BackgroundLeishmania infantum is the parasite responsible for the disease in humans known as zoonotic visceral leishmaniasis (ZVL). Dogs are considered the main domestic reservoir of ZVL and sand flies are the proven vectors. The use of systemic insecticides in dogs has been studied as an alternative strategy to control ZVL in endemic areas. One systemic insecticide in dogs, fluralaner, has a proven anti-sand fly effect in membrane-fed studies. However, the efficacy and duration on sand flies directly feeding from dogs treated with fluralaner remains unknown.MethodsDirect feeding bioassays were performed on 10 beagle dogs that had been randomly assigned to two groups: one with five dogs orally treated with Bravecto® (fluralaner) and other five as a control. About 30 females of Phlebotomus papatasi were allowed to directly feed from dogs at seven days before the administration of the treatment and Days 3, 17, 31, 45 and 73 post-treatment. Sand fly mortality after feeding was observed every 24 h for 5 days. The Kaplan-Meyer method, Henderson-Tilton formula and a negative binomial mixed model were used to respectively calculate: (i) mortality and its 95% confidence interval (CI); (ii) efficacy of the insecticide at killing sand flies in 24 h; and (iii) differences in the risk of sand fly death at 24 h after feeding.ResultsControl sand fly mortality 24 h after feeding was always ≤ 20% and mortality in the fluralaner group ranged from 2% (95% CI: 0–4%) 7 days before treatment to 100% at 3 days post-treatment. Fluralaner efficacy was 100, 93, 94 and 75% at Days 3, 17, 31 and 45, respectively (P < 0.0001). The increase in the risk of sand fly death was 32.9 (95% CI: 4–263), 76 (95% CI: 8–705), 95.8 (95% CI: 9–1029) and 10.6 times (95% CI: 1.43–79) on Days 3, 17, 31 and 45, respectivelyConclusionsThe efficacy of fluralaner, orally administered to dogs, against sand-flies was above 90% for 31 days. Fluralaner administered to dogs should be further evaluated as a control strategy in ZVL endemic areas.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-3231-8) contains supplementary material, which is available to authorized users.

Leishmania infantum causes zoonotic visceral leishmaniasis (ZVL) in the Mediterranean basin and South America. The parasite has been shown to co-infect HIV patients and an outbreak in central Spain was reported in the last decade. Therfore, ZVL is a public health problem, dogs being the parasite's reservoir. We have developed a DNA vaccine based on the L . infantum activated protein kinase A receptor (LACK) using different plasmid vectors and vaccinia virus strains as vehicles. Recently, we have generated an antibiotic resistance marker-free plasmid vector called pPAL. Homologous pPAL-LACK prime-boost vaccination protects Beagle dogs as well as a heterologous plasmid-virus regime. For both reasons, pPAL improves safety. IL12 was described to trigger Th1 response through IFN-γ production in infected dogs, being a good candidate for cytokine therapy in conventional treatment-unresponsive dogs. Herein, we report a complete protection study in dogs through inoculation of genes encoding for the p35 and p40 subunits which compose canine IL12 in combination with the LACK gene. A homologous plasmid-plasmid regime using independent pPAL constructs for each gene was inoculated in a 15-day interval. The infectious challenge using L . infantum promastigotes was successful. The outcome was pPAL-LACK vaccine protection suppression by IL12 administration. The important implications of this finding are discussed in the manuscript.

