The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96·8%), whereas 85 sera were positive with DAT (sensitivity=91·4%) and 89 sera were positive with whole cell ELISA (sensitivity=95·7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89·2%) and whole cell ELISA was negative in 114 cases (specificity=87·7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.

A surface agglutination/immobilization antigen was purified from the novel agglutination/immobilization serotype (serotype G37) of the ciliated protozoan Cryptocaryon irritans, a parasite of seawater fishes. Serum from fish immunized with C. irritans theronts had agglutination/immobilization activity against theronts in vitro. However, fish and rabbit antisera raised against serotype G32 (reported previously) caused little agglutination/immobilization of serotype G37 theronts. Immunological analysis indicated that the 37 kDa theront surface membrane protein may be the agglutination/immobilization antigen of this serotype. The full-length 37 kDa antigen cDNA contained 1171 base pairs, encoding a 331–amino acid protein with hydrophobic N- and C-termini, which are characteristically found in proteins containing a C-terminal glycosylphosphatidylinositol anchor. In addition, the genetically characterized nucleotide sequences of the first internal transcribed spacer region of ribosomal DNA of these 2 serotypes were compared. The internal transcribed spacer rDNA sequence of serotype G32 was identical to that of isolates from Pingtung, Taiwan, and from the USA. On the other hand, the sequences of serotype G37 were not identical to those of any C. irritans isolate.

A fish-infecting Microsporidia Potaspora morhaphis n. gen., n. sp. found adherent to the wall of the coelomic cavity of the freshwater fish, Potamorhaphis guianensis, from lower Amazon River is described, based on light microscope and ultrastructural characteristics. This microsporidian forms whitish xenomas distinguished by the numerous filiform and anastomosed microvilli. The xenoma was completely filled by several developmental stages. In all of these stages, the nuclei are monokaryotic and develop in direct contact with host cell cytoplasm. The merogonial plasmodium divides by binary fission and the disporoblastic pyriform spores of sporont origin measure 2·8±0·3×1·5±0·2 μm. In mature spores the polar filament was arranged into 9–10 coils in 2 layers. The polaroplast had 2 distinct regions around the manubrium and an electron-dense globule was observed. The small subunit, intergenic space and partial large subunit rRNA gene were sequenced and maximum parsimony analysis placed the microsporidian described here in the clade that includes the genera Kabatana, Microgemma, Spraguea and Tetramicra. The ultrastructural morphology of the xenoma, and the developmental stages including the spores of this microsporidian parasite, as well as the phylogenetic analysis, suggest the erection of a new genus and species.

The treatment of Neospora caninum infection in the bovine host is still at an experimental stage. In contrast to the in vivo situation, a wide range of compounds have been intensively investigated in cell-culture-based assays. Tools to demonstrate efficacy of treatment have remained conventional including morphological and cell biological criteria. In this work, we present a molecular assay that allows the distinction between live and dead parasites. Live parasites can be detected by measuring the mRNA level of specific genes, making use of the specific mRNA available in live cells. The NcGra2 gene of N. caninum, which is known to be expressed in both tachyzoites and bradyzoites, was used to establish a quantitative real-time RT-PCR, for monitoring parasite viability. Validation of the system in vitro was achieved using Neospora-infected cells that had been treated for 2–20 days with 30 μg/ml toltrazuril. NcGRA2-RT-real time PCR demonstrated that a 10-day toltrazuril-treatment exerted parasitostatic activity, as assessed by the presence of NcGRA2-transcripts, whereas after a 14-day treatment period no NcGRA2-transcripts were detected, showing that the parasites were no longer viable. Concurrently, extended culture for a period of 4 weeks in the absence of the drug following the 14-day toltrazuril treatment did not lead to further parasite proliferation, confirming the parasiticidal effect of the treatment. This assay has the potential to be widely used in the development of novel drugs against N. caninum, with a view to distinguishing between parasiticidal and parasitostatic efficacy of given compounds.

