Performance of a real time PCR for leishmaniasis diagnosis using a L. (L.) infantum hypothetical protein as target in canine samples.

Visceral leishmaniasis represents an important public health issue in different parts of the world, requiring that measures be put in place to control the spread of the disease worldwide. The canine leishmaniasis diagnosis is not easy based on clinical signs, since dogs may not develop the infection with recognizable signs. Thus, the laboratorial diagnosis is essential to ascertain the incidence and prevalence of canine leishmaniasis especially in areas with major control efforts. Although, the diagnosis can be performed by the use of different approaches, the molecular methods such as PCR have become an indispensable tool for leishmaniases diagnosis. A TaqMan assay for real-time PCR (Linj31-qPCR) was developed to determine the parasite occurrence in clinical cases of leishmaniasis. The assay targets an L. (L.) infantum hypothetical protein region. The specificity of the assay was verified by using Leishmania World Health Organization reference strains including parasites belonging to subgenus L. (Leishmania), subgenus L. (Viannia), other Leishmania species and Trypanosoma cruzi. The sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for diagnosis was ascertained by testing 277 samples from dogs in regions endemic for visceral and/or cutaneous leishmaniasis and from regions in which leishmaniasis was not endemic in São Paulo State, Brazil. Diagnosis of canine visceral leishmaniasis (CVL) was determined on these animals by conventional PCR and three serological tests. The dog samples were divided into four groups. I, dogs with CVL (n = 101); II, dogs with other diseases and without CVL (n = 97); III, dogs with American cutaneous leishmaniasis (n = 7), and, IV, dogs without CVL (n = 72) from areas where leishmaniasis was not endemic as control group. Results indicated that Linj31-qPCR was able to identify parasites belonging to subgenus L. (Leishmania) with no cross-amplification with other parasite subgenera. The Linj31-qPCR detected Leishmania parasites DNA in 98% of samples from Group I. In conclusion this methodology can be used as routine diagnostic tools to detect parasites from subgenus Leishmania.

Persistent cutaneous canine leishmaniasis caused by Leishmania (Viannia) braziliensis in an area with predominance of Nyssomyia neivai in the state of São Paulo, Brazil.

American cutaneous leishmaniasis (ACL) is a neglected zoonotic disease caused mainly by Leishmania (Viannia) braziliensis, which is endemic throughout Brazil. Canine ACL cases were investigated in a rural area of Monte Mor, São Paulo, where a human ACL case had been confirmed. Dogs were evaluated through clinical and laboratory diagnosis including serology, cytological tissue preparations and PCR on skin lesions, lymph node and bone marrow samples. Entomological investigations on sandflies trapped in the surroundings of the study area were performed for 14 months. Nyssomyia neivai was the predominant phlebotomine species, comprising 94.65% of the captured specimens (832 out of 879). This species was the most abundant in all trapping sites, including human homes and dog shelters. Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri and Expapilata firmatoi were also captured. Two of the three dogs examined were positive for anti-Leishmania IgG in ELISA using the antigen Fucose mannose ligand and skin samples were positive for L. (V.) braziliensis in PCR, but all the samples collected were negative for L. (L.) infantum. One of the dogs had a confirmed persistent infection for more than one year.

Efficacy of afoxolaner (NexGard®) on the treatment of myiasis caused by the New World screwworm fly Cochliomyia hominivorax (Diptera: Calliphoridae) in naturally infested dogs.

New World screwworm (NWS) myiasis is an infestation by Cochliomyia hominivorax larvae that consume the living tissue of warm-blooded animals, including humans. Domestic dogs are among the potential hosts of these flies that lay their eggs on the edges of wounds. NWS myiasis cases can be fatal if untreated. Treatment with parasiticides must be fast-acting, long-lasting and show 100% efficacy, since open wounds can be reinfested. Afoxolaner is a molecule from the isoxazoline family with proven ectoparasiticide action against fleas, ticks and mites in dogs. Fourteen healthy client-owned dogs, naturally infested by C. hominivorax larvae, were treated with afoxolaner (NexGard®) as per label recommendations, providing at least the minimum dosage of 2.5 mg/kg. Maggot infestations were classified as light (fewer than 10 larvae), mild (from 10 to 20 larvae) or severe (more than 20 larvae), according to the number of larvae found in the wound and/or collected from the ground after treatment. Twenty-four hours post-treatment, infested lesions were carefully inspected and collected larvae were counted and classified as live or dead. All maggots were identified as second and third instar larvae of C. hominivorax and were found dead within 24 h after treatment, demonstrating 100% larvicidal efficacy against C. hominivorax.

