RESUMEN

Canine visceral leishmaniasis (CVL) diagnosis is still a challenge in endemic areas with limited diagnostic resources. This study proposes a score with the potential to distinguish positive CVL cases from negative ones. We studied 265 dogs that tested positive for CVL on ELISA and parasitological tests. A score ranging between 0 and 19 was recorded on the basis of clinical signs. Dogs with CVL had an overall higher positivity of the majority of clinical signs than did dogs without CVL or with ehrlichiosis. Clinical signs such as enlarged lymph nodes (83.93%), muzzle/ear lesions (55.36%), nutritional status (51.79%), bristle condition (57.14%), pale mucosal colour (48.21%), onychogryphosis (58.93%), skin lesion (39.28%), bleeding (12.50%), muzzle depigmentation (41.07%), alopecia (39.29%), blepharitis (21.43%), and keratoconjunctivitis (42.86%) were more frequent in dogs with CVL than in dogs with ehrlichiosis or without CVL. Moreover, the clinical score increased according to the positivity of all diagnostic tests (ELISA, p < 0.001; parasite culture, p = 0.0021; and smear, p = 0.0003). Onychogryphosis (long nails) [odds ratio (OR): 3.529; 95% confidence interval (CI): 1.832-6.796; p < 0.001], muzzle depigmentation (OR: 4.651; 95% CI: 2.218-9.750; p < 0.001), and keratoconjunctivitis (OR: 5.400; 95% CI: 2.549-11.441; p < 0.001) were highly associated with CVL. Interestingly, a score cut-off value ≥ 6 had an area under the curve of 0.717 (p < 0.0001), sensitivity of 60.71%, and specificity of 73.64% for CVL diagnosis. The clinical sign-based score for CVL diagnosis suggested herein can help veterinarians reliably identify dogs with CVL in endemic areas with limited diagnostic resources.

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RESUMEN

Canine visceral leishmaniasis (CVL) diagnosis is still a challenge in endemic areas with limited diagnostic resources. This study proposes a score with the potential to distinguish positive CVL cases from negative ones. We studied 265 dogs that tested positive for CVL on ELISA and parasitological tests. A score ranging between 0 and 19 was recorded on the basis of clinical signs. Dogs with CVL had an overall higher positivity of the majority of clinical signs than did dogs without CVL or with ehrlichiosis. Clinical signs such as enlarged lymph nodes (83.93%), muzzle/ear lesions (55.36%), nutritional status (51.79%), bristle condition (57.14%), pale mucosal colour (48.21%), onychogryphosis (58.93%), skin lesion (39.28%), bleeding (12.50%), muzzle depigmentation (41.07%), alopecia (39.29%), blepharitis (21.43%), and keratoconjunctivitis (42.86%) were more frequent in dogs with CVL than in dogs with ehrlichiosis or without CVL. Moreover, the clinical score increased according to the positivity of all diagnostic tests (ELISA, p < 0.001; parasite culture, p = 0.0021; and smear, p = 0.0003). Onychogryphosis (long nails) [odds ratio (OR): 3.529; 95% confidence interval (CI): 1.832-6.796; p < 0.001], muzzle depigmentation (OR: 4.651; 95% CI: 2.218-9.750; p < 0.001), and keratoconjunctivitis (OR: 5.400; 95% CI: 2.549-11.441; p < 0.001) were highly associated with CVL. Interestingly, a score cut-off value ≥ 6 had an area under the curve of 0.717 (p < 0.0001), sensitivity of 60.71%, and specificity of 73.64% for CVL diagnosis. The clinical sign-based score for CVL diagnosis suggested herein can help veterinarians reliably identify dogs with CVL in endemic areas with limited diagnostic resources.

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The aim of this study was to identify and classify the pulmonary alterations in dogs naturally infected by Leishmania (Leishmania) infantum according to clinical manifestation. After diagnosis to VL by parasitological exam, 24 infected dogs were subdivided in two groups: 10 dogs with until four clinical signs; and 14 dogs with five or more clinical signs. As control were evaluated 5 healthy dogs. Pulmonary tissue samples were stained by hematoxylin-eosin (H-E) and