Leishmania (Viannia) braziliensis: epidemiology of canine cutaneous leishmaniasis in the State of Paraná (Brazil)

The present study examines the role that dogs play in the maintenance of the Leishmania cycle in the State of Paraná, Southern Brazil. Dogs were examined in three regions where cutaneous leishmaniasis is endemic or epidemic (R1-Vale da Ribeira; R2-Central region of Paraná State and R3-Northern region). To determine serum prevalence rates ELISA was used. In regions endemic for Trypanosoma cruzi (R1 and R3), serum from dogs seroreactive towards Leishmania antigen was subjected to T. cruzi adsorption in order to eliminate cross-reaction with common antigen epitopes. Concomitantly, dogs with cutaneous lesions were biopsied to isolate and identify parasites using RAPD. Leishmania were classified by the phenetic method using the Jaccard coefficient of similarity, and grouped by Unweighted Pair-Group Method using an Arithmetic Average (UPGMA). A total of 410 dogs were studied. In R1 (Vale da Ribeira) 159 dogs were evaluated of which 10 had anti-Leishmania antibody. In R2 (Central Paraná), 39 animals were examined of which 8 were seropositive. In R3 (the North) 212 dogs were evaluated of which 39 animals were seropositive. Thirteen dogs had cutaneous lesions and the parasites were isolated from a dog with mucocutaneous lesion in R1, two animals with simple skin lesions in R2 and 10 dogs with multiple lesions in R3. The identification of the parasite by molecular methods showed it to be L. (Viannia) braziliensis. Based on this information, the role of domestic dogs in Leishmania infection of cutaneous leishmaniasis in Paraná is discussed.     

Over-expression of Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase activity

During the infectious cycle, protozoan parasites undergo various developmental transitions and switch virulence factors in response to extracellular signals in insect vectors and human hosts. Despite the importance of environmental sensing in parasite pathogenicity, little is known about the pathways that transduce extracellular signals into stage-specific gene expression. Here, we used a transgenic approach to gain insight into localisation and activity of three green fluorescence protein (GFP)-tagged Leishmania major mitogen-activated protein kinases, LmaMPK4, 7 and 10. The GFP-LmaMPKs in both L. major and Leishmania donovani transgenic lines showed predominant cytoplasmic localisation and the over-expression had no effect on promastigote morphology, growth and the ability to differentiate into stationary-phase metacyclics for L. major and axenic amastigotes for L. donovani. We isolated the GFP-tagged MPKs from parasite extracts and tested their phosphotransferase activity across various culture conditions. For all three GFP-LmaMPKs, kinase activity was low or absent in promastigote extracts but significantly increased in L. major promastigotes after exposure to pH 5.5 and 34 degrees C, and in axenic L. donovani amastigotes. Enhanced activity correlated with increased GFP-LmaMPK phosphorylation as judged by phospho-specific fluorescent staining of the immuno-precipitated kinases. We could extend these findings to the endogenous LmaMPK10, which accumulated in the phospho-protein fraction of axenic amastigotes but not promastigotes, and thus follows the stage-specific phosphorylation profile of episomally expressed GFP-LmaMPK10. These results provide evidence for the functional conservation of Leishmania MAP kinases in parasite environmental sensing and underscore the potential of transgenic approaches to gain insight into signaling events during the Leishmania life cycle.     

Parasites, emerging disease and wildlife conservation

In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.     

First report of genetic hybrids between two very divergent Leishmania species: Leishmania infantum and Leishmania major

The genus Leishmania includes many pathogenic species which are genetically very distant. The possibility of genetic exchange between different strains is still an important and debated question. Very few genetic hybrids (i.e., offspring of genetically dissimilar species) have been described in Leishmania. In this study, we report the first example of genetic hybrids occurring between two divergent Leishmania species, Leishmania infantum and Leishmania major. These two species have distinct geographical distributions and are transmitted by different vector species to different mammalian reservoir hosts. These hybrid strains were isolated in Portugal from immunocompromised patients and characterized by molecular and isoenzymatic techniques. These approaches showed that these chimeric strains probably contained the complete genome of both L. major and L. infantum. We believe this is the first report of genetic hybrids between such phylogenetically and epidemiologically distant species of Leishmania. This raises questions about the frequency of such cross-species genetic exchange in natural conditions, modalities of hybrid transmission, their long term maintenance as well as the consequences of these transfers on phenotypes such as drug resistance or pathogenicity.     

The amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection and persist

Leishmania are dimorphic protozoan parasites that live as flagellated forms in the gut of their sandfly vector and as aflagellated forms in their mammalian hosts. Although both parasite forms can infect macrophages and dendritic cells, they elicit distinct responses from mammalian cells. Amastigotes are the parasites forms that persist in the infected host; they infect cells recruited to lesions and disseminate the infection to secondary sites. In this review I discuss studies that have investigated the mechanisms that Leishmania amastigotes employ to harness the host cell's response to infection. It should be acknowledged that our understanding of the mechanisms deployed by Leishmania amastigotes to modulate the host cell's response to infection is still rudimentary. Nonetheless, the results show that amastigote interactions with mammalian cells promote the production of anti-inflammatory cytokines such as IL-10 and TGF-beta while suppressing the production of IL-12, superoxide and nitric oxide. An underlying issue that is considered is how these parasites that reside in sequestered vacuolar compartments target host cell processes in the cytosol or the nucleus; does this occur through the release of parasite molecules from parasitophorous vacuoles or by engaging and sustaining signalling pathways throughout the course of infection?     

Intradermal inoculations of low doses of Leishmania major and Leishmania amazonensis metacyclic promastigotes induce different immunoparasitic processes and status of protection in BALB/c mice

In order to simulate the natural long term parasitisms which may occur in mammals infected with Leishmania, cutaneous leishmaniases due to Leishmania major or Leishmania amazonensis were induced using a model based on the inoculation of 10-1000 metacyclic promastigotes into the ear dermis of BALB/c mice. The final outcome of these parasitisms was dependent upon the number of inoculated parasites. Only some of the mice inoculated with ten parasites displayed cutaneous lesions, whereas most mice infected with 100 metacyclics and all mice infected with 1000 metacyclics developed progressive lesions. We found, using the latter experimental conditions, that the onset of the pathology was associated with: (a) parasite multiplication in the inoculation site and the draining lymph node correlating with an increase of the lymph node cell number, especially in L. major-infected mice; and (b) the detection of lymph node cells, at least in part CD4(+) T lymphocytes, able to produce high levels of interferon-gamma, interleukin (IL)-4, IL-10 and IL-13. Thereafter, mice infected by L. major harboured few parasites in the ear and had a 100-fold reduction in lymph node parasite load between 23 and 40 weeks post-inoculation. In contrast, the parasite loads of L. amazonensis-infected mice remained stable in the ear and increased in nodes during the same period of time. Only L. major-infected mice that exhibited cutaneous lesions in the primary site were resistant to the re-inoculation of 1000 metacyclic promastigotes, whereas all L. amazonensis-primary infected mice remained susceptible to a second homologous challenge. These results are the first to document that a status of resistance to re-infection, referred to concomitant immunity, is coupled to the development of primary progressive lesions in L. major-infected BALB/c mice. Such a protective status is absent in L. amazonensis-infected BALB/c mice.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

Adhesion of MRP8/14 to amastigotes in skin lesions of Leishmania major-infected mice

In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that MRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular MRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for MRP14 are expressed commonly in both developmental stages.     

Occurrence of Cystosporogenes sp. (Protozoa, Microsporidia) in a multi-species insect production facility and its elimination from a colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae)

We have isolated a microsporidium from a laboratory colony of the eastern spruce budworm, Choristoneura fumiferana (Clem.) (Lepidoptera: Tortricidae). Light and electron microscopic investigations showed that gross pathology and ultrastructure of our isolate are similar to those described for Cystosporogenes legeri from the European grape vine moth, Lobesia botrana. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods revealed perfect homology with the C. legeri sequence. The microsporidian was infectious to other Choristoneura species, as well as Malacosoma disstria, Lymantria dispar, and Lambdina fiscellaria. Incubation of infected egg masses at 41 degrees C for 20 min followed by 30 min in 33% formaldehyde did not reduce disease incidence in larval offspring. Exposure of one or two generations to fumagillin at 6000 ppm or higher eliminated infection in adult moths, but also reduced colony fitness. A clean colony was established by conducting individual matings and selecting disease-free offspring.     

Evidence incriminating midges (Diptera: Ceratopogonidae) as potential vectors of Leishmania in Australia

The first autochthonous Leishmania infection in Australia was reported by Rose et al. (2004) and the parasite was characterised as a unique species. The host was the red kangaroo (Macropus rufus) but the transmitting vector was unknown. To incriminate the biological vector, insect trapping by a variety of methods was undertaken at two field sites of known Leishmania transmission. Collected sand flies were identified to species level and were screened for Leishmania DNA using a semi-quantitative real-time PCR. Collections revealed four species of sand fly, with a predominance of the reptile biter Sergentomyia queenslandi (Hill). However, no Leishmania-positive flies were detected. Therefore, alternative vectors were investigated for infection, giving startling results. Screening revealed that an undescribed species of day-feeding midge, subgenus Forcipomyia (Lasiohelea) Kieffer, had a prevalence of up to 15% for Leishmania DNA, with high parasitemia in some individuals. Manual gut dissections confirmed the presence of promastigotes and in some midges material similar to promastigote secretory gel, including parasites with metacyclic-like morphology. Parasites were cultured from infected midges and sequence analysis of the Leishmania RNA polymerase subunit II gene confirmed infections were identical to the original isolated Leishmania sp. Phylogenetic analysis revealed the closest known species to be Leishmania enriettii, with this and the Australian species confirmed as members of Leishmania sensu stricto. Collectively the results strongly suggest that the day-feeding midge (F. (Lasiohelea) sp. 1) is a potential biological vector of Leishmania in northern Australia, which is to our knowledge the first evidence of a vector other than a phlebotomine sand fly anywhere in the world. These findings have considerable implications in the understanding of the Leishmania life cycle worldwide.     