Vaccination against canine leishmaniasis in Brazil.

Prevention of canine Leishmania infantum infection is critical to management of visceral leishmaniasis in people living in endemic areas of Brazil. A bill (PL 1738/11), currently under consideration, proposes to establish a national vaccination policy against canine leishmaniasis in Brazil. However, there is no solid scientific evidence supporting the idea that this could reduce transmission from infected vaccinated dogs to sand flies to a level that would significantly reduce the risk of L. infantum infection or visceral leishmaniasis in humans. Thus, we advocate that insecticide-impregnated collars should the first line protective measure for public health purposes and that vaccines are applied on a case-by-case, optional basis for individual dog protection.

Use of miltefosine to treat canine visceral leishmaniasis caused by Leishmania infantum in Brazil.

BACKGROUND: Visceral leishmaniasis (VL) is an infectious disease with a variety of clinical signs. The main form of parasite transmission to humans and other mammalian hosts is through the bite of infected arthropod females with Lutzomyia longipalpis serving as the main vector in the Americas. Dogs are the main urban domestic reservoirs of the parasite and the main source of vector infection due to their high prevalence in endemic areas and the large number of parasites in the skin of infected animals. Although miltefosine has been used in Europe since 2002 for treatment of VL infected dogs, in the Americas the treatment of dogs has not been recommended. Therefore, this study aimed to evaluate efficacy of miltefosine observing a reduction of clinical signs in infected dogs and the infectiveness to the vector by Leishmania (L.) infantum. METHODS: To our knowledge, this is the first controlled study using qPCR and xenodiagnosis to evaluate the efficacy of miltefosine (Milteforan®, Virbac) as a single treatment in Brazil. Thirty-five adult dogs with canine visceral leishmaniasis (CVL), confirmed by clinical and laboratory tests, were included in this study. They received miltefosine at a dose of 2 mg/kg every 24 h for 28 days. The dogs were observed over a three-month period, during which clinical evaluations based on a scoring system were conducted at pre-established times. Parasite load was assessed by cytology and real-time polymerase chain reaction (qPCR). Transmissibility to the vector was evaluated by xenodiagnosis. RESULTS: At the end of the period, the following were observed: (i) the remission of clinical signs with a reduction in clinical scores for 94.2% of the animals; (ii) a statistically significant reduction (98.7%) in parasitic load by qPCR; and (iii) a reduction in infectivity to sand flies. After treatment, 74.2% of the animals remained or had become non-infectious. CONCLUSIONS: Our study indicates that the use of miltefosine administered orally for 4 weeks contributes to a clinical improvement and reduction in infectivity of dogs to L. infantum.

Persistent cutaneous canine leishmaniasis caused by Leishmania (Viannia) braziliensis in an area with predominance of Nyssomyia neivai in the state of São Paulo, Brazil / Leishmaniose cutânea canina persistente causada por Leishmania (Viannia) braziliensis em uma área com predomínio de Nyssomyia neivai no estado de São Paulo, Brasil

