Introduction. Leishmania donovani, the causative agent of visceral leishmaniasis in the Indian subcontinent, has been reported to be genetically homogeneous. In order to support ongoing initiatives to eliminate the disease, highly discriminative tools are required for documenting the parasite population and dynamics. Methods. Thirty-four clinical isolates of L. donovani from Nepal were analysed on the basis of size and restriction endonuclease polymorphisms of PCR amplicons from kinetoplast minicircle DNA, 5 nuclear microsatellites, and nuclear loci encoding glycoprotein 63, cysteine proteinase B, and hydrophilic acylated surface protein B. We present and validate a procedure allowing standardized analysis of kDNA fingerprint patterns. Results. Our results show that parasites are best discriminated on the basis of kinetoplast minicircle DNA (14 genotypes) and 1 microsatellite defining 7 genotypes, while the remaining markers discriminated 2 groups or were monomorphic. Combination of all nuclear markers revealed 8 genotypes, while extension with kDNA data yielded 18 genotypes. Conclusion. We present tools that allow discrimination of closely related L. donovani strains circulating in the Terai region of Nepal. These can be used to study the micro-epidemiology of parasite populations, determine the geographical origin of infections, distinguish relapses from re-infection, and monitor the spread of particular variants.

It has been observed that fluorescent membrane-impermeant molecules can enter the cercariae as they penetrate mouse skin. The hypothesis to be tested was that such molecules, which included Lucifer Yellow and a variety of fluorescent dextrans, entered the parasite through the nephridiopore and excretory tubules as well as through the surface membrane. FITC-labelled poly-L-lysine (molecular weight 10 kDa), added at 4°C during syringe transformation, was found to enter the nephridiopore and labelled the excretory bladder and sometimes the excretory tubules. This finding indicates that macromolecules (10 kDa) can enter the nephridiopore. It was found that linoleic acid (a normal constituent of skin) greatly stimulated uptake of Lucifer Yellow and dextrans into the excretory/subtegumental region of 2-h-old schistosomula. This correlated with an increased uptake of membrane-impermeant propidium iodide at 37°C. Since increased uptake of propidium iodide occurs when membranes become permeable, the surface membrane could also be a pathway of transport of the membrane-impermeant molecules into the schistosomulum.

Muscle tissue from 37 red deer from Norway was examined for sarcocysts. Sarcocysts from 2 reindeer were obtained for comparative studies. Cysts were excised and morphologically classified by light microscopy, scanning electron microscopy, and DNA sequence analysis. Five Sarcocystis species, Sarcocystis hjorti n. sp., Sarcocystis hardangeri, Sarcocystis ovalis, Sarcocystis rangiferi, and Sarcocystis tarandi, were found. All 5 species have previously been identified from either reindeer or moose by their sarcocyst morphology and/or ssu rRNA gene sequence. S. hjorti was the most prevalent species. Multiple variants of the ssu rRNA gene and the first internal transcribed spacer were found in S. rangiferi and S. tarandi from both red deer and reindeer. Phylogenetic analyses indicated that S. tarandi occurs in both red deer and reindeer, but it could not be clearly demonstrated whether the sequence variation within S. rangiferi between hosts was due to different paralogues or/and different species. DNA sequencing was necessary for definitive species identification, since the hair-like protrusions on the cysts of S. hjorti were not always recognizable by light microscopy and since different cervids harbour Sarcocystis species with highly similar cyst morphology of which at least some are not intermediate host specific.

We used 12 microsatellite markers developed for Leishmania braziliensis to genotype 28 strains of the main species of the Leishmania guyanensis complex (i.e. L. guyanensis and L. panamensis) collected in Ecuador and Peru. The important heterozygote deficits observed in these populations are similar with the previous data obtained in L. braziliensis and raise again the debate on the reproductive mode of these protozoan parasites. The data showed genetic polymorphism and geographical differentiation giving information on population structure of the L. guyanensis complex. Regarding the two species, this study enhances again the debate on the taxonomic status of the different isolates belonging to L. guyanensis s.l. since the results showed substantial heterogeneity within this species complex. In conclusion, this study increases the number of available microsatellite loci for L. guyanensis species complex and raises fundamental biological questions. It confirms that microsatellite markers constitute good tools for population genetic studies on parasites of this complex.