Tetracapsuloides bryosalmonae is the myxozoan that causes the commercially and ecologically important proliferative kidney disease of salmonid fish species. Immunohistochemistry and electron microscopy were used to examine the development of this parasite within the kidney of the brown trout Salmo trutta. The main replicative phase of T. bryosalmonae is a cell doublet composed of a primary cell and a single secondary cell. Engulfment of one secondary cell by another to form a secondary-tertiary doublet (S-T doublet) heralded the onset of sporogony whereupon the parasite migrated to the kidney tubule lumen. Within the tubule, the parasite transformed into a pseudoplasmodium and anchored to the tubule epithelial cells via pseudopodial extensions. Within each pseudoplasmodium developed a single spore, composed of 4 valve cells, 2 polar capsules and 1 sporoplasm. The pseudoplasmodia formed clusters suggesting that large numbers of spores develop within the fish. This examination of T. bryosalmonae suggests that the main replicative phase of freshwater myxozoans within vertebrates is via direct replication of cell doublets rather than through the rupturing of extrasporogonic stages, while tertiary cell formation relates only to sporogony. Taken in conjunction with existing phylogenetic data, 5 distinct sporogonial sequences are identified for the Myxozoa.

Protease expression among TCI and TCII field isolates was analysed. Gelatin-containing gels revealed hydrolysis bands with molecular masses ranging from 45 to 66 kDa. The general protease expression profile showed that TCII isolates presented higher heterogeneity compared to TCI. By utilizing protease inhibitors, we showed that all active proteases at acid pH are cysteine-proteases and all proteases active at alkaline pH are metalloproteases. However, the expression of cruzipain, the T. cruzi major cysteine-protease, did not reproduce a heterogeneous TCII cysteine zymogram profile. Dendogram analyses based on presence/absence matrices of proteases and cruzipain bands showed a TCI separation from the TCII group with 50–60% similarity. We suggest that the observed cysteine protease diversification contributes to differential host infection between TCI and II genotypes.

Trypanosomatid parasites possess 2 distinct iron-containing superoxide dismutases (Fe-SODs) designated SODA and SODC, both of which are targeted to their mitochondria. In contrast to SODAs that carry typical mitochondrial transit peptides, SODCs have highly unusual mitochondrial targeting signals. Our analyses clearly show that these pre-sequences are bipartite possessing a signal peptide-like domain followed by a transit peptide-like domain. Consequently, they resemble N-terminal extensions of proteins targeted to multi-membrane plastids, suggesting that trypanosomatids once contained a eukaryotic alga-derived plastid. Further support for this hypothesis comes from striking similarities in length, hydropathy profile, and amino acid composition of SODC pre-sequences to those of Euglena and dinoflagellate plastid proteins. To account for these data, we propose that the Trypanosomatidae initially possessed a gene encoding a mitochondrial Fe-SOD with a classical mitochondrial transit peptide. Before or after plastid acquisition, a gene duplication event gave rise to SODA and SODC. In a subsequent evolutionary step a signal peptide was linked to SODC, enabling its import into the plastid. When the trypanosomatid plastid subsequently was lost, natural selection favoured adaptation of the SODC N-terminal signal as a mitochondrial transit peptide and re-targeting to the mitochondrion.

To understand possible factors controlling transmission of trematode larvae between first and second intermediate hosts we examined the impact of ambient fauna on parasite transmission in a marine intertidal parasite-host association. Cockle hosts (Cerastoderma edule) kept together with selected co-occurring macrozoobenthic species in mesocosms acquired a lower parasite load compared to cockles kept alone, when targeted by cercariae of the trematode Himasthla elongata. The reduction of parasite load in the cockles differed between the 7 macrozoobenthic species tested and was between 35 and 91%. Three different types of reduction could be distinguished: (1) predators (Carcinus maenas, Crangon crangon) actively preying upon cercariae, (2) non-host filter feeders (Crepidula fornicata, Mya arenaria, Crassostrea gigas) filtering cercariae but not becoming infected and (3) alternative hosts (Mytilus edulis, Macoma balthica) becoming infected by the cercariae and thus distracting cercariae from the target hosts. In addition, interference competition may occur in the form of disturbance of cockles by ambient organisms resulting in lower filtration rates and subsequently lower parasite loads. Our results suggest that the species composition and relative abundance of the ambient fauna of parasite-host systems play an important role in controlling trematode transmission rates in benthic marine systems.

