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the study period. We suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms.

An attenuated line of Leishmania major (L. major H-line) has been established by culturing promastigotes in vitro under gentamicin pressure. A modification of the previously described method for the generation of attenuated L. major is described, giving rise to attenuated parasites after 8 rather than 12 subpassages. No lesions developed in BALB/c mice infected with L. major H-line, whereas L. major wild-type (WT) induced a Th2 like response with progressive lesions. Analysis of splenocyte IFN-γ and IL-4 production following stimulation with promastigotes shows that the L. major H-line preferentially induces Th1-like responses and possibly down-regulates Th2 responses in BALB/c mice. L. major H-line parasites remained localized in the skin and draining lymph node, whereas L. major WT parasites disseminated into the visceral organs of BALB/c mice. Mice infected with L. major H-line acquired some resistance against L. major WT. These results show that the attenuated cell line of L. major is not only avirulent but that it may also modulate the host immune response.