Abstract American cutaneous leishmaniasis (ACL) is a neglected zoonotic disease caused mainly by Leishmania (Viannia) braziliensis, which is endemic throughout Brazil. Canine ACL cases were investigated in a rural area of Monte Mor, São Paulo, where a human ACL case had been confirmed. Dogs were evaluated through clinical and laboratory diagnosis including serology, cytological tissue preparations and PCR on skin lesions, lymph node and bone marrow samples. Entomological investigations on sandflies trapped in the surroundings of the study area were performed for 14 months. Nyssomyia neivai was the predominant phlebotomine species, comprising 94.65% of the captured specimens (832 out of 879). This species was the most abundant in all trapping sites, including human homes and dog shelters. Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri and Expapilata firmatoi were also captured. Two of the three dogs examined were positive for anti-Leishmania IgG in ELISA using the antigen Fucose mannose ligand and skin samples were positive for L. (V.) braziliensis in PCR, but all the samples collected were negative for L. (L.) infantum. One of the dogs had a confirmed persistent infection for more than one year.

Resumo A leishmaniose tegumentar Americana (LTA) é uma doença zoonótica negligenciada, causada principalmente por Leishmania (Viannia) braziliensis, sendo endêmica em todo o Brasil. Foram investigados casos de LTA canina em uma área rural da cidade de Monte Mor, São Paulo, onde foi confirmado um caso humano de LTA. Os cães foram avaliados por diagnóstico clínico e laboratorial, incluindo sorologia, esfregaços microscópicos e PCR de amostras em lesões de pele, linfonodos e medula óssea. Também foram realizadas investigações entomológicas durante 14 meses, usando-se armadilhas luminosas para flebotomíneos nas proximidades da área de estudo. Nyssomyia neivai foi a espécie de flebotomíneo predominante com 94,65% dos espécimes capturados (832 de 879). Essa espécie foi a mais abundante em todos os locais de captura, incluindo-se abrigos para humanos e cães. Foram também capturadas as espécies Ny. whitmani, Migonemyia migonei, Pintomyia monticola, Evandromyia cortellezzii, Pi. fischeri e Expapilata firmatoi. Dos três cães examinados, dois apresentaram IgG anti-Leishmania positivo no ELISA, usando-se o antígeno "Fucose mannose ligand", PCR da lesão de pele positivo para L. (V.) braziliensis e negativo em todas amostras para L. (L.) infantum. Um dos cães apresentou infecção persistente por mais de um ano.

Control of visceral leishmaniasis in Brazil: recommendations from Brasileish.

On 26 October 2012, veterinary medicine clinicians and researchers, members of Brasileish - Study Group about Animal Leishmaniasis - met at the Regional Council of Veterinary Medicine of Minas Gerais, in the city Belo Horizonte, in order to discuss many aspects of the situation of canine visceral leishmaniasis (CVL) in Brazil. In the face of recent national and international scientific evidence, we, the members of Brasileish, have elaborated some recommendations for the management and control of CVL in Brazil.

Decrease of the incidence of human and canine visceral leishmaniasis after dog vaccination with Leishmune in Brazilian endemic areas.

Leishmune, the first prophylactic vaccine licensed against canine visceral leishmaniasis (CVL), has been used in Brazil since 2004, where seropositive dogs are sacrificed in order to control human visceral leishmaniasis (VL). We demonstrate here that vaccination with Leishmune does not interfere with the serological control campaign (110,000 dogs). Only 1.3% of positivity (76 among 5860) was detected among Leishmune uninfected vaccinees. We also analyzed the possible additive effect of Leishmune vaccination over dog culling, on the decrease of the incidence of CVL and VL in two Brazilian endemic areas, from 2004 to 2006. In Araçatuba, a 25% of decline was seen in CVL with a 61% decline in human cases, indicating the additive effect of Leishmune vaccination of 5.7% of the healthy dogs (1419 dogs), on regular dog culling. In Belo Horizonte (BH), rising curves of canine and human incidence were observed in the districts of Barreiro, Venda Nova and Noroeste, while the canine and human incidence of Centro Sul, Leste, Nordeste, Norte, Pampulha and Oeste, started to decrease or maintained a stabilized plateau after Leishmune vaccination. Among the districts showing a percent decrease of human incidence (-36.5%), Centro Sul and Pampulha showed the highest dog vaccination percents (63.27% and 27.27%, respectively) and the lowest dog incidence (-3.36% and 1.89%, respectively). They were followed by Oeste, that vaccinated 25.30% of the animals and experienced an increase of only 12.86% of dog incidence and by Leste and Nordeste, with lower proportions of vaccinees (11.72% and 10.76%, respectively) and probably because of that, slightly higher canine incidences (42.77% and 35.73%). The only exception was found in Norte district where the reduced human and canine incidence were not correlated to Leishmune vaccination. Much lower proportions of dogs were vaccinated in Venda Nova (4.35%), Noroeste (10.27%) and Barreiro (0.09%) districts, which according to that exhibited very increased canine incidences (24.48%, 21.85% and 328.57%, respectively), and pronounced increases in human incidence (14%, 4% and 17%, respectively). The decrease of canine (p=-0.008) and human incidences (p=-0.048) is directly correlated to the increase of the number of vaccinated dogs, confirming the additive control effect of Leishmune vaccination over dog culling, reducing the parasite reservoir, protecting dogs and, in this way, reducing the risk of transmission of VL to humans and becoming a new effective control tool.

