A series of 2277 Leishmania strains from Old World visceral leishmaniasis foci, isolated between 1973 and 2008, were studied by isoenzyme analysis. The strains were obtained from humans, domestic and wild carnivores, rodents and phlebotomine sandflies, and came from 36 countries. In all, 60 different zymodemes were identified and clustered by a phenetic analysis into 3 different groups corresponding to the typically visceralizing species L. donovani (20 zymodemes, 169 strains), L. archibaldi (3 zymodemes, 46 strains) and L. infantum (37 zymodemes, 2,062 strains). The taxonomic position of these isoenzymatic groups is discussed in view of contradictory results obtained from recent molecular studies.

The role of Mycobacterium w (Mw) vaccine as an immunomodulator and immunoprophylactant in the treatment of mycobacterial diseases (leprosy and pulmonary tuberculosis) is well established. The fact that it shares common antigens with leishmanial parasites prompted its assessment as an immunostimulant and as an adjunct to known anti-leishmanials that may help in stimulating the suppressed immune status of Leishmania donovani-infected individuals. The efficacy of Mw vaccine was assessed as an immunomodulator, prophylactically either alone or in combination with anti-leishmanial vaccine, as well as therapeutically as an adjunct to anti-leishmanial treatment in L. donovani-infected hamsters, representing a chronic human Visceral Leishmaniasis (VL) model. Similarly, its efficacy was also evaluated in L. donovani-infected BALB/c mice, representing an acute VL model. The preliminary studies revealed that Mw was ineffective as an immunostimulant and/or immunoprophylactant in hamsters infected with L. donovani, as estimated by T-cell immunological responses. However, in the BALB/c mice-VL model it appeared as an effective immunostimulant but a futile prophylactic agent. It is therefore inferred that, contrary to its role in managing tuberculosis and leprosy infections, Mw vaccine has not been successful in controlling VL infection, emphasizing the need to find detailed explanations for the failure of this vaccine against the disease.

The potato cyst nematodes (PCN) Globodera pallida and G. rostochiensis are major pests of potatoes. The G. pallida (and G. rostochiensis) life cycle includes both diapause and quiescent stages. Nematodes in dormancy (diapause or quiescent) are adapted for long-term survival and are more resistant to nematicides. This study analysed the mechanisms underlying diapause and quiescence. The effects of several compounds (8Br-cGMP, oxotremorine and atropine) on the activation of hatching were studied. The measurements of some morphometric parameters in diapaused and quiescent eggs after exposure to PRD revealed differences in dorsal gland length, subventral gland length and dorsal gland nucleolus. In addition, the expression of 2 effectors (IVg9 and cellulase) was not induced in diapaused eggs in water or PRD, while expression was slightly induced in quiescent eggs. Finally, we performed a comparative study to identify orthologues of C. elegans diapause related genes in plant-parasitic nematodes (G. pallida, Meloidogyne incognita, M. hapla and Bursaphelenchus xylophilus). This analysis suggested that it was not possible to identify G. pallida orthologues of the majority of C. elegans genes involved in the control of dauer formation. All these data suggest that G. pallida may use different mechanisms to C. elegans in regulating the survival stage.

Some Trypanosoma and Leishmania species are multi-host parasites whose distribution overlaps in several parts of the Brazilian Amazon basin. Despite being a common trait among wild mammals, mixed infections and their consequences for the host's health and parasite transmission are still a poorly known phenomenon. Here we describe a triple mixed infection – Trypanosoma cruzi, T. rangeli and Leishmania infantum – in a bone marrow sample from an anteater Tamandua tetradactyla captured in a house backyard from the endemic Abaetetuba municipality in the Amazon basin. T. cruzi was also isolated from blood samples. The mini-exon multiplex PCR characterization detected the infection by T. rangeli and T. cruzi (TcI genotype), while L. infantum infection was confirmed by an ITS-PCR followed by amplicon sequencing. This is the first description of T. rangeli isolation from bone marrow and the first report of L. infantum infection in xenarthrans. The implications of this finding are discussed considering the influence of mixed infections in the role of this mammal species as a putative reservoir host of these 3 trypanosomatid species.

We tested for the effect of age of a rodent host (Meriones crassus) on reproductive performance of fleas in terms of number and quality of offspring and predicted that fleas would perform better on juvenile and old than on subadult and adult hosts. The number of flea offspring was evaluated via egg and new imago production, while their quality was estimated via duration of development, resistance to starvation and body size. Although fleas produced more eggs when they exploited adults than when they exploited juvenile, subadult and old hosts, significantly more new imago emerged from fleas fed on juvenile and old hosts than on subadult and adult hosts. Fleas performed better when they fed on juvenile and/or old hosts than on subadult and adult hosts in 2 of 3 measures of offspring quality (duration of development and body size). Nevertheless, when offspring quality was estimated via resistance to starvation of a new imago, fleas demonstrated good performance in young (juvenile and subadult) hosts, while they performed poorly in old hosts. Thus, general reproductive performance of fleas was better when they exploited young and old hosts than when they exploited median age cohorts. However, the effect of host age on flea reproductive performance was manifested somewhat differently between (a) male and female hosts and (b) male and female flea offspring.