The sterol-binding antibiotic nystatin inhibits entry of non-opsonized Leishmania donovani into macrophages

Leishmania donovani is an obligate intracellular parasite that infects macrophages of the vertebrate host resulting in visceral leishmaniasis in humans, a major public health problem worldwide. The molecular mechanisms involved in internalization of Leishmania are still poorly characterized. We report here that cholesterol sequestration by the sterol-binding antifungal polyene antibiotic nystatin markedly inhibits binding and entry of non-opsonized L. donovani promastigotes into macrophages. Interestingly, these effects are not observed when serum-opsonized L. donovani are used for infectivity studies thus pointing the essential role of cholesterol in mediating entry of the parasite via the non-opsonic pathway. Based on our earlier results where leishmanial infectivity was shown to be sensitive to physical depletion of cholesterol from macrophages, these results indicate that the mere sequestration of cholesterol in the host plasma membrane is sufficient to inhibit the binding and entry of non-opsonized L. donovani. These results represent the first report on the effect of a cholesterol-sequestering agent on the entry of Leishmania parasites to host macrophages. More importantly, these findings offer the possibility of reevaluating the mechanism behind the effectiveness of current therapeutic strategies to treat leishmaniasis.     

Pathogenic effects of bacteria isolated from larvae of Hylesia metabus Crammer (Lepidoptera: Saturniidae)

Hylesia metabus larvae are susceptible to several pathogens indigenous to the area in which they are found. Some larvae show symptoms characteristic of bacterial infection; they become flaccid and lethargic, and show a marked loss of appetite. We isolated and identified 29 bacterial strains from live, dead and experimentally infected H. metabus larvae, and evaluated their pathogenic activity. The bacteria which caused mortality in the larvae were: Pseudomonas aeruginosa (60-93.3%), Proteus vulgaris (20%), Alcaligenes faecalis, Planococcus sp. and Bacillus megaterium (10%), at doses of 3-4 x 10(7). Although P. aeruginosa is a well-known insect pathogen, this is the first report of its pathogenic activity on H. metabus. The potential risk to humans and low virulence make it unlikely that P. aeruginosa could be used in an augmentative biological control programme. However its natural incidence may be enhanced using parasites and predators of H. metabus as carriers.     

Patterns of intermediate host use and levels of association between two conflicting manipulative parasites.

For many parasites with complex life cycles, manipulation of intermediate host phenotypes is often regarded as an adaptation to increase the probability of successful transmission. This phenomenon creates opportunities for either synergistic or conflicting interests between different parasite species sharing the same intermediate host. When more than one manipulative parasite infect the same intermediate host, but differ in their definitive host, selection should favour the establishment of a negative association between these manipulators. Both Polymorphus minutus and Pomphorhynchus laevis exploit the amphipod Gammarus pulex as intermediate host but differ markedly in their final host, a fish for P. laevis and a bird for P. minutus. The pattern of host use by these two conflicting manipulative parasites was studied. Their incidence and intensity of infection and their distribution among G. pulex were first examined by analysing three large samples of gammarids collected from the river Tille, Eastern France. Both parasites had low prevalence in the host population. However, temporal fluctuation in the level of parasitic infection was observed. Overall, prevalence of both parasite species was higher in male than in female G. pulex. We then assessed the degree of association between the two parasites among their intermediate hosts, using two different methods: a host-centred measure and a parasite-centred measure. Both measures gave similar results; showing random association between the two acanthocephalan species in their intermediate hosts. We discuss our results in relation to the selective forces and ecological constraints that may determine the pattern of association between conflicting manipulative parasites.     