For over 50 years it has been known that there are considerable differences in the severity and rate of progression of both Trypanosoma brucei rhodesiense and T. b. gambiense infection between individuals. Yet research into the factors, whether parasite or host, which control virulence in Human African trypanosomiasis is in its infancy. In this paper we review the clinical evidence for virulence variation and the epidemiological and experimental data that give clues as to the mechanisms involved. Evidence will be presented for both asymptomatic forms of T. b. gambiense infection and low virulence forms of T. b. rhodesiense infection in humans. While in both cases the mechanisms remain to be elucidated, the overall infection virulence phenotype is determined by both parasite and host genotype.

Monocystis sp. are sporocyst-forming apicomplexan parasites common in seminal vesicles of the earthworm Lumbricus terrestris where they may account for temporary castration. This study describes the internal transcribed spacer (ITS) region of the ribosomal cistron of Monocystis sp. This region, including ITS-1, the 5·8S ribosomal RNA gene, and ITS-2, was PCR amplified, cloned, and sequenced for Monocystis sp. isolated from the seminal vesicles of several wild-caught L. terrestris. Our analysis revealed substantial polymorphisms, also within single host organisms, indicating intra-host diversity of parasites. These genetic markers are the first that allow distinction of Monocystis sp. genotypes, opening new avenues for the study of parasite diversity within and between hosts.

To investigate the phylogeny of benign Theileria parasites, we determined the complete major piroplasm surface protein (MPSP) gene sequences for 6 benign theilerial organisms, including the first from tick. Sequences were analysed alongside published sequences for 39 benign Theileria parasites, using Bayesian inference and maximum parsimony. All MPSP sequences were 852 nucleotides, except for Gansu, Wuchangbuf, VB01, and VB01; Gansu contained 873 nucleotides, and the other 3 had 855. Deduced amino acid sequences contained 284 residues, except for Gansu (291) and Wuchangbuf, VB01, and VB01 (285 each). Pairwise comparisons showed identities among 45 theilerial MPSP sequences ranging from 70·9 to 99·8% for nucleotide and 71·0 to 100% for amino acid sequences. Our results clearly indicate that all global parasites, excluding Brisbane, were classified into 1 of 8 types; 6 types of Theileria exist in Korea. Each type, excluding Type 6, has several type-specific amino acid sequences. The phylogenetic tree derived from the nucleotide sequences showed 2 sister-group relationships, Type 2+Type 7 and Type 3+Brisbane, with a new branching pattern: (Type 6 (Type 8 ((Type 2, Type 7), (Type 1, (Type 4, (Type 5, (Type 3, Brisbane))))))). Our sequence data showed no geographical influence on worldwide Theileria parasite distribution.

Mesocestodes corti has the capacity to develop from the tetrathyridium (larva) stage to adult worm in vitro by trypsin and serum stimulation. Consequently, it has been used as an experimental model system for studying cestode development, host-parasite relationships and anthelmintic drugs. We describe morphological features in 5 different developmental stages of M. corti obtained in vitro, including larvae from the peritoneal cavity of infected mice, trypsin- and serum-stimulated larvae, elongated parasites as well as segmented and mature worms. It is unambiguously confirmed that sexually mature worms are obtained as a result of this in vitro process of differentiation. Defined cellular regions are present in all stages of development studied, some of them surrounded by a basal lamina. Glycogen is present in the larvae obtained from the mouse peritoneal cavity and in parasites encapsulated in the mouse host liver. Glycogen distribution in the parasite changes on trypsin and serum stimulation to differentiate. We propose that changes in the distribution of neutral polysaccharides in the parenchyma of the parasite at different stages of development and degradation of polysaccharides in the transition from segmented to adult worm are related to energy needs necessary for the cellular processes leading to the mature specimen.

