Detection of Leishmania DNA in wild foxes and associated ticks in Patagonia, Argentina, 2000 km south of its known distribution area

Indexación: Web of Science; PubMedBackground: Zoonotic Visceral Leishmaniasis (ZVL) is a vector-borne disease affecting humans and other mammals and caused by the protozoan parasite Leishmania (Leishmania) infantum (syn. L. chagasi), belonging to the L. donovani complex. The regions in Northern Argentina (above 32 degrees S) are its southern distribution limit in South America. Results: We detected Leishmania sp. DNA (most likely belonging to the L. donovani complex) in 37.5 % of 32 grey foxes (Pseudalopex griseus) captured in Argentinean Patagonia (48 degrees S and 50 degrees S). Eleven monosexual pools of Amblyomma tigrinum ticks from eight different foxes (six grey foxes and two culpeo foxes P. culpaeus) were also positive. The southernmost known distribution limit for L. infantum, and the southernmost reported capture of a phlebotominae, had previously been 2000 and 750 km north of our study area, respectively. Conclusions: This finding is significant because it markedly extends the distribution area of leishmaniasis; supports the existence of a sylvatic cycle in the absence of dogs; and has implications in transmission, indicating that either sand fly distribution is broader than currently thought or non-sand fly Leishmania maintenance is possible. Additional molecular, parasitological, epidemiological and entomological studies are still needed.http://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-016-1515-

Development of a LAMP assay for detection of Leishmania infantum infection in dogs using conjunctival swab samples

Background: Leishmania infantum infections in dogs play a crucial role in the transmission of pathogens causing visceral leishmaniasis to humans in the Gansu province, northwest China. To be able to control zoonotic transmission of the parasite to humans, a non-invasive loop-mediated isothermal amplification (LAMP) assay to specifically detect L. infantum infections in dogs was developed. Methods: The primers used in the LAMP assay were designed to target kinetoplast DNA minicircle sequences of the L. infantum isolate MCAN/CN/90/SC and tested using DNA isolated from promastigotes of different Leishmania species. The LAMP assay was evaluated with conjunctional swab samples obtained from 111 and 33 dogs living in an endemic and a non-endemic region of zoonotic visceral leishmaniasis in the Gansu province, respectively. The LAMP assay was also compared with conventional PCR, ELISA and microscopy using conjunctional swab, serum and bone marrow samples from the dogs, respectively. Results: The LAMP assay detected 1 fg of L. infantum DNA purified from cultured promastigotes which was 10-fold more sensitive than a conventional PCR test using Leishmania genus-specific primers. No cross reaction was observed with DNA isolated from promastigotes of L. donovani, L. major, L. tropica, and L. braziliensis, and the L. infantum reference strain MHOM/TN/80/IPT1. The L. infantum-positive rates obtained for field-collected samples were 61.3%, 58.6%, 40.5% and 10.8% by LAMP, PCR, ELISA and microscopy, respectively. As only one out of the 33 samples from control dogs from the non-endemic region of zoonotic visceral leishmaniasis was positive by the LAMP assay and the PCR test, the observed true negative rate (specificity) was 97% for both methods. Conclusion: This study has shown that the non-invasive, conjunctional swab-based LAMP assay developed was more sensitive in the detection of leishmaniasis in dogs than PCR, ELISA and microscopy. The findings indicate that the LAMP assay is a sensitive and specific method for the field surveillance of domestic dogs, particularly of asymptomatic canines, in ZVL-endemic areas in western China

Insights on adaptive and innate immunity in canine leishmaniosis

Canine leishmaniosis (CanL) is caused by the parasite Leishmania infantum and is a systemic disease, which can present with variable clinical signs, and clinicopathological abnormalities. Clinical manifestations can range from subclinical infection to very severe systemic disease. Leishmaniosis is categorized as a neglected tropical disease and the complex immune responses associated with Leishmania species makes therapeutic treatments and vaccine development challenging for both dogs and humans. In this review, we summarize innate and adaptive immune responses associated with L. infantum infection in dogs, and we discuss the problems associated with the disease as well as potential solutions and the future direction of required research to help control the parasite

Interactions between BRCA2 and RAD51 for promoting homologous recombination in Leishmania infantum.