Effects of temperature on fecundity in vitro, egg hatching and reproductive development of Benedenia seriolae and Zeuxapta seriolae (Monogenea) parasitic on yellowtail kingfish Seriola lalandi

Globally, aquaculture industries involved with commercial culture of kingfish (Seriola spp.) experience outbreaks of monogenean parasites, which can cause heavy stock losses. In Australia and New Zealand, aquaculturists of kingfish Seriola lalandi incur financial losses caused by two monogenean species: Benedenia seriolae and Zeuxapta seriolae which parasitise the skin and gills, respectively. This study provides information on some basic temperature-dependent life-cycle parameters of these problematic monogeneans on S. lalandi. Hatching times and age at maturity were inversely related to water temperature within the range experienced by wild kingfish in New Zealand (13-21 degrees C). Mature B. seriolae in vitro laid on average 37 eggs/day that hatched over approximately 4 days; peak hatching occurred 9, 11 and 22 days post-deposition at temperatures of 21, 17.5 and 13+/-1.0 degrees C, respectively. Z. seriolae in vitro laid on average 246 eggs/day that hatched over 2 days; peak hatching occurred 7, 9 and 15 days post-deposition at these respective temperatures. B. seriolae matured within 20, 25 and 48 days p.i. at 21, 18 and 13 degrees C. Z. seriolae matured within 25, 37 and >52 days p.i. at the same temperatures. This research describes stages in the reproductive development of B. seriolae and Z. seriolae and discusses the inclusion of basic parasitic life-cycle parameters into management strategies designed to maximise treatment efficacy and limit monogenean epizootics in sea-cage kingfish culture.     

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell and murine macrophage susceptibility to axenic amastigotes for the screening of leishmanicidal compounds

This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 parasites/murine macrophage. The parasite burden was maximal at 72 h post-infection (h.p.i.) for THP-1 cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i. Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 μM in THP-1 and murine macrophages at 72 h.p.i.Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macrophages and indicate the suitability of this model to screen compounds for leishmanicidal activity.     

Temporal association of entomopathogenic nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) and bacteria

Galleria mellonella L. larvae were infected with three species (seven strains) of Steinernema spp. or three species (three strains) of Heterorhabditis spp. Infected larvae were incubated at 22, 27, and 32 degrees C. Larvae were dorsally dissected every 6h over a 48-h period. Hemolymph was collected and streaked on tryptic soy agar plates. Several non-symbiotic bacterial species were identified from infected insect cadavers: Enterobacter gergoviae, Vibrio spp., Pseudomonas fluorescens type C, Serratia marcescens, Citrobacter freundii, and Serratia proteomaculans. At 18-24 h incubation, the nematode-associated symbiont occurred almost exclusively. Bacterial associates generally appeared outside the 18-24 h window. Infective juveniles of Steinernema feltiae (Filipjev) (27), Steinernema riobrave Cabanillas, Poinar, and Raulston (Oscar), or Steinernema carpocapsae (Weiser) (Kapow) were left untreated, or surface sterilized using thimerosal, then pipetted under sterile conditions onto tryptic soy agar plates. Several additional species of associated bacteria were identified using this method compared with the less extensive range of species isolated from infected G. mellonella. There was no difference in bacterial species identified from non-sterile or surface sterilized nematodes, suggesting that the bacteria identified originated from either inside the nematode or between second and third stage juvenile cuticles. Infective juveniles of S. feltiae (Cowles), S. carpocapsae (Cowles), and H. bacteriophora Poinar (Cowles) were isolated from field samples. Nematodes were surface-sterilized using sodium hypochlorite, mixed with G. mellonella hemolymph, and pipetted onto Biolog BUG (with blood) agar. Only the relevant symbionts were isolated from the limited number of samples available. The nematodes were then cultured in the laboratory for 14 months (sub-cultured in G. mellonella 7-times). Other Enterobacteriaceae could then be isolated from the steinernematid nematodes including S. marcescens, Salmonella sp., and E. gergoviae, indicating the ability of the nematodes to associate with other bacteria in laboratory culture.     

Potentially human pathogenic Acanthamoeba isolated from a heated indoor swimming pool in Switzerland

Some free-living amoebae, including some species of the genus Acanthamoeba, can cause infections in humans and animals. These organisms are known to cause granulomatous amebic encephalitis (GAE) in predominantly immune-deficient persons. In the present study, we isolated a potentially human pathogenic Acanthamoeba isolate originating from a public heated indoor swimming pool in Switzerland. The amoebae, thermophilically preselected by culture at 37 °C, subsequently displayed a high thermotolerance, being able to grow at 42 °C, and a marked cytotoxicity, based on a co-culture system using the murine cell line L929. Intranasal infection of Rag2-immunodeficient mice resulted in the death of all animals within 24 days. Histopathology of brains and lungs revealed marked tissue necrosis and hemorrhagic lesions going along with massive proliferation of amoebae. PCR and sequence analysis, based on 18S rDNA, identified the agent as Acanthamoeba lenticulata. In summary, the present study reports on an Acanthamoeba isolate from a heated swimming pool suggestive of being potentially pathogenic to immunocompromised persons.     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.