Comparative evaluation of enzyme-linked immunosorbent assay based on crude and purified antigen in the diagnosis of canine visceral leishmaniasis in symptomatic and oligosymptomatic dogs

This study evaluated the performance of crude total antigen (CTA) and fucose-mannose ligand antigen (FML) in an enzyme-linked immunosorbent assay for diagnosis of canine visceral leishmaniasis (CVL). The assays used sera from known negative controls (n = 30), clinically symptomatic (n = 30) and oligosymptomatic (n = 30) parasitologically proven infection (by microscopy). Aspirates of popliteal lymph node from infected canines were colleted to score parasitism and compared with the ELISA results. The study indicated that FML used in ELISA provided high sensitivity for detecting oligosymptomatic dogs (90%) and CTA showed greater sensitivity than FML for symptomatic canines (90%). In oligosymptomatic dogs, specificity was 100% for CTA-ELISA, but in symptomatic dogs, FML specificity was higher (96.7%) than CTA-ELISA (93.3%). A significant correlation was observed between the degree of parasitism and the results obtained in CTA-ELISA. Since no available antigen offers 100% specificity and sensitivity for CVL diagnosis, the choice of antigen used must depend on the aim of the investigation. (C) 2008 Elsevier B.V. All rights reserved.

Morphological, immunohistochemical and molecular study of renal lesions in canine visceral leishmaniasis (CVL)

Canine visceral leishmaniasis (CVL) is an anthropozoonosis characterized by a clinically chronic progressive disease. Non lymphoid organs are also affected, especially the kidneys. Dogs with leishmaniasis usually die with renal failure despite treatment. Haematoxylin-eosin (HE) staining in kidney tissue sections has low sensitivity for parasite identification. Immunohistochemistry (IHC) and polymerase chain reaction (PCR) are efficient methods for Leishmania sp. antigen and DNA detection in cases of low parasite burden. The present study aims to identify renal lesions of CVL and correlate them with microscopic findings determined by histochemistry, IHC and PCR. Both IHC and PCR provided similar positivity for amastigote identification, 3/20 animals (15%), thus increasing detection of the parasite in renal tissues when compared with histopathologic examination. The lesion most commonly observed with visceral leishmaniasis-positive canine kidney tissue was membranoproliferative glomerulonephritis, followed by interstitial nephritis without correlation to the number of amastigotes.

Relationship between dog culling and incidence of human visceral leishmaniasis in an endemic area

Domestic dogs are the main reservoirs of visceral leishmaniasis (VL) in urban areas; one of the control measures adopted in Brazil is the elimination of this reservoir. In order to test the relationship between the euthanasia rate of the canine reservoir and the incidence of the disease in humans, data on dog culling from the Centre for Zoonosis Control of Aracatuba, São Paulo, Brazil, during the period from 1999 to 2008 and visceral leishmaniasis human cases registered in the same period were analyzed. Reduction of human VL incidence was statistically correlated to dog euthanasia rate (P=0.0211; r(2)=0.616) when it was analyzed for the period of two years after application of this measure. Other factors that may influence this relationship are considered. (C) 2010 Elsevier B.V. All rights reserved.

Dog culling and replacement in an area endemic for visceral leishmaniasis in Brazil

Measures employed to control visceral leishmaniasis in Brazil have focused on vector control by residual insecticide spraying and diagnosis of infection with elimination of positive dogs. We describe dog culling and replacement in a Brazilian endemic area (the Alvorada District, Aracatuba, SP) in order to better understand dog population dynamics when elimination of the dog reservoir is adopted as the main control measure. From August 2002 to July 2004, 60.9% of the estimated dog population for the area was culled with a mean age of 34 months old. The presence of anti-Leishmania sp. antibodies was recorded for only 26.7% of the euthanized canines. Replacement was observed in 38.8% of the cases, some of them by 2 or more dogs and in a mean time of 4 months. Dogs were replaced mostly by puppies of both sexes with a mean age of 6.8 months. From August 2002 to April 2005 we were able to follow-up 116 of these dogs, during a mean time of 8.7 months. Canine visceral leishmaniasis seropositivity by ELISA was observed in 42.2% of the followed dogs, 30.6% of which were already positive at the first evaluation. By the end of the follow-up period 37% of the dogs were submitted to euthanasia, with a mean age of 18.3 months. In the studied CVL endemic area of Brazil, euthanasia and the subsequent replacement ratio were high, increasing the dog population turnover and leading to a younger population that might be more susceptible to a variety of other infectious diseases in addition to CVL. Dog culling as a control strategy for VL should be reassessed. (C) 2008 Elsevier B.V. All rights reserved.