Objective. Bilharziosis is one of the most important helminthal infections in humans and is caused by blood flukes of the genus Schistosoma. Three different life stages of the parasite occur within the mammalian host: schistosomula located in the skin, pre-adults located in the lung and adult worms located in the portal venous system. Erythrocytes are a major source of nutrient supply for adults. However, sources of nutrition for the developing stages are still unclear. Methods. To investigate whether schistosomula, pre-adults and adults of Schistosoma mansoni ingest human serum albumin (HSA) in vitro, these life stages were incubated with aminofluorescein-labelled human serum albumin (Afl-HSA) for 5 h. To test the uptake of albumin in vivo, the albumin conjugate was given intravenously to S. mansoni infected NMRI mice 24 h before harvesting the 3 life stages. Results. In comparison to the control group schistosomula, pre-adults, and adults showed an accumulation of Afl-HSA within the oesophagus and intestinal caecum in vitro and in vivo. Conclusion. Our findings suggest that albumin seems to be a major source of energy supply for the early schistosomal life stages and an additive energy support for adult worms. Since albumin has been used successfully as a drug carrier for chemotherapeutic substances against malignant disorders, further studies will focus on albumin as a carrier for anthelminthics in a drug-targeting model.

Salivarian trypanosomes pose a substantial threat to livestock, but their full diversity is not known. To survey trypanosomes carried by tsetse in Tanzania, DNA samples from infected proboscides of Glossina pallidipes and G. swynnertoni were identified using fluorescent fragment length barcoding (FFLB), which discriminates species by size polymorphisms in multiple regions of the ribosomal RNA locus. FFLB identified the trypanosomes in 65 of 105 (61·9%) infected proboscides, revealing 9 mixed infections. Of 7 different FFLB profiles, 2 were similar but not identical to reference West African Trypanosoma vivax; 5 other profiles belonged to known species also identified in fly midguts. Phylogenetic analysis of the glycosomal glyceraldehyde phosphate dehydrogenase gene revealed that the Tanzanian T. vivax samples fell into 2 distinct groups, both outside the main clade of African and South American T. vivax. These new T. vivax genotypes were common and widespread in tsetse in Tanzania. The T. brucei-like trypanosome previously described from tsetse midguts was also found in 2 proboscides, demonstrating a salivarian transmission route. Investigation of mammalian host range and pathogenicity will reveal the importance of these new trypanosomes for the epidemiology and control of animal trypanosomiasis in East Africa.

Treatment of liver fibrosis associated with Schistosoma japonicum ova-induced granulomas remains a challenging proposition. Paeoniflorin (PAE, C23H28O11) has anti-inflammatory, anti-allergic, and immunoregulatory effects and it is commonly used in Chinese Herbal prescriptions to treat hepatic disorders. The present study was carried out to investigate the effects of PAE on hepatic fibrosis of mice infected with S. japonicum and to explore its possible mechanism. Upon pathological examination of PAE-treated mice, the size of egg granuloma, fibrosis scores, the concentration of IL-13 and hydroxyproline in liver were significantly reduced compared with the model mice. In the primary culture of hepatic stellate cells (HSCs), PAE inhibited IL-13-induced collagen synthesis. These results suggested that PAE might alleviate the hepatic granulomas and fibrosis caused by S. japonicum and the inhibitory effect of PAE on hepatic fibrosis might be associated with its ability to decrease the level of IL-13 and to interfere with the IL-13 signalling molecule in HSCs.

Anti-trypanosomiasis vaccination still remains the best theoretical option in the fight against a disease that is continuously hovering between its wildlife reservoir and its reservoir in man and livestock. While antigentic variation of the parasite surface coat has been considered the major obstacle in the development of a functional vaccine, recent research into the biology of B cells has indicated that the problems might go further than that. This paper reviews past and current attempts to design both anti-trypanosome vaccines, as well as vaccines directed towards the inhibition of infection-associated pathology.

African trypanosomes have emerged as promising unicellular model organisms for the next generation of systems biology. They offer unique advantages, due to their relative simplicity, the availability of all standard genomics techniques and a long history of quantitative research. Reproducible cultivation methods exist for morphologically and physiologically distinct life-cycle stages. The genome has been sequenced, and microarrays, RNA-interference and high-accuracy metabolomics are available. Furthermore, the availability of extensive kinetic data on all glycolytic enzymes has led to the early development of a complete, experiment-based dynamic model of an important biochemical pathway. Here we describe the achievements of trypanosome systems biology so far and outline the necessary steps towards the ambitious aim of creating a ‘Silicon Trypanosome’, a comprehensive, experiment-based, multi-scale mathematical model of trypanosome physiology. We expect that, in the long run, the quantitative modelling enabled by the Silicon Trypanosome will play a key role in selecting the most suitable targets for developing new anti-parasite drugs.

