Trypanosoma (Herpetosoma) grosi, which naturally parasitizes Apodemus spp., can experimentally infect Mongolian jirds (Meriones unguiculatus). Three isolates from A. agrarius, A. peninsulae, and A. speciosus (named SESUJI, HANTO, and AKHA isolates, respectively) of different geographical origin (AKHA from Japan, and the others from Vladivostok), exhibited different durations of parasitaemia in laboratory jirds (2 weeks for HANTO, and 3 weeks for the others). To assess the genetic background of these T. grosi isolates, their small (SSU) and large subunit (LSU) ribosomal RNA genes (rDNA) were sequenced along with those of 2 other Herpetosoma species from squirrels. The SSU rDNA sequences of these 3 species along with available sequences of 3 other Herpetosoma trypanosomes (T. lewisi, T. musculi and T. microti) seemed to reflect well the phylogenetic relationship of their hosts. Three isolates of T. grosi exhibited base changes at 2–6 positions of 2019-base 18S rDNA, at 5–29 positions of 1817/1818-base 28Sα rDNA, or 1–5 positions of 1557–1559-base 28Sβ rDNA, and none was separated from the other 2 isolates by rDNA nucleotide sequences. Since base changes of Herpetosoma trypanosomes at the level of inter- and intra-species might occur frequently in specified rDNA regions, the molecular analysis on these regions of rodent trypanosomes could help species/strain differentiation and systematic revision of Herpetosoma trypanosome species, which must be more abundant than presently known.

Sand fly saliva plays an important role in Leishmania transmission. We characterized the host antibody response to saliva from 3 sand fly species. Specific IgG was observed in sera from experimentally bitten mice as well as in sera from individuals living in the endemic area of Leishmania tropica in Sanliurfa, Turkey. Sera of Sanliurfa inhabitants showed high IgG levels against saliva of Phlebotomus sergenti and P. papatasi, the 2 most abundant sand fly species in this area, but did not react with saliva of the New World sand fly, Lutzomyia longipalpis. Patients with active Le. tropica lesions possessed significantly higher anti-P. sergenti IgG levels than the healthy individuals from the same place while anti-P. papatasi IgG levels were equal in both groups. Major protein bands in P. papatasi and P. sergenti saliva reacted with both, human and mice sera; in P. papatasi, however, mouse IgG recognized preferentially the 42 kDa protein band while the human IgG reacted strongly with the 30 kDa band. Our data suggest that the antibody response to sand fly saliva could be used for monitoring the exposure of humans and other hosts to sand flies and might be used as a marker of risks for Leishmania transmission in endemic areas.

By sampling individual rainbow trout, Oncorhynchus mykiss, at a fish farm we showed that Argulus coregoni were aggregated within their host population. The relative significance of susceptibility and exposure generating the observed pattern was tested using experimental infections. We examined, whether rainbow trout developed protective resistance mechanisms against the louse following a challenge infection and if there was variation between individual trout in their susceptibility to A. coregoni metanauplii. Fish were exposed to 20 A. coregoni for 5, 25, 50, 85 or 120 min and the numbers attaching recorded. Three weeks later, developing argulids were removed and the experiment repeated with a standardized exposure of 20 metanauplii. Prior exposure of fish with A. coregoni did not reduce the total infection intensity compared to naïve fish, but fish gained infection more rapidly. We suggest that there is no protective acquired resistance of pre-exposed rainbow trout to subsequent Argulus exposure. The possibility that an immunosuppressive mechanism by argulids was acting enabling the higher attachment rate could be refuted since control individuals, not previously exposed to lice, gained the infection at a similar rate as the fish challenged twice. Our results do not indicate clear differences in susceptibility among individual fish but the transmission of metanauplii on fish seemed to be opportunistic and non-selective. Our results support the view that variation in exposure time, rather than differences in susceptibility of individual hosts, might be the key factor in generating the aggregated distribution of Argulus on their hosts.