Leishmania parasites can escape the immune response by invading cell types lacking leishmanicidal mechanisms. Silent persistence of Leishmania parasites in the host organism is responsible for asymptomatic carriage and relapses after cured leishmaniasis. Here, we studied the interaction between Hepatic Stellate Cells (HSC) and Leishmania. An original model of human HSC in primary culture infected with L. donovani was developed. The presence of intracellular parasites was studied and quantified using optical and confocal microscopy. HSC characteristics were studied using microscopy, methylene blue assay, long-term cultures and qPCR. We showed for the first time that human HSC are permissive to L. donovani infection, with no modification of HSC survival, growth rate and proinflammatory and fibrogenic characteristics. Intracellular parasites did not replicate but HSC had no effect on their survival. Indeed, after a 40-day culture, infected HSC cultures transferred on NNN medium yielded new promastigotes that were able to proliferate and efficiently infect new cells. HSC are permissive to L. donovani, with neither parasite killing nor apparent cell damage. Thus, HSC could act as potent sanctuary cells for Leishmania in the liver, which could partially explain parasite reactivation after an asymptomatic carriage or a cured visceral leishmaniasis.

Heliciculture is an excellent alternative to obtain edible snails but its viability is seriously threatened by pathogens. A parasitological survey was conducted in 3 mixed system-based heliciculture farms in Galicia (NW Spain), with the species Tetrahymena rostrata, Tetrahymena limacis, Tetratrichomonas limacis, Cryptobia helicogenae, Brachylaima aspersae (metacercariae and sporocysts), Alloionema appendiculatum, Nemhelix bakeri, and Riccardoella limacum being commonly found infecting Helix aspersa aspersa (petit-gris) snails. With the exception of C. helicogenae, N. bakeri, and B. aspersae sporocysts, all species were also detected in Helix aspersa maxima (gros-gris) snails, although generally with lower parameters. Most monoxenous infections, and consequently multiple parasitism, exhibited a rising trend during the first 2 months of intensive mating, with tendencies being slowed down or even reversed during the third month as a result of accumulated mortality and a sampling-derived reduction in host density. No parasites were vertically transmitted and infections were initially acquired from invading gastropod and micromammal reservoirs during fattening. Finally, artificial hibernation reduced significantly the prevalence of most species. These results confirm the importance of parasites in heliciculture and emphasize the need to prevent the entry of wild reservoirs into the farms and to rapidly remove the carcasses of dead snails from the reproduction units and fattening pens.

Members of the ATP-binding cassette (ABC) transporter family (P-glycoproteins, Half-transporters and Multidrug Resistant Proteins) potentially play a role in the development of anthelmintic resistance. The aim of this study was to investigate the possible involvement of ABC transporters in anthelmintic resistance in the bovine parasite, Cooperia oncophora. Partial sequences of 15 members of the ABC transporter protein family were identified, by mining a transcriptome dataset combined with a degenerate PCR approach. Reverse transcriptase PCR showed that most of the ABC transporters identified were constitutively transcribed throughout the life cycle of C. oncophora. Constitutive differences in gene transcript levels between a susceptible and resistant isolate were only observed for Con-haf-9 and Con-mrp-1 in eggs of the resistant isolate, while no differences were observed in L3 or the adult life stage. Analysis of resistant adult worms, collected from calves 14 days after treatment with either ivermectin or moxidectin, showed a significant 3- to 5-fold increase in the transcript levels of Con-pgp-11 compared to non-exposed worms. Interestingly, a 4-fold transcriptional up-regulation of Con-pgp-11 was also observed in L3 of the resistant isolate, after in vitro exposure to different concentrations of ivermectin, whereas this effect was not observed in exposed L3 of the susceptible isolate. The results suggest that the worms of this particular resistant isolate have acquired the ability to up-regulate Con-pgp-11 upon exposure to macrocyclic lactones. Further work is needed to understand the genetic basis underpinning this process and the functional role of PGP-11.