Observations on the feeding habits of Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae: Phlebotominae) in Campo Grande, an endemic area of visceral leishmaniasis in Mato Grosso do Sul, Brazil

Sand flies were captured weekly with CDC light traps from December 2003 to November 2005 in three areas of Campo Grande, in the Brazilian state of Mato Grosso do Sul. These areas incorporated two patches of remnant forest and five houses. The blood meals of engorged female sand flies were identified using the avidin-biotin system of immunoenzymatic ELISA capture. Most (327/355) of the females analysed were Lutzomyia longipalpis, of which 66.4% reacted with human blood, 64.8% with that of birds and 8.9% with that of dogs. Females that had taken human blood predominated in the residential areas and two forest patches. The following combinations of blood were also detected for L. longipalpis in some of the samples analysed: bird + human (43.4%), bird + human + dog (6.1%). The combination bird + human + dog + pig was also found for Nyssomyia whitmani. Dogs and pigs appear to have little attractiveness for L. longipalpis. The results obtained demonstrate the eclecticism and high anthropophily of L. longipalpis and raise new questions with regard to the importance of dogs in VL epidemiology and the possible role of man as a source of infection for sand flies. (C) 2008 Elsevier B.V. All rights reserved.

Immune response pattern of the popliteal lymph nodes of dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Surveillance of canine visceral leishmaniasis in a disease-free area

A leishmaniose é uma importante parasitose re-emergente observada no mundo, particularmente em países tropicais. Não há ainda relatos de casos autóctones no estado do Paraná. Não há até o momento referência de vigilância no reservatório canino, tais como Curitiba e região metropolitana do estado. O objetivo do estudo foi determinar a soroprevalência da leishmaniose visceral em cães entregues ao Centro de Controle de Zoonoses de São José dos Pinhais, Paraná para eutanásia. A detecção sorológica da presença de anticorpos contra Leishmania sp. foi realizada por (ELISA) indireto e pela Reação de Imunofluorescência Indireta (RIFI). Além disso, impressão de linfonodo poplíteo coletadas ao acaso de 50 cães com sinais clínicos suspeitos para leishmaniose visceral e analisados sob microscopia óptica para detecção de formas amastigotas, foram negativas. Amostras de soro de 364 animais foram testadas, e os resultados mostraram somente uma amostra positiva (0,0027%), reagente ao ELISA e negativa à RIFI, entretanto, o cão não apresentava sinais clínicos. A vigilância ao acaso em uma população de vários locais de uma área metropolitana pode ser uma forma de prevenção da disseminação da doença. Com base nos resultados observados, Curitiba e região metropolitana foram consideradas de baixo risco para a leishmaniose visceral.

Performance of IFAT, ELISA, direct parasitological examination and PCR on lymph node aspirates for canine visceral leishmaniasis diagnosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Leishmania spp. and/or Trypanosoma cruzi diagnosis in dogs from endemic and nonendemic areas for canine visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

First Autochthonous Case of Canine Visceral Leishmaniasis in the Center of Rio Grande do Sul State, Brazil

Background: Leishmaniasis is one of the most important vector-borne diseases of humans. This parasitic disease can be caused by many species of Leishmania. In humans, different species of the parasite are associated with different forms of the disease, cutaneous and visceral. Among domesticated animals, dogs are the most important species in the epidemiology of this disease. Leishmania chagasi, an important zoonosis, is well established as the agent of visceral leishmaniasis in Brazil. The disease is endemic in north, northeast, midwest and southeast, and is transmitted to mammals by hematophagous insects such as the Lutzomyia longipalpis. In 2008, our research group has diagnosed a case of canine leishmaniasis in the municipality of Uruguaiana and subsequently there were several cases in the city and the neighbor municipality of Sao Borja. Most Brazilian states are endemic for leishmaniasis, with the exception of Rio Grande do Sul. In southern Brazil, the reports of humans and dogs infected by Leishmania spp. are the source of endemic area in the country. Therefore, the aim of this study is register the first clinical case of canine visceral leishmaniasis in the municipality of Santa Maria, RS.Case: In october 2010, a veterinary clinic of Santa Maria received a canine, female, Doberman, with two years of age. The animal had severe skin lesions on the head and limbs, pale mucous membranes, and enlarged lymph nodes. According to the owner, the animal showed progressive weight loss and anorexia for more than five days. During the clinical examination the blood was collected for hemogram and cytology of lymph nodes was performed by puncture aspiration with a fine needle. In the erythrogram, it was observed a decrease in the total number of erythrocytes (2.8 x 10(6)/mu L), hematocrit (21%), hemoglobin (6.8 g/dL) and platelets (98 x 10(3)/mu L). In the leucogram, any alteration was observed. The cytology of lymph nodes showed amastigotes forms, suggestive of the Leishmania spp. Based on this finding; we performed the blood collection for PCR, to confirm parasitism and to determine the species of Leishmania. At the molecular test was used PCR-specific for L. chagasi, and the result was positive.Discussion: This is the first autochthonous clinical case in the central region of the RS, non-endemic area for leishmaniasis. In serological studies of visceral leishmaniasis it was diagnosed in five asymptomatic dogs in the municipalities of Santa Maria, Julio de Castilhos and Itaara, however not confirmed by molecular analysis. In the municipalities of Cruz Alta and Uruguaiana cases of L. chagasi have been reported in dogs which previously resided in Leishmania sp. endemic areas. The municipality of Sao Borja had the first record of L. longipalpis in the RS during the leishmaniasis outbreak in 2008-2009. In the central region of the RS vector has not been found, but because in this first autochthonous case dog in Santa Maria believe that the parasite is present and/or doing other insect transmission of leishmaniasis. Clinical signs associated with hematologic and coagulation disorders observed in the canine are commonly described in symptomatic dogs in endemic regions. This case of autochthonous leishmaniasis reinforces the idea of the vector presence in Santa Maria, center of the RS. We believe that canine leishmaniasis is an emerging disease in the southern region of Brazil.