Between 20% and 60% of the population of most countries are infected with the protozoan Toxoplasma gondii. Subjects with clinically asymptomatic life-long latent toxoplasmosis differ from those who are Toxoplasma free in several behavioural parameters. Case-control studies cannot decide whether these differences already existed before infection or whether they were induced by the presence of Toxoplasma in the brain of infected hosts. Here we searched for such morphological differences between Toxoplasma-infected and Toxoplasma-free subjects that could be induced by the parasite (body weight, body height, body mass index, waist-hip ratio), or could rather correlate with their natural resistance to parasitic infection (fluctuating asymmetry, 2D[ratio ]4D ratio). We found Toxoplasma-infected men to be taller and Toxoplasma-infected men and women to have lower 2D[ratio ]4D ratios previously reported to be associated with higher pre-natal testosterone levels. The 2D[ratio ]4D ratio negatively correlated with the level of specific anti-Toxoplasma antibodies in Toxoplasma-free subjects. These results suggest that some of the observed differences between infected and non-infected subjects may have existed before infection and could be caused by the lower natural resistance to Toxoplasma infection in subjects with higher pre-natal testosterone levels.

While purine transport has been widely studied in protozoa, almost nothing is known about their capacity to salvage pyrimidines. Here, we report a Leishmania major transporter with high affinity for uracil (Km=0·32±0·07 μM) which we designated LmU1. This transporter displayed a high degree of specificity, as it had virtually no affinity for cytosine, thymine or purine nucleobases, nor did it transport pyrimidine nucleosides. Highest affinity was for 5-fluorouracil. The results show that the permeant binding site of LmU1 interacts strongly with the keto groups of uracil, as shown by a low affinity for 2-thio- and 4-thiouracil. LmU1 appears to further bind uracil through a weak hydrogen bond with N(1)H of the pyrimidine ring in addition to a stronger H-bond with N(3)H. Substrate binding and selectivity were strikingly similar to that of the U1 transporter in the related kinetoplastid Trypanosoma brucei. Uracil analogues likely to be transported by LmU1 were also screened for antileishmanial activity, with 5-fluorouracil displaying strong activity against promastigotes and intracellular amastigotes. Overall, the results show that, like purine nucleobase transport, pyrimidine nucleobase transport function is very similar in L. major and T. brucei insect forms.

The protozoa Leishmania spp. are obligate intracellular parasites that inhabit the macrophages of their host. Since macrophages are specialized for the identification and destruction of invading pathogens, both directly and by triggering an innate immune response, Leishmania have evolved a number of mechanisms for suppressing some critical macrophage activities. In this review, we discuss how various species of Leishmania distort the host macrophage's own signalling pathways to repress the expression of various cytokines and microbicidal molecules (nitric oxide and reactive oxygen species), and antigen presentation. In particular, we describe how MAP Kinase and JAK/STAT cascades are repressed, and intracellular Ca2+ and the activities of protein tyrosine phosphatases, in particular SHP-1, are elevated.

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

Fatal murine cerebral malaria is an encephalitis and not simply a local manifestation in the brain of a systemic process. Histopathologically, murine cerebral malaria has been characterized by monocyte adherence to the endothelium of the microvasculature, activation of microglial cells, swelling of endothelial cell nuclei, microvasculature damage, and breakdown of the blood-brain barrier with cerebral oedema. Brain parenchymal cells have been proposed to be actively involved in the pathogenesis of murine cerebral malaria. We, therefore, compared the neurochemical characteristics of Plasmodium berghei ANKA-infected mice with controls to determine whether cerebral malarial infection significantly impairs specific neuronal populations. Between 6 and 7 days after infection, we found a significant loss of neurones containing substance P, with preservation of cells containing somatostatin, neuropeptide Y and calbindin in the striatum of infected mice compared with controls. In the cortex of infected mice, we found a significant reduction in the number of cells containing substance P, somatostatin and neuropeptide Y. The number of calbindin-containing neurones was unchanged. This study found significant changes in the neurochemical characteristics of the cortex and striatum of mice infected with P. berghei ANKA, which may contribute to their cerebral symptoms.

Lymphocytes infected with the protozoan parasite Theileria parva are transformed to permanently proliferating cells, an event underlying the pathology of the disease. However, the molecular signalling mediating this process is complex and poorly understood. Here, we show that down-regulation of JNK signalling by transient over expression of a dominant-negative mutant of JNK (JNK-APF) significantly increases Annexin-V-phycoerythrin (V-PE) labelling on infected B cell populations observed using flow cytometry. To establish whether this increase was specifically due to apoptosis, we used a novel single-cell imaging method: micro-rotation (MR)-imaging, designed to allow high-resolution 3-dimensional imaging of single cells in suspension. With this method we visualized subcellular patterns of V-PE uptake and chromatin organization in lymphocytes co-transfected with JNK-APF and GFP-tagged histone-H2B. This single-cell approach allowed us to clearly reveal characteristic apoptotic phenotypes, whose patterns reflected progressive states of programmed cell death due to JNK down-regulation. Our results strongly suggest a role for JNK in the survival of Theileria-infected B cells, and demonstrate the powerful utility of a new and unique 3-dimensional imaging method for living cells in suspension.