The domestic dog's involvement with different members of the Trypanosomatidae family has been the focus of several studies due to this animal's close proximity to man. Recently this animal has been infected by a new Trypanosoma species (T. caninum), described in Rio de Janeiro and 19 similar isolates were later obtained. The objective of this study was to identify these isolates. All samples were isolated from intact skin cultures and analysed morphologically, by biochemical isoenzyme electrophoresis assays and by several molecular PCR assays. Additionally, anti-Leishmania sp. antibodies were assessed using the indirect Immunofluorescence Antibody Test (IFAT) in all animals. The methodologies employed to identify the isolates, including partial nucleotide sequences of 18S rRNA gene, indicated patterns identical to T. caninum and patterns different from the other species, including T. cruzi and T. rangeli samples. A phylogenetic tree constructed with the partial 18S ribosomal sequence shows that T. caninum is clustered with T. pestanai. Ten (52·6%) animals presented anti-Leishmania sp. antibodies with titres varying from 1:40 to 1:320. Thus, the hypothesis that this protozoan has disseminated among the dogs in Rio de Janeiro must be considered. The importance of a correct diagnosis in those animals and the possible consequences in the areas where visceral leishmaniasis is found are discussed here.

No vaccines or safe chemotherapy are available for Chagas disease. Pentamidine and related di-cations are DNA minor groove-binders with broad-spectrum anti-protozoal activity. Therefore our aim was to evaluate the in vitro efficacy of di-cationic compounds – DB1645, DB1582, DB1651, DB1646, DB1670 and DB1627 – against bloodstream trypomastigotes (BT) and intracellular forms of Trypanosoma cruzi. Cellular targets of these compounds in treated parasites were also analysed by fluorescence and transmission electron microscopy (TEM). DB1645, DB1582 and DB1651 were the most active against BT showing IC50 values ranging between 0·15 and 6·9 μm. All compounds displayed low toxicity towards mammalian cells and DB1645, DB1582 and DB1651 were also the most effective against intracellular parasites, with IC50 values ranging between 7·3 and 13·3 μm. All compounds localized in parasite nuclei and kDNA (with greater intensity in the latter structure), and DB1582 and DB1651 also concentrated in non-DNA-containing cytoplasmic organelles possibly acidocalcisomes. TEM revealed alterations in mitochondria and kinetoplasts, as well as important disorganization of microtubules. Our data provide further information regarding the activity of this class of compounds upon T. cruzi which should aid future design and synthesis of agents that could be used for Chagas disease therapy.

Up to 5% of untreated female Onchocerca volvulus filariae develop potentially fatal pleomorphic neoplasms, whose incidence is increased following ivermectin treatment. We studied the occurrence of 8 filarial proteins and of Wolbachia endobacteria in the tumor cells. Onchocercomas from patients, untreated and treated with antibiotics and anthelminthics, were examined by immunohistology. Neoplasms were diagnosed in 112 of 3587 female and in 2 of 1570 male O. volvulus. The following proteins and other compounds of O. volvulus were expressed in the cells of the neoplasms: glutathione S-transferase 1, lysosomal aspartic protease, cAMP-dependent protein kinase, alpha-enolase, aspartate aminotransferase, ankyrin E1, tropomyosin, heat shock protein 60, transforming growth factor-beta, and prostaglandin E2. These findings prove the filarial origin of the neoplasms and confirm the pleomorphism of the tumor cells. Signs indicating malignancy of the neoplasms are described. Wolbachia were observed in the hypodermis, oocytes, and embryos of tumor-harbouring filariae using antibodies against Wolbachia surface protein, Wolbachia HtrA-type serine protease, and Wolbachia aspartate aminotransferase. In contrast, Wolbachia were not found in the cells of the neoplasms. Further, neoplasm-containing worms were not observed after more than 10 months after the start of sufficient treatment with doxycycline or doxycycline plus ivermectin.