In most organisms, the primary function of homologous recombination (HR) is to allow genome protection by the faithful repair of DNA double-strand breaks. The vital step of HR is the search for sequence homology, mediated by the RAD51 recombinase, which is stimulated further by proteins mediators such as the tumor suppressor BRCA2. The biochemical interplay between RAD51 and BRCA2 is unknown in Leishmania or Trypanosoma. Here we show that the Leishmania infantum BRCA2 protein possesses several critical features important for the regulation of DNA recombination at the genetic and biochemical level. A BRCA2 null mutant, generated by gene disruption, displayed genomic instability and gene-targeting defects. Furthermore, cytological studies show that LiRAD51 can no longer localize to the nucleus in this mutant. The Leishmania RAD51 and BRCA2 interact together and the purified proteins bind single-strand DNA. Remarkably, LiBRCA2 is a recombination mediator that stimulates the invasion of a resected DNA double-strand break in an undamaged template by LiRAD51 to form a D-loop structure. Collectively, our data show that LiBRCA2 and LiRAD51 promote HR at the genetic and biochemical level in L. infantum, the causative agent of visceral leishmaniasis

Detection of Leishmania infantum by PCR, serology and cellular immune response in a cohort study of Brazilian dogs

The sensitivity and specificity of PCR, serology (ELISA) and lymphoproliferative response to Leishmania antigen for the detection of Leishmania infantum infection were evaluated in a cohort of 126 dogs exposed to natural infection in Brazil. For PCR, Leishmania DNA from bone-marrow was amplified with both minicircle and ribosomal primers. The infection status and time of infection of each dog were estimated from longitudinal data. The sensitivity of PCR in parasite-positive samples was 98%. However, the overall sensitivity of PCR in post-infection samples, from dogs with confirmed infection, was only 68%. The sensitivity of PCR varied during the course of infection, being highest (78–88%) 0–135 days post-infection and declining to around 50% after 300 days. The sensitivity of PCR also varied between dogs, and was highest in sick dogs. The sensitivity of serology was similar in parasite-positive (84%), PCR-positive (86%) and post-infection (88%) samples. The sensitivity of serology varied during the course of infection, being lowest at the time of infection and high (93–100%) thereafter. Problems in determining the specificity of serology are discussed. The sensitivity and specificity of cellular responsiveness were low. These data suggest that PCR is most useful in detecting active or symptomatic infection, and that serology can be a more sensitive technique for the detection of all infected dogs

Heterogeneities in leishmania infantum infection : using skin parasite burdens to identify highly infectious dogs

Background: The relationships between heterogeneities in host infection and infectiousness (transmission to arthropod vectors) can provide important insights for disease management. Here, we quantify heterogeneities in Leishmania infantum parasite numbers in reservoir and non-reservoir host populations, and relate this to their infectiousness during natural infection. Tissue parasite number was evaluated as a potential surrogate marker of host transmission potential. Methods: Parasite numbers were measured by qPCR in bone marrow and ear skin biopsies of 82 dogs and 34 crab-eating foxes collected during a longitudinal study in Amazon Brazil, for which previous data was available on infectiousness (by xenodiagnosis) and severity of infection. Results: Parasite numbers were highly aggregated both between samples and between individuals. In dogs, total parasite abundance and relative numbers in ear skin compared to bone marrow increased with the duration and severity of infection. Infectiousness to the sandfly vector was associated with high parasite numbers; parasite number in skin was the best predictor of being infectious. Crab-eating foxes, which typically present asymptomatic infection and are non-infectious, had parasite numbers comparable to those of non-infectious dogs. Conclusions: Skin parasite number provides an indirect marker of infectiousness, and could allow targeted control particularly of highly infectious dogs

Analysis by RNA-seq of transcriptomic changes elicited by heat shock in Leishmania major