We monitored temporal changes in the magnitude of altered host behaviour in minnows (Pimephales promelas) experimentally infected with metacercariae of a brain-encysting trematode (Ornithodiplostomum ptychocheilus). This parasite develops and then encysts in a region of the brain that mediates the optomotor response (OMR), an innate behaviour that links visual stimuli with motor performance. The OMR of infected and uninfected minnows was evaluated between 0 and 10 weeks post-infection (p.i.), an interval spanning the development period of metacercariae to infectivity in birds. Trials involved monitoring the time an individual minnow spent following a spinning drum that had been painted with alternating black and white stripes. At 2 and 4 weeks p.i., infected minnows followed the drum 40% less often than controls. Differences between controls and infected fish declined thereafter, and were undetectable by 10 weeks p.i. Both control and infected fish habituated equally rapidly to the spinning drum. However, the difference in performance between controls and infected fish was 29% for experienced fish and 48% for fish that had never experienced the drum. Because maximum parasite-induced reduction in OMR coincided with the period of maximum parasite development, the behavioural effects are most likely due to unavoidable pathology in the brain associated with developing larvae.

The development of a new species, Bacillidium vesiculoformis n. sp. (Microspora, Mrazekiidae), is described from the freshwater oligochaete Nais simplex (Oligochaeta, Naididae). Initial stages of parasite development consist of a monokaryotic merogony within a haemocyte of the intestinal blood sinus. The resulting hypertrophied haemocyte is attached to the chloragocytes of the sinus by fine cytoplasmic extensions with the sinus around the cell becoming greatly enlarged. The meronts within the haemocyte form diplokaryotic sporonts that undergo sporogenesis directly within the cytoplasm of the host cell. The infected cell becomes packed with spores and developmental stages, causing it dramatically to increase in size, eventually rupturing the oligochaete and cell. Sporogony appears to be disporoblastic. Released spores were observed to have an adhesive quality. Transmission studies conducted with mature spores failed to transmit the parasite horizontally although vertical transmission was observed. Phylogenetic analysis of the parasite demonstrated that B. vesiculoformis clustered with microsporidian parasites of bryozoa and two other microsporidians, Janacekia debaiseuxi and an unidentified Bacillidium sp.

Microsporidia are obligate intracellular parasites that utilize a unique mechanism to infect host cells, which is one of the most sophisticated infection mechanisms in biology. Microsporidian spores contain a long coiled polar tube that extrudes from the spores and penetrates the membranes of new host cells. We have initiated a study to investigate the invasive process and intracellular fate of the microsporidium Encephalitozoon cuniculi. Here we show that relatively few cells were infected through the traditional penetration of the polar tube from outside. Rather, phagocytosis of spores occurred at least 10 times more frequently than injection of sporoplasms. Some spores extruded their polar tube inside the cells following phagocytosis. Membranes of the vacuoles surrounding the internalized spores were positive for late endosomal and lysosomal markers. Spores that remained inside these compartments disappeared within 3 days. Thus, our studies demonstrate that in addition to the unique way in which microsporidia infect host cells, E. cuniculi spores can also gain access to host cells by phagocytosis. The presence of intracellular spores that have extruded their polar tube shows that some spores germinate after phagocytosis, thus escaping from the phagosomes that mature into lysosomes.

Periods of low host density impose a constraint on parasites with direct transmission, challenging their permanence in the system. The microsporidium Octosporea bayeri faces such constraint in a metapopulation of its host, the cladoceran Daphnia magna, where ponds frequently lose their host population due to ponds drying out in summer and freezing in winter. We conducted experiments aimed to investigate the mechanisms of transmission of O. bayeri, and discuss how these mechanisms could contribute to the parasite's permanence in the system. Spores accumulate in the fat cells and the ovaries of the host, and vary in morphology, possibly corresponding to 3 different spore types. Horizontal transmission occurred through the release of spores from dead hosts, with the proportion of infected hosts depending on the spore dose. Further, spores are able to persist outside the host both in dry and wet conditions. Vertical transmission occurred to both parthenogenetic and sexual offspring. The former were invariably infected, while the sexually produced resting eggs (=ephippia) had a less efficient transmission. The parasite may be carried by the ephippia, and thus disperse to new ponds together with the host. Together, these mechanisms may allow the parasite to endure periods of harsh environmental conditions both outside and inside the host.

