Canine visceral leishmaniasis (CVL) is the major source of human visceral leishmaniasis (VL) and is transmitted from dogs to sand flies to humans. To control the spread of this disease, early and accurate detection of infected dogs is critical but challenging. Here we demonstrate the potential of the Dual-Path Platform (DPP(®)) CVL rapid test for detecting K26/K39-reactive antibodies in sera from clinically symptomatic (n=60) and asymptomatic (n=60) Leishmania infantum-infected dogs. For the specificity evaluation, assays were performed using known negative diagnostic serum samples (n=59) and cross-reaction control sera (n=11) from animals born in a VL-free area of Brazil. The diagnostic kit displayed high specificity (96%) but low sensitivity (47%) in identifying parasite-positive dogs without signs of CVL. However, the test sensitivity was significantly higher (98%) in diseased cases, indicating that this convenient test may be useful to identify the most infectious dogs. Efforts should be pursued to obtain a more sensitive DPP-multiplexed test parameter (i.e. based on simultaneous yet separate antibody detection of carefully selected multiple antigens of diagnostic utility) for effective serodiagnosis of early-infected dogs, as this will likely allow more efficient canine removal regimens than those used in practice by public health services.

DNA barcoding has been an effective tool for species identification in several animal groups. Here, we used DNA barcoding to discriminate between 47 morphologically distinct species of Brazilian sand flies. DNA barcodes correctly identified approximately 90% of the sampled taxa (42 morphologically distinct species) using clustering based on neighbor-joining distance, of which four species showed comparatively higher maximum values of divergence (range 4.23–19.04%), indicating cryptic diversity. The DNA barcodes also corroborated the resurrection of two species within the shannoni complex and provided an efficient tool to differentiate between morphologically indistinguishable females of closely related species. Taken together, our results validate the effectiveness of DNA barcoding for species identification and the discovery of cryptic diversity in sand flies from Brazil.

BackgroundBecause domestic dogs are reservoir hosts for visceral leishmaniasis (VL) in Brazil, one of the approaches used to reduce human disease incidence is to cull infected dogs. However, the results of controlled intervention trials based on serological screening of dogs and killing of seropositive animals are equivocal. A prophylactic vaccine to protect dogs from being infectious to the sand fly vector could be an effective strategy to provide sustained control. Here, we investigated whether a currently licensed commercial subunit rA2 protein–saponin vaccine (Leish-tec®) had an additional effect to dog culling on reducing the canine infectious populations.Methodology/Principal findingsThis prospective study was conducted in an L. infantum highly endemic area of southeast Brazil. At the onset of the intervention, all of the eligible dogs received through subcutaneous route a three-dose vaccine course at 21-day intervals and a booster on month 12. For the purpose of comparison, newly recruited healthy dogs were included as the exposed control group. To ascertain vaccine-induced protection, dogs were screened on clinical and serological criteria every 6 months for a 2-year follow-up period. Antibody-based tests and histopathological examination of post-mortem tissue specimens from euthanized animals were used as a marker of infection. The standardized vaccine regime, apart from being safe, was immunogenic as immunized animals responded with a pronounced production of anti-A2-specific IgG antibodies. It should be noted the mean seroconversion time for infection obtained among immunized exposed dogs (~ 18 months), which was twice as high as that for unvaccinated ones (~ 9 months). After two transmission cycles completed, the cumulative incidence of infection did differ significantly (P = 0.016) between the vaccinated (27%) and unvaccinated (42%) dogs. However, the expected efficacy for the vaccine in inducing clinical protection was not evident since 43% of vaccine recipients developed disease over time. Our estimates also indicated that immunoprophylaxis by Leish-tec® vaccine in addition to dog culling might not have an impact on bringing down the incidence of canine infection with L. infantum in areas of high transmission rates.Conclusions/SignificanceLeish-tec® as a prophylactic vaccine showed promise but needs to be further optimized to be effective in dogs under field conditions, and thereby positively impacts human incidence.

Sand fly (Diptera: Psychodidae) taxonomy is complex and time-consuming, which hampers epidemiological efforts directed toward controlling leishmaniasis in endemic regions such as northeastern Brazil. Here, we used a fragment of the mitochondrial cytochrome c oxidase I (COI) gene to identify sand fly species in Maranhão State (northeastern Brazil) and to assess cryptic diversity occurring at different spatial scales. For this, we obtained 148 COI sequences of 15 sand fly species (10 genera) from Maranhão (fine spatial scale), and joined them to COI sequences from other Brazilian localities (distant about 2,000 km from Maranhão, broad spatial scale) available in GenBank. We revealed cases of cryptic diversity in sand flies both at fine (Lutzomyia longipalpis (Lutz and Neiva) and Evandromyia termitophila (Martins, Falcão and Silva)) and broad spatial scales (Migonemyia migonei (França), Pressatia choti (Floch and Abonnenc), Psychodopygus davisi (Root), Sciopemyia sordellii (Shannon and Del Ponte), and Bichromomyia flaviscutellata (Mangabeira)). We argue that in the case of Bi. flaviscutellata, the cryptic diversity is associated with a putative new species. Cases in which DNA taxonomy was not as effective as morphological identification possibly involved recent speciation and/or introgressive hybridization, highlighting the need for integrative approaches to identify some sand fly species. Finally, we provide the first barcode sequences for four species (Brumptomyia avellari (Costa Lima), Evandromyia infraspinosa (Mangabeira), Evandromyia evandroi (Costa Lima and Antunes), and Psychodopygus complexus (Mangabeira)), which will be useful for further molecular identification of neotropical species.

Reserva Biológica de Duas Bocas (2,910 ha) is one of the largest Atlantic forest remnants in the State of Espírito Santo, Southeastern Brazil. We recorded non-volant tetrapods in this area from May 2007 through April 2008, using pitfalls, live traps, camera traps, and diurnal and nocturnal opportunistic searches. In addition, we compiled available museum and literature records from this area. We documented 52 species of amphibians, 24 species of non-avian reptiles, and 39 species of non-volant mammals. Out of these 115 species, 47 are new records for this area and six other species had their geographic ranges expanded with the present study. Furthermore, we present the record of predation of the tree frog Hypsiboas faber by the snake Chironius bicarinatus. Out of the species listed, five species are listed as threatened with extinction in the State of Espírito Santo, and many others have uncertain conservation status. Reserva Biológica de Duas Bocas is an important wildlife refuge, especially considering the expansion of urban areas in its surroundings.