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.

The aim of this study was to evaluate cytokine expression in 22 Leishmania infantum naturally infected dogs, in order to correlate this parameter with the clinical status of infected animals. After 4 and 8 months from the first diagnosis of Leishmania infection, clinical and laboratory examination of dogs was performed and peripheral blood mononuclear cells (PBMC) were isolated. The cytokine profile was analysed in terms of IFN-gamma, IL-4, IL-10 and TNF-alpha mRNA expression in cultured PBMC by a semi-quantitative reverse transcriptase-PCR. Thirteen out of 22 Leishmania-infected dogs remained asymptomatic in the follow-up, while 9 showed clinical signs of leishmaniasis. IL-4, IL-10, TNF-alpha and IFN-gamma mRNA levels were not significantly different in asymptomatic compared to symptomatic animals 4 months from the diagnosis of Leishmania infection, but were significantly higher in symptomatic versus asymptomatic dogs after 8 months from diagnosis. In addition, IL-4, IL-10 and TNF-alpha mRNA levels significantly increased only in symptomatic dogs at 8 months, in comparison to the levels found at 4 months. These results show a mixed Th1 and Th2 cytokine response in Leishmania-infected dogs, with higher cytokine expression in dogs with manifest clinical disease, during the second follow-up after 8 months from the first diagnosis of infection.

Vaccination remains the best hope for control of all forms of leishmaniasis, and the development of a safe and effective vaccine is a critical global public-health priority. Our previous work showed that immunization with non-persistent phosphomannomutase-deficient (ΔPMM) Leishmania major parasites confers significant protection in susceptible BALB/c mice due to increased T-cell numbers and suppression of IL-10 and IL-13 early during infection. Here, we complemented the ΔPMM L. major parasites with human PMM2 to determine whether we could further improve the protection. Complemented ΔPMM parasites have restored glycoconjugate biosynthesis, while retaining avirulence of the parental knockout strain. Immunization with hPMM2 add-back parasites showed similar Th1/Th2 cytokine profiles to that observed in ΔPMM-vaccinated mice. However, the numbers of the activated CD4+CD44hi and CD8+CD44hi T cells recruited to the draining lymph nodes early after Leishmania infection were reduced, leading to decreased protection following hPMM2-immunization. Thus, the magnitude of T-cell responses early in the infection and the absence of mannose-rich glycoconjugates determine the protective outcome of anti-leishmanial immunity.

A new Coccidia species is reported from the natural population of Pied Flycatcher (Ficedula hypoleuca) in northern Germany. Sporulated oocysts were found in faeces from 6 of 8 sampled adults. The spherical oocysts of the new Isospora species have a brownish, smooth, bi-layered wall. Average size of sporulated oocysts was 19·4×19·3 μm (17·5–22·8 μm×17·5–22·8 μm) with a shape index (length/width) of 1·0. The sporulated oocysts have no micropyle or residuum, but enclose several small polar granules that often cluster into 2–3 dumbbell-shaped formations. Sporocysts are slightly elongated, rounded at the end opposite the Stieda body, 15·3 μm×9·2 μm in size (13·8–16·1 μm× 8·5–10·3 μm), and have a shape index of 1·7 (1·6–1·8). The Stieda body has a prominent knob-like cap, whereas the substieda body is absent. Sporocysts contain a small compact sporocyst residuum and 4 sporozoites. COI haplotypes identical to those isolated from faecal oocysts were PCR amplified from the blood of 13-day-old nestlings, suggesting that the newly described species has extra-intestinal stages in blood. This represents the first description of a new avian Isospora species supported by molecular sequence data from the same oocysts that are described morphologically.

For trophically transmitted parasites, transitional larval size is often related to fitness. Larger parasites may have higher establishment success and/or adult fecundity, but prolonged growth in the intermediate host increases the risk of failed transmission via natural host mortality. We investigated the relationship between the larval size of an acanthocephalan (Acanthocephalus lucii) and a trait presumably related to transmission, i.e. altered colouration in the isopod intermediate host. In natural collections, big isopods harboured larger worms and had more modified (darker) abdominal colouration than small hosts. Small isopods infected with a male parasite tended to have darker abdominal pigmentation than those infected with a female, but this difference was absent in larger hosts. Female size increases rapidly with host size, so females may have more to gain than males by remaining in and growing mutually with small hosts. In experimental infections, a large total parasite volume was associated with darker host respiratory operculae, especially when it was distributed among fewer worms. Our results suggest that host pigment alteration increases with parasite size, albeit differently for male and female worms. This may be an adaptive strategy if, as parasites grow, the potential for additional growth decreases and the likelihood of host mortality increases.