Identification of parasite species is particularly challenging in larval and juvenile hosts, and this hampers the understanding of parasite acquisition in early life. The work described here employs a new combination of methods to identify parasite species and study parasite succession in fry of perch (Perca fluviatilis) from Lake Constance, Germany. Classical morphological diagnostics are combined with sequence comparisons between parasite life-stages collected from various hosts within the same ecosystem. In perch fry at different stages of development, 13 different parasite species were found. Incomplete morphological identifications of cestodes of the order Proteocephalidea, and trematodes of the family Diplostomatidae were complemented with sequences of mitochondrial DNA (cytochrome oxidase 1) and/or nuclear (28 s rDNA) genes. Sequences were compared to published data and used to link the parasites in perch to stages from molluscs, arthropods and more easily identifiable developmental stages from other fishes collected in Lake Constance, which both aided parasite identification and clarified transmission pathways. There were distinct changes in parasite community composition and abundance associated with perch fry age and habitat shifts. Some parasites became more abundant in older fish, whereas the composition of parasite communities was more strongly affected by the ontogenetic shifts from the pelagic to the littoral zone.

Testosterone is involved in the development and expression of physiological, morphological and behavioural traits. High levels are often associated with high infection risk and/or intensity, suggesting a trade-off between sexual traits and immunity. Classically invoked mechanisms are immunological or behavioural, i.e., testosterone increases susceptibility or resistance to parasites via an impact on immunity or modulates behaviours involved in parasite transmission. However, studies report contrasted patterns. Given its modes of action and the diversity of host-parasite interactions, testosterone should not act similarly on all interactions. To reduce host and context diversity, we studied 3 viruses in the same cat population: the aggressively transmitted Feline Immunodeficiency virus (FIV), and the Feline Calicivirus (FCV) and Herpesvirus (FHV) both transmitted during friendly contacts. Testosterone had a strong effect on the probability of being positive to FIV whereas its effect was significantly weaker on FCV and FHV. These findings demonstrate that testosterone can be differentially associated with parasites of the same type (viruses). The difference we observed was consistent with a behavioural-mediated effect (increased aggressiveness), supporting the idea that the testosterone effect on infection risk is at least partially driven by behavioural mechanisms in our system. Further investigations (e.g., individual immunity measures) are required to confirm this hypothesis.

The intra-erythrocytic protozoan Babesia bovis is an economically important pathogen that causes an acute and often fatal infection in adult cattle. Babesiosis limitation depends on the early activation of macrophages, essential cells of the host innate immunity, which can generate an inflammatory response mediated by cytokines and nitric oxide (NO). Herein, we demonstrate in bovine macrophages that lipids from B. bovis attenuated R1A strain (LA) produced a stronger NO release, an early TNFα mRNA induction and 2-fold higher IL-12p35 mRNA levels compared to the lipids of virulent S2P strain (LV). Neither LA nor LV induced anti-inflammatory IL-10. Regarding signalling pathways, we here report that LA induced a significant phosphorylation of p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) whereas LV only induced a reduced activation of ERK1/2. Besides, NF-κB was activated by LA and LV, but LA produced an early degradation of the inhibitor IκB. Interestingly, LV and the majority of its lipid fractions, exerted a significant inhibition of concanavalin A-induced peripheral blood mononuclear cell proliferation with respect to LA and its corresponding lipid fractions. In addition, we determined that animals infected with R1A developed a higher increase in IgM anti-phosphatidylcholine than those inoculated with S2P. Collectively, S2P lipids generated a decreased inflammatory response contributing to the evasion of innate immunity. Moreover, since R1A lipids induced a pro-inflammatory profile, we propose these molecules as good candidates for immunoprophylactic strategies against babesiosis.

Aggregated distributions among individual hosts are a defining feature of metazoan parasite populations. Heterogeneity among host individuals in exposure to parasites or in susceptibility to infection is thought to be the main factor generating aggregation, with properties of parasites themselves explaining some of the variability in aggregation levels observed among species. Here, using data from 410 samples of helminth parasites on fish hosts, I tested the contribution of (i) within-sample variation in host body size, taken as a proxy for variability in host susceptibility, and (ii) parasite taxon and developmental stage, to the aggregated distribution of parasites. Log-transformed variance in numbers of parasites per host was regressed against log mean number across all samples; the strong relationship (r2 = 0·88) indicated that aggregation levels are tightly constrained by mean infection levels, and that only a small proportion of the observed variability in parasite aggregation levels remains to be accounted for by other factors. Using the residuals of this regression as measures of ‘unexplained’ aggregation, a mixed effects model revealed no significant effect of within-sample variation in host body size or of parasite taxon or stage (i.e. juvenile versus adult) on parasite aggregation level within a sample. However, much of the remaining variability in parasite aggregation levels among samples was accounted for by the number of individual hosts examined per sample, and species-specific and study-specific effects reflecting idiosyncrasies of particular systems. This suggests that with most differences in aggregation among samples already explained, there may be little point in seeking universal causes for the remaining variation.

We have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.