The effect of sainfoin (Onobrychis viciifolia) hay consumption on the pathophysiology and local cellular responses of growing lambs during infection with Trichostrongylus colubriformis was investigated. Thirty-two lambs, 16 weeks of age, were allocated to 1 of 4 treatment groups (n=8) that were offered either grass (G) or sainfoin (S) hay while concurrently either infected (+), or not (−) with 12 000 L3 T. colubriformis larvae per week for 6 weeks. Liveweight gains were affected by diet (P=0·002) and reduced by infection (P<0·005). Faecal egg count was reduced in S+ compared to G+from days 35 to 42 (P=0·001); however, total egg output, worm burdens at day 42 and worm fecundity were similar between diets (P>0·05). Feeding sainfoin appeared to enhance immune cell development with tissue eosinophils, mast cells and pan T cells present in greater concentrations in S+ than in G+ animals. However, further studies are required to determine if the enhanced immune cell development is a consequence of a greater nutrient supply or a direct influence of sainfoin metabolites on local inflammatory responses to the gastrointestinal nematode T. colubriformis.

In previous studies, ultrastructural observations revealed a large number of gap junctions (GJs) in the neck and immature proglottid tissues of Taenia solium tapeworms. In these helminths, cytoplasmic glycogen sacs are connected by numerous discrete GJs to other cells throughout the maturing strobilar tissue. Discontinuous sucrose gradients were used to purify membrane fractions containing GJs, which were identified by ultrastructural analysis. A trans-membrane peptide sequence from a highly conserved innexin region was used to construct a 20-amino acid synthetic peptide and used to raise polyclonal antibodies in rabbits that recognized both a 55 and a 67 kDa protein in a Western blot of the GJ-enriched pellet. Immunohistochemistry of larval and adult worm sections incubated with antiserum to the synthetic peptide and a secondary anti-rabbit IgG bound to fluorescein, revealed strong binding to the tegumentary surface of the worm, as well as patchy fluorescent areas in the parenchyma. The results indicate that both the tegument of cysticerci and adult T. solium contain innexin-rich membranes, which may function as a tegumentary transport system for small molecules.

This study set out to determine whether the sex ratio of fleas collected from host bodies is a reliable indicator of sex ratio in the entire flea population. To answer this question, previously published data on 18 flea species was used and it was tested to see whether a correlation exists between the sex ratio of fleas collected from host bodies and the sex ratio of fleas collected from host burrows. Across species, the female:male ratio of fleas on hosts correlated strongly with the female:male ratio of fleas in their burrows, with the slope of the regression overlapping 1. Controlling for flea phylogeny by independent contrasts produced similar results. It was also ascertained whether a host individual is a proportional random sampler of male and female fleas and whether the sex ratio in flea infrapopulations depends on the size of infrapopulations and on the gender and age of a host. Using field data, the sex ratio in infrapopulations of 7 flea species parasitic on 4 rodent species was analysed. Populations of 3 species (Nosopsyllus iranus, Parapulex chephrenis and Xenopsylla conformis) were significantly female-biased, whereas male bias was found in 1 species (Synosternus cleopatrae). In general, the sex ratio of fleas collected from an individual rodent did not differ significantly from the sex ratio in the entire flea population. Neither host gender, and age nor number of fleas co-occurring on a host affected (a) the sex ratio in flea infrapopulations and (b) the probability of an infrapopulation to be either female- or male-biased.