Seasonal occurrence of metazoan ectoparasites is described for the first time in marine European flounder, Platichthys flesus (L.). The parasitofauna, in this study monitored during 1 year, was found to be similar to that previously recorded for flounder. Moreover, specimens of Caligus sp. Müller, 1785 and Lepeophtheirus pectoralis (Copepoda: Caligidae), Acanthochondria cornuta (Copepoda: Chondracanthidae), Holobomolochus confusus (Copepoda: Bomolochidae) and Nerocila orbignyi (Isopoda: Cymothoidae), and also, a praniza larva (Isopoda: Gnathiidae), were isolated. From these, L. pectoralis and A. cornuta were the dominant parasites in all samples of flounder, while Caligus sp., H. confusus, N. orbignyi and the gnathiid praniza seemed to infect the flounder only occasionally. As far as the seasonality of infections is concerned, it differed considerably from that described for estuarine environments. Indeed, both prevalence and abundance of L. pectoralis and A. cornuta reached significant peaks in the summer, whereas the literature identifies the autumn as the season of maximum infection on estuarine flounder. Thus, the former period seems more favourable for the occurrence of epizooties of L. pectoralis and A. cornuta in flounder culturing systems running on seawater and operated in the studied or similar environments.

Harnessing the full utility of extensive gene sequences recently available for the economically important sea louse, Lepeophtheirus salmonis, requires the adaptation of modern molecular biology approaches to this non-model organism. Using a putative microsomal prostaglandin E synthase type-2 (PGES2) as a candidate gene, we investigated gene-knockdown by double-stranded RNA interference (dsRNAi) in the small free-living and the larger parasitic stages of L. salmonis. dsRNA was administered to nauplius and copepodid stages by immersion for 7 h. Pre-adult and adults received dsRNA by intra-haemocoelic injection. The extent, speed and persistence of the knockdown effects were determined by RT-PCR. LsPGES2 was abundantly expressed in all life stages, including the non-parasitic stages. Administration of dsRNA to nauplius and copepodids by immersion had no effect on mortality rates and moulting through to copepodids was observed. Dramatic knockdown of LsPGES2 was observed within 7 h and persisted for at least 48 h. Injection of dsRNA had no effect on mortality in pre-adults and adults, but knockdown of LsPGES2 was apparent within 24 h, reaching 95% over the 72 h and was persistent for at least 120 h. The methods developed resulted in rapid and persistent knockdown in L. salmonis suitable for studies in the different stadia.

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0·9% at the 18S rDNA and 10·7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38·2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

An attempt was made to evaluate the immunoprophylactic efficacy of Brugia malayi transglutaminase (BmTGA) as a DNA vaccine, for human lymphatic filariasis. BmTGA was cloned and characterized in the DNA vaccine vector pVAX1. Further, the tissue distribution study of the DNA construct, pVAX-TGA was carried out in mice and the DNA vaccine was shown to be efficiently distributed to all the organs, was accessible to the immune system, and at the same time was metabolized quickly and did not pose problems of toxicity. Intramuscular immunization in mice showed significant antibody production and splenocyte proliferation upon antigenic stimulation. The immune responses were biased towards the Th1 arm, as evaluated in terms of isotype antibody distribution and cytokine profile. Thus, analysis of the humoral and cellular immune responses indicated that BmTGA is a potent immunogen. However, protection studies as determined by the micropore chamber method using live microfilarial larvae, showed that the DNA vaccine could confer only partial protection in the mouse model. We conclude that despite the induction of sufficient humoral and cellular immune responses, BmTGA as a DNA vaccine could not confer much protection against subsequent challenge and other aspects of the immune responses need to be further investigated.