BackgroundZoonotic visceral leishmaniasis (ZVL) caused by Leishmania (Leishmania) infantum is an important disease in humans and dogs. Different mammal species are reservoirs but dogs are considered to be the main one. Phlebotomine sand flies are the proven vector. Four systemic insecticides approved for their use in dogs were previously selected based on their potential to be used in endemic countries as part of the control programs of ZVL. These insecticides are proved to be safe and effective against the on-label insects and parasites, but there is no information about their activity against phlebotomine sand flies.MethodsThe phlebotomine mortality of four systemic insecticides in dogs was evaluated using two randomized clinical trials. For the first trial, thirty dogs were randomly allocated into five groups: four treatments and one control, of equal size. The treatments evaluated were: Guardian®SR, Elanco (moxidectin); Comfortis®, Elanco (spinosad); Bravecto®, Merck Animal Health (fluralaner); and NexGard®, Merial (afoxolaner). Blood from dogs was taken at days 2, 4, 21 and 31 post-treatment (trial 1). The compound that showed the highest efficacy was selected for a second trial (trial 2) with 20 dogs sampled at days 0, 2, 4, 7, 14, 18, 32, 39, 51 and 84 post-treatment. Membrane feeding bioassays with Phlebotomus papatasi were used to evaluate the phlebotomine mortality efficacy of the different treatments. Phlebotomine mortality was observed every 24 h following the membrane feeding during 5 days. A mixed model for a negative binomial logistic regression, and a Cox proportional hazard mixed model were used to estimate phlebotomine mortality due to different treatments.ResultsFluralaner was the only compound that showed significant phlebotomine mortality. Fluralaner maintained the phlebotomine mortality between 60–80% for 30 days after treatment. In trial 1 we found that fluralaner increased the risk of death by 1.9 times (95% CI: 1.02–3.6) and 1.7 times (95% CI: 1.09–2.6) at days 2 and 4 after treatment. The Cox model resulted in an increase of 1.47 (95% CI: 1.1–1.96) times in hazard risk at day 2 and 1.89 (95% CI: 1.35–2.45) at day 4 after treatment.In trial 2 we found that fluralaner increased the risk of death by 1.64 times (95% CI: 1.16–2.54) and 1.97 times (95% CI: 1.23–3.17) at days 14 and 32. The hazard risk was also increased by 1.92 (95% CI: 1.4–2.64) times at day 14 after treatment. Phlebotomine survival including all experimental days was significantly lower in the fluralaner group in both trials.ConclusionsA single oral treatment of fluralaner in dogs induces phlebotomine mortality. Systemic insecticides in dogs should be considered as a potential preventive measure of ZVL.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2820-x) contains supplementary material, which is available to authorized users.

Early diagnosis of renal damage in Leishmania infected dogs may allow appropriate treatments and prevent some deaths. This study investigates neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker of kidney disease in dogs experimentally infected with Leishmania infantum. Serum, urine, and kidney samples were collected from 30 infected beagle dogs and six uninfected control dogs. Based on proteinuria and azotemia values, dogs were initially classified. NGAL was measured in urine and serum samples. Then, the urinary NGAL to creatinine ratio (uNGAL/C) was calculated. Kidney samples were taken for histopathological studies, and the dogs were classified according to the severity of glomerular and tubulointerstitial lesions. In Leishmania-infected dogs, the uNGAL/C was significantly higher in proteinuric non-azotemic dogs compared with non-proteinuric non-azotemic dogs (p = 0.038). Serum NGAL (sNGAL) concentration did not differ between groups. Microscopic studies revealed several degrees of glomerulonephritis and slight focal lymphoplasmacytic interstitial nephritis in 89% and 55% of infected dogs, respectively. Urinary protein to creatinine ratio (UPC) and uNGAL/C were significantly higher in dogs with affected glomeruli compared to infected dogs without renal lesions (p = 0.045 and p = 0.043, respectively). The results show that uNGAL/C correlates with proteinuria and the presence of moderate glomerular lesions in non-azotemic dogs experimentally infected with L. infantum.

Canine leishmaniasis (CanL) diagnosis is not fully resolved. Currently, two specific methodologies are in continuous development, the detection of the parasite DNA or RNA in target organs and the detection of specific antibodies against Leishmania sp. For a correct diagnosis, it has been shown that the joint use of this type of test is necessary. In this work, a Sybr Green and a TaqMan Probe based on real time PCRs (qPCR) was performed for the detection of Leishmania sp. in order to correlate the results with clinicopathological and serological evaluations (IFA, ELISA and DAT) to propose an optimal biological sample to be used to detect the parasite in both early and late stages of the infection. A total of four samples were processed: conjunctival swabs, popliteal lymph node aspirates, bone marrow aspirates, and peripheral blood from experimentally infected dogs belonging to a larger study. Our results indicated that a single non-invasive sample (conjunctival swab) and the application of both types of qPCR would be reliable for determining Leishmania infection as well as the disease stage in dogs, thus avoiding bone marrow, lymph node aspirate or blood samples collection.