Besides their medical relevance, Leishmania is an adequate model for studying post-transcriptional mechanisms of gene expression. In this microorganism, mRNA degradation/stabilization mechanisms together with translational control and post-translational modifications of proteins are the major drivers of gene expression. Leishmania parasites develop as promastigotes in sandflies and as amastigotes in mammalians, and during host transmission, the parasite experiences a sudden temperature increase. Here, changes in the transcriptome of Leishmania major promastigotes after a moderate heat shock were analysed by RNA-seq. Several of the up-regulated transcripts code for heat shock proteins, other for proteins previously reported to be amastigote-specific and many for hypothetical proteins. Many of the transcripts experiencing a decrease in their steady-state levels code for transporters, proteins involved in RNA metabolism or translational factors. In addition, putative long noncoding RNAs were identified among the differentially expressed transcripts. Finally, temperature-dependent changes in the selection of the spliced leader addition sites were inferred from the RNA-seq data, and particular cases were further validated by RT-PCR and Northern blotting. This study provides new insights into the post-transcriptional mechanisms by which Leishmania modulate gene expressionThis work was supported by grants (to B.A. and J.M.R.) from Ministerio de Economía, Industria y Competitividad, project number SAF2017-86965-R (co-funded with FEDER funds), and by the Network of Tropical Diseases Research RICET (RD16/0027/0008), co-funded with FEDER funds. The CBMSO receives institutional grants from the Fundación Ramón Areces and from the Fundación Banco de Santande

Evolutionary and geographical history of the Leishmania donovani complex with a revision of current taxonomy.

Leishmaniasis is a geographically widespread severe disease, with an increasing incidence of two million cases per year and 350 million people from 88 countries at risk. The causative agents are species of Leishmania, a protozoan flagellate. Visceral leishmaniasis, the most severe form of the disease, lethal if untreated, is caused by species of the Leishmania donovani complex. These species are morphologically indistinguishable but have been identified by molecular methods, predominantly multilocus enzyme electrophoresis. We have conducted a multifactorial genetic analysis that includes DNA sequences of protein-coding genes as well as noncoding segments, microsatellites, restriction-fragment length polymorphisms, and randomly amplified polymorphic DNAs, for a total of approximately 18,000 characters for each of 25 geographically representative strains. Genotype is strongly correlated with geographical (continental) origin, but not with current taxonomy or clinical outcome. We propose a new taxonomy, in which Leishmania infantum and L. donovani are the only recognized species of the L. donovani complex, and we present an evolutionary hypothesis for the origin and dispersal of the species. The genus Leishmania may have originated in South America, but diversified after migration into Asia. L. donovani and L. infantum diverged approximately 1 Mya, with further divergence of infraspecific genetic groups between 0.4 and 0.8 Mya. The prevailing mode of reproduction is clonal, but there is evidence of genetic exchange between strains, particularly in Africa

Leishmania manipulation of sand fly feeding behavior results in enhanced transmission.

In nature the prevalence of Leishmania infection in whole sand fly populations can be very low (<0.1%), even in areas of endemicity and high transmission. It has long since been assumed that the protozoan parasite Leishmania can manipulate the feeding behavior of its sand fly vector, thus enhancing transmission efficiency, but neither the way in which it does so nor the mechanisms behind such manipulation have been described. A key feature of parasite development in the sand fly gut is the secretion of a gel-like plug composed of filamentous proteophosphoglycan. Using both experimental and natural parasite-sand fly combinations we show that secretion of this gel is accompanied by differentiation of mammal-infective transmission stages. Further, Leishmania infection specifically causes an increase in vector biting persistence on mice (re-feeding after interruption) and also promotes feeding on multiple hosts. Both of these aspects of vector behavior were found to be finely tuned to the differentiation of parasite transmission stages in the sand fly gut. By experimentally accelerating the development rate of the parasites, we showed that Leishmania can optimize its transmission by inducing increased biting persistence only when infective stages are present. This crucial adaptive manipulation resulted in enhanced infection of experimental hosts. Thus, we demonstrate that behavioral manipulation of the infected vector provides a selective advantage to the parasite by significantly increasing transmission