Setaria cervi, a bovine filarial parasite contains significant acid phosphatase (AcP) activity in its various life stages. Two forms of AcP were separated from somatic extract of adult female parasite using cation exchange, gel filtration and concavalin affinity chromatography. One form having a molecular mass of 79 kDa was characterized as dual specific protein tyrosine phosphatase (ScDSP) based on substrate specificity and inhibition studies. With various substrates tested, it showed significant activity in the order of phospho-L-tyrosine>pNPP>ADP>phospho-L-serine. Inhibition by orthovanadate, fluoride, molybdate, and zinc ions further confirms protein tyrosine phosphatase nature of the enzyme. Km and Vmax determined with various substrates were found to be 16·66 mM, 25·0 μM/ml/min with pNPP; 20·0 mM, 40·0 μM/ml/min with phospho-L-tyrosine and 27·0 mM, 25·0 μM/ml/min with phospho-L-serine. KI with pNPP and sodium orthovanadate (IC50 33·0 μM) was calculated to be 50·0 mM. Inhibition with pHMB, silver nitrate, DEPC and EDAC suggested the presence of cysteine, histidine and carboxylate residues at its active site. Cross-reactivity with W. bancrofti-infected sera was demonstrated by Western blotting. ScDSP showed elevated levels of IgE in chronic filarial sera using ELISA. Under in vitro conditions, ScDSP resulted in increased effector function of human eosinophils when stimulated by IgG, which showed a further decrease with increasing enzyme concentration. Results presented here suggest that S. cervi DSP should be further studied to determine its role in pathogenesis and the persistence of filarial parasite.

Changes in the cardiac β-adrenergic system in early stages of Trypanosoma cruzi infection have been described. Here, we studied an early (135 days post-infection–p.i.) and a late stage (365 days p.i.) of the cardiac chronic form of the experimental infection (Tulahuen or SGO-Z12 strains), determining plasma epinephrine and norepinephrine levels, β-receptor density, affinity and function, cardiac cAMP concentration and phosphodiesterase activity, cardiac contractility, and the presence of β-receptor autoantibodies. Tulahuen-infected mice presented lower epinephrine and norepinephrine levels; lower β-receptor affinity and density; a diminished norepinephrine response and higher cAMP levels in the early stage, and a basal contractility similar to non-infected controls in the early and augmented in the late stage. The Tulahuen strain induced autoantibodies with weak β-receptor interaction. SGO-Z12-infected mice presented lower norepinephrine levels and epinephrine levels that diminished with the evolution of the infection; lower β-receptor affinity and an increased density; unchanged epinephrine and norepinephrine response in the early and a diminished response in the late stage; higher cAMP levels and unchanged basal contractility. The SGO-Z12 isolate induced β-receptor autoantibodies with strong interaction with the β-receptors. None of the antibodies, however, acted a as β-receptor agonist. The present results demonstrate that this system is seriously compromised in the cardiac chronic stage of T. cruzi infection.

Cane toads (Bufo marinus) were introduced to Australia in 1935 and have since spread widely over the continent, generating concern regarding ecological impacts on native predators. Most Australian cane toad populations are infected with lung nematodes Rhabdias pseudosphaerocephala, a parasite endemic to New World (native-range) cane toad populations; presumably introduced to Australia with its toad host. Considering the high intensities and prevalence reached by this parasite in Australian toad populations, and public ardour for developing a control plan for the invasive host species, the lack of experimental studies on this host-parasite system is surprising. To investigate the extent to which this lungworm influences cane toad viability, we experimentally infected metamorph toads (the smallest and presumably most vulnerable terrestrial phase of the anuran life cycle) with the helminth. Infected toads exhibited reduced survival and growth rates, impaired locomotor performance (both speed and endurance), and reduced prey intake. In summary, R. pseudosphaerocephala can substantially reduce the viability of metamorph cane toads.

Immunopathological and ultrastructural studies were carried out on the gut of 30 specimens of powan Coregonus lavaretus (L.) from Lake Piediluco, Italy. The digestive tracts of 10 (33·3%) of the powan were found to harbour an acanthocephalan Dentitruncus truttae (Sinzar 1955). The numerous trunk spines of D. truttae reduced the number of mucosal folds near the parasite site of infection. The acanthocephalan induced hyperplasia and hypertrophy of the intestinal mucous cells and many worms were surrounded with an adherent mucous gel. Near the site of acanthocephalan attachment, the number of mucous cells was significantly higher (P<0·01) in comparison to those found in uninfected intestines. Rodlet cells (RCs) were present in the epithelial layer in both infected and uninfected fish, with no significant difference in the numbers observed (P>0·05). In infected intestine, mast cells were more abundant than in uninfected gut (P<0·01). Migration of the mast cells and their intense degranulation at the site of infection were suggested. Immunohistochemical tests applied to sections of intestinal tissue of both infected and uninfected powan revealed that the parasitized C. lavaretus had a larger number of mast cells positive for met-enkephalin and serotonin antisera.