Activation of casein kinase II (CK2) was one of the first observations made on how Theileria parasites manipulate host cell signal transduction pathways and we argue that CK2 induction may in fact contribute to many of the different activation events that have been described since 1993 for Theileria-infected lymphocytes such as sustained activation of transcription factors c-Myc and NF-κB. CK2 also contributes to infected lymphocyte survival by inhibiting caspase activation and is probably behind constitutive PI3-K activation by phosphorylating PTEN. Finally, we also discuss how CK2A may act not only as a kinase, but also as a stimulatory subunit for the protein phosphatase PP2A, so dampening down the MEK/ERK and Akt/PKB pathways and for all these reasons we propose CK2 as a central player in Theileria-induced lymphocyte transformation.

Blastocrithidia culicis is an insect trypanosomatid that presents bacterial endosymbionts. The cell-associated and secreted proteinases of the endosymbiont-bearing and aposymbiotic strains were compared through the incorporation of proteinaceous substrates into sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Few qualitative changes could be detected in the proteolytic zymograms in the 2 strains studied when gelatin, casein, haemoglobin or bovine serum albumin (BSA) were tested. However, the level of proteolytic activities was significantly higher in the aposymbiotic strain. Some of the B. culicis proteins reacted in Western blots with antibodies raised against gp63, a zinc-metalloproteinase, and cruzipain, a cysteinyl-proteinase, which are virulence factors of the human pathogenic trypanosomatids, Leishmania spp. and Trypanosoma cruzi, respectively. The anti-cross-reacting determinant (CRD) antibody recognized 2 polypeptides (50 and 58 kDa) in the spent culture media and in the supernatant from glycosylphosphatidylinositol-phospholipase C (GPI-PLC)-treated cells, suggesting that these proteins are GPI-anchored to the plasma membrane. In addition, the anti-gp63 reacted with the 50 kDa protein. The identification of protein homologues in trypanosomatids with distinct life-cycles may help to determine the importance of proteinases in trypanosomatids.

We studied the role of Trypanosoma cruzi reinfection in regard to inflammatory and cytokine response at the inoculation site, lymph node and heart. We reinfected Balb/c mice intradermically into the hind foot-pad with natural infective metacyclic trypomastigotes. They were followed from 24 h to 30 days after the last reinfection. At the inoculation site 24 h after the last re-infection, the infiltrating inflammatory cells increased dramatically with respect to baseline inflammation, reaching maximum infiltrates for the third day. In contrast, parasite DNA was undetectable 24 h after inoculation, despite poor cytokine induction, only IFN-gamma, IL-12 and TGF-β were noticeable on days 7 and 15, whereas in the lymph nodes draining the inoculation site positive expression of IL-2, IL-4, IL-12 and TGF-beta were found to be induced as soon as 24 h after re-entry of parasite. In the heart, the inflammatory response increased immediately 24 h after re-entry of parasites, reaching its maximum on the 7th day and returning to baseline on day 30. In conclusion, although the inflammatory response is triggered in both compartments by re-entry of parasites, the inflammatory process returns almost to baseline after 30 days, leaving a persistent low-grade inflammation.

Data from a long-term study of the intestinal helminth parasite community of eels, Anguilla anguilla, stocked into the shallow eutrophic Neusiedler See, Austria, were collected over an 8 year period (1994–2001). In total, 720 eels from 2 sampling sites were examined. The parasite community showed characteristics similar to those in the natural eel populations in rivers of the UK and mainland Europe: it was species poor, with only 5 species (Acanthocephalus lucii, Acanthocephalus anguillae, Raphidascaris acus, Proteocephalus macrocephalus, Bothriocephalus claviceps) comprising the component community and a maximum infracommunity richness of 4 species. Over the period, the intestinal parasite community of the sampling site in Illmitz, which was originally dominated by A. lucii, changed. As levels of A. anguillae increased to a point at which it dominated the community, diversity increased whilst dominance of a single species decreased. By contrast the community in the southern sampling site remained rather constant with a continuous high infection level of A. anguillae and low abundance of A. lucii. Both acanthocephalan species exhibited higher infection levels in larger eels and in different seasons of the year and the infection parameters were significantly different between the years of study. The significant differences in the infection levels of the 2 acanthocephalan species at the 2 sampling sites were surprising as both acanthocephalan species use the same intermediate host, Asellus aquaticus, and the sampling sites were in close proximity and were similar in terms of water quality, host size and invertebrate abundance. Differences in the fish communities of the 2 sampling sites and eel movements rather than interspecific competition are discussed as possible explanations for the differences in the parasite communities of the 2 sampling sites.