The FML-vaccine (Leishmune) against canine visceral leishmaniasis: a transmission blocking vaccine.

Transmission blocking vaccines are one of the control strategies for vector-transmitted protozoan diseases. Antibodies raised in the vaccinated host prevent the development of the parasite in the insect vector, interrupting the epidemiological cycle. The FML antigen of Leishmania donovani in combination with saponin (FML-vaccine and Leishmune) induced 92-97% of protections against zoonotic visceral leishmaniasis. We assayed the ability of FML to inhibit Leishmania donovani and Leishmania chagasi procyclic promastigote-binding to dissected Lutzomyia longipalpis midguts. We found a dose-dependent inhibition, more pronounced on L. donovani (80%) than on L. chagasi promastigotes (p<0.001). On the other hand, the Fab-IgG serum fraction of Leishmune vaccinated dogs (IgG2 predominant), also inhibited parasite binding in a dose-response (p<0.0001) with an equally potent effect against L. donovani or L. chagasi (p = 0.061). The transmission blocking properties of the Leishmune vaccine was also assessed by an in vivo membrane assay, with sand flies fed with 1.5 x 10(7) amastigotes, human blood and, vaccinated or normal control dog sera. Significantly higher values were found in rate of infection (p<0.025) and intensity of infection (number of parasites/insect) (p<0.05) of control sand flies, making a very reduced infection index (20.7%) in the vaccine group. Our results disclosed that the Leishmune vaccine is a TBV, and that the dog antibodies present in sera, even 12 months after vaccination, lead to a significant effective protection of 79.3%.

IgG1/IgG2 antibody dichotomy in sera of vaccinated or naturally infected dogs with visceral leishmaniosis.

Canine antibody IgG, IgG1 and IgG2 anti-FML responses were investigated in dogs vaccinated with the fucose-mannose ligand (FML)-vaccine of Leishmania donovani and in dogs with naturally acquired visceral leishmaniosis. While similar levels of total IgG antibodies were seen in the seropositive naturally infected dogs and in vaccinees, significant differences between the groups were found regarding their IgG1/IgG2 anti-FML antibody composition (P<0.005). Higher IgG1 absorbencies were seen in infected dogs, while the IgG2 subtype was predominant in pre-immune sera, and in vaccinated animals, both after the first and the third dose (P<0.005). The average ratio between IgG1/IgG2 was then 1.124 for infected animals and 0.733 for FML-vaccinees. Also, a significant increase in IgG2 antibodies was observed from the first to the third vaccine injection (P<0.005). In the infected dogs, a high correlation between their IgG absorbance (Abs) values and the number of symptoms (P=0.017) was disclosed. Thus, the analysis of IgG subclasses disclosed a dichotomous response to visceral leishmaniosis: IgG1 associated to natural infection and IgG2 associated to a humoral response subsequent to the FML-vaccine treatment. An IgG1/IgG2>or=1 would characterize the sera of visceral leishmaniasis infected animals evoluting towards the overt disease while ratios