Evaluation of renal and hepatic functions in dogs naturally infected by visceral leishmaniasis submitted to treatment with meglumine antimoniate

The present study aimed to evaluate the renal and hepatic responses in eight dogs with visceral leishmaniasis submitted to treatment with meglumine antimoniate and to verify the occurrence of possible side effects. Urinalysis, hepatic and renal function tests were carried out in all animals at up to seven moments. After the end of a six-month observation period, all dogs were euthanized. Before the beginning of the experiment urinary and biochemical alterations were observed in four dogs due to the changes caused by the parasite itself. These alterations included the presence of renal cells, cylindruria, proteinuria, azotemia, hyperproteinemia, and hypoalbuminemia. One dog died on the third day after treatment because an aggravation of the clinical picture, probably due to the medication. During the course of the study, an increase in hepatic enzymes was verified in two animals. Sixty days after the beginning of the treatment four dogs showed remission of clinical signs. The other three were asymptomatic with persistent biochemical alterations. From these, two presented recurrence of clinical signs about 150 days after the beginning of the treatment while in the other, hyperproteinemia persisted. Meglumine antimoniate was not efficient to treat dogs with severe renal dysfunction and the side effects observed were pain at the site of injection and the probable transient hepatotoxicity, evidenced by biochemical examinations, but without the presence of clinical signs. (c) 2006 Elsevier Ltd. All rights reserved.

Elimination and replacement of canine reservoir in an area endemic for visceral leishmaniasis

This work reviews studies about dog culling in Brazilian endemic areas for visceral leishmaniasis, as well as studies on dog replacement after euthanasia in areas where it is indicated as a control measure for this zoonosis.

Anti-leishmania antibodies in cerebrospinal fluid from dogs with visceral leishmaniasis

Visceral leishmaniasis in Brazil is caused by Leishmania (Leishmania) chagasi and the dog is its most important reservoir. The clinical features in dogs include loss of weight, lymphadenopathy, renal failure, skin lesions, fever, hypergammaglobulinemia, hepatosplenomegaly, anemia, and, rarely, neurological symptoms. Most infected animals develop active disease, characterized by high anti-leishmania antibody titers and depressed lymphoproliferative ability. Antibody production is not primarily important for protection but might be involved in the pathogenesis of tissue lesions. An ELISA test was used to determine if there is an association between neurological symptoms and the presence of anti-L. chagasi antibodies in cerebrospinal fluid (CSF). Thirty serum and CSF samples from symptomatic mixed breed dogs (three with neurological symptoms) from a region of high incidence of visceral leishmaniasis in Brazil were examined for antibody using total parasite antigen and anti-dog IgG peroxidase conjugate. A high level of L. chagasi antibodies was observed in sera (mean absorbance ± SD, 1.939 ± 0.405; negative control, N = 20, 0.154 ± 0.074) and CSF (1.571 ± 0.532; negative control, N = 10, 0.0195 ± 0.040) from all animals studied. This observation suggests that L. chagasi can cause breakdown of filtration barriers and the transfer of antibodies and antigens from the blood to the CSF compartment. No correlation was observed between antibody titer in CSF and neurological symptoms.

Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune((R)) vaccine

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune (R) vaccine, formulated with an increased adjuvant concentration (1 mg of saponin rather than 0.5 mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune (R) vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune (R)-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p < 0.0001), a higher and stable IgG2 and a decreasing IgG I response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune (R) immumotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p < 0.0001), the parasitological evidence (p = 0.038) and a decrease in Leishinania-specific CD4+ lymphocyte proportions (p = 0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune (R) vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune (R) vaccine was subjected to a safety analysis and found to be well tolerated and safe. (c) 2007 Elsevier Ltd. All rights reserved.

Pro-inflammatory cytokines predominate in the brains of dogs with visceral leishmaniasis: A natural model of neuroinflammation during systemic parasitic infection

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. To investigate the pathogenesis of brain alterations occurring during visceral leishmaniasis infection, the expression of the cytokines IL-1β, IL-6, IL-10, IL-12p40, IFN-γ, TGF-β and TNF-α and their correlations with peripheral parasite load were evaluated in the brains of dogs naturally infected with Leishmania infantum. IL-1β, IFN-γ and TNF-α were noticeably up-regulated, and IL-10, TGF-β and IL-12p40 were down-regulated in the brains of infected dogs. Expression levels did not correlate with parasite load suggestive that the brain alterations are due to the host's immune response regardless of the phase of the disease. These data indicate the presence of a pro-inflammatory status in the nervous milieu of dogs with visceral leishmaniasis especially because IL-1β and TNF-α are considered key factors for the initiation, maintenance and persistence of inflammation. © 2012 Elsevier B.V.