In this study with the filarial model Litomosoides sigmodontis, we demonstrate that the worms ingest host red blood cells at a precise moment of their life-cycle, immediately after the fourth moult. The red blood cells (RBC) were identified microscopically in live worms immobilized in PBS at 4 °C, and their density assessed. Two hosts were used: Mongolian gerbils, where microfilaraemia is high, and susceptible BALB/c mice with lower microfilaraemia. Gerbils were studied at 12 time-points, between day 9 post-inoculation (the worms were young 4th stage larvae) and day 330 p.i. (worms were old adults). Only the very young adult filarial worms had red blood cells in their gut. Haematophagy was observed between days 25 and 56 p.i. and peaked between day 28 and day 30 p.i. in female worms. In males, haematophagy was less frequent and intense. Similar kinetics of haematophagy were found in BALB/c mice, but frequency and intensity tended to be lower. Haematophagy seems useful to optimize adult maturation. These observations suggest that haematophagy is an important step in the life-cycle of L. sigmodontis. This hitherto undescribed phenomenon might be characteristic of other filarial species including human parasites.

Manuls or Pallas' cats (Felis manul, syn. Otocolobus manul) are endangered wild cats from Central Asia kept and bred in many zoos. Despite good breeding success young cats frequently die from acute toxoplasmosis. From 1998 to 2002, a breeding pair in the Schönbrunn Zoo in Vienna, Austria, gave birth to 24 kittens; 58% of kittens died between the 2nd and the 14th week of life, mostly due to acute toxoplasmosis. The epidemiology of toxoplasmosis in Pallas' cats was examined and a control strategy to protect the kittens from fatal toxoplasmosis was developed. One 12-week-old kitten from a litter of 6 born in 2001 died of generalized toxoplasmosis. This kitten had shed T. gondii oocysts that were bioassayed in mice. Toxoplasma gondii was isolated in tissue culture inoculated with tissues of these mice. The surviving animals were immediately treated with clindamycin for 16 weeks; they acquired a natural infection and seroconverted by the end of this time without clinical signs.

We addressed the question whether diaplacental transmission of Neospora caninum can be controlled by metaphylactic chemotherapy using toltrazuril or enrofloxacin. Female C57/BL6 mice, infected on day 10 of pregnancy, were medicated for 6 consecutive days p.i. with 52·5 mg toltrazuril or – as an out-group control medication – 16·7 mg enrofloxacin per kg body weight per day. Other control groups received either infection but no medication or vice versa. Toltrazuril treatment significantly reduced pre- and perinatal losses (10 deliveries of healthy newborns, versus 1 abortion and 4 failures) when compared to control-enrofloxacin (2 deliveries, versus 1 abortion, 7 failures and 2 pre-parturient deaths of dams) and non-treated animals (3 deliveries, versus 6 abortions, 8 failures and 4 pre-parturient deaths). Simultaneously, PCR-based parasite detection in the brain of mothers, histopathological findings as well as clinical fatality were significantly less frequent in toltrazuril-treated dams. The overall toltrazuril treatment efficacy was determined as 87%, that of enrofloxacin-treatment as 17%. The progenies of toltrazuril-treated dams also exhibited a very low rate of PCR-positivity in their brain (3 out of 39), whereas untreated dams delivered litters with mostly PCR-positive brains (12 out of 14) and a relatively high death rate post-partum (5 out of 19 newborns died). Mice subjected to a second mating delivered newborns all negative by N. caninum-PCR, indicating that diaplacental tachyzoite passage does not occur in a later, repeated pregnancy. Overall, our experiments showed that toltrazuril-treatment of an acute N. caninum-infection – induced during pregnancy – results in a clear reduction of fetal losses and a marked reduction of diaplacental passage of the parasite to the fetal brain, whereas enrofloxacin, as an out-group control substance, failed to show the same effect.