The epidemiology of schistosomiasis japonicum over small areas remains poorly understood, and this is particularly true in China. We aimed to identify high-risk areas for schistosomiasis and associated risk factors in the Poyang Lake region, China. A cross-sectional study was conducted and 60 of 920 persons (6·5%) were found to be infected with Schistosoma japonicum. Locations of households and snail habitats were determined using a hand-held global positioning system. We mapped the data in a geographical information system and used spatial scan statistics to explore clustering of infection, logistic regression and Bayesian geostatistical models to identify risk factors for each individual's infection status and multinomial logistic regression to identify risk factors for living in a cluster area. The risk of schistosomiasis was spatially clustered and higher in fishermen and males, not in persons who lived in close proximity to snail habitats and infected water sources. This study has demonstrated significant spatial variation in the prevalence of schistosomiasis at a small spatial scale. The results suggest that demographic factors (gender, occupation) rather than the distance to infected water are driving human transmission at small-scale spatial levels. Such information can be used to plan locally targeted interventions based on anthelminthic drug administration, snail control and sanitation improvement.

Trypanosoma cruzi (Tc), the causative agent of Chagas disease, is a diverse species with 2 primary genotypes, TcI and TcII, with TcII further subdivided into 5 subtypes (IIa–e). This study evaluated infection dynamics of 4 genetically and geographically diverse T. cruzi strains in 2 South American reservoirs, degus (Octodon degus) and grey short-tailed opossums (Monodelphis domestica). Based on prior suggestions of a genotype-host association, we hypothesized that degus (placental) would more readily become infected with TcII strains while short-tailed opossums (marsupial) would be a more competent reservoir for a TcI strain. Individuals (n=3) of each species were intraperitoneally inoculated with T. cruzi trypomastigotes of TcIIa [North America (NA)-raccoon (Procyon lotor) origin], TcI [NA-Virginia opossum (Didelphis virginiana)], TcIIb [South America (SA)-human], TcIIe (SA-Triatoma infestans), or both TcI and TcIIa. Parasitaemias in experimentally infected degus peaked earlier (7–14 days post-inoculation (p.i.)) compared with short-tailed opossums (21–84 days p.i.). Additionally, peak parasitaemias were higher in degus; however, the duration of detectable parasitaemias for all strains, except TcIIa, was greater in short-tailed opossums. Infections established in both host species with all genotypes, except for TcIIa, which did not establish a detectable infection in short-tailed opossums. These results indicate that both South American reservoirs support infections with these isolates from North and South America; however, infection dynamics differed with host and parasite strain.

The gender-biased pattern of parasite infestation has been shown to be a complicated phenomenon that cannot be explained by a single mechanism but rather involves several different mechanisms. We asked what are the factors that affect the manifestation and extent of gender-biased parasitism and studied the relationship between parasite-related (mean abundance, mean species richness and total species richness of all parasites), host-related (rodent density and proportion of reproductive males and females both separately and together) and environment-related (mean daily maximal and minimal temperatures, rainfall and relative humidity) factors and the magnitude of gender-biased infestation of a South African rodent Rhabdomys pumilio by ixodid ticks, gamasid mites, lice and fleas. We found that spatial variation in gender differences in parasite infestation was affected by parasite-, host- and environment-related factors, although the set of factors affecting gender differences in infestation differed among higher taxa of ectoparasites. Gender differences in infestation by fleas and lice were affected mainly by parasite-related factors, whereas gender differences in infestation by ticks and, in part, by mites were affected mainly by host-related and environmental factors. In addition, spatial variation in most measures of gender difference in mite infestation remained unexplained.

A histopathological study was performed to clarify the characteristics of granuloma formation and liver fibrosis in Schistosoma mekongi infection in comparison with S. japonicum infection. Mice were exposed to S. mekongi (Laotian strain) and S. japonicum (Japanese strain) cercariae, and were dissected at 6, 8, 12, 16, and 20 weeks post-exposure. In the liver, granulomas in S. mekongi infection were cellular, initially organized with foam cells, and continuously appeared in the intralobular area, while granulomas in S. japonicum infection were fibrous and did not continuously appear in the intralobular area. Portal fibrosis was not seen in S. mekongi infection, but was commonly seen in S. japonicum infection in the later weeks. Granulomas in the small intestine were seen mainly in the submucosa with foam cells in S. mekongi infection and without foam cells in S. japonicum infection. The lung granulomas contained mainly histiocytes in both S. mekongi and S. japonicum infection. The absence of portal fibrosis in S. mekongi infection allows schistosome eggs to infiltrate into the intralobular area continuously, which can be what lies behind the ultrasonographic differences; the echogenic network pattern as was seen in S. japonicum infection, has not been noted in S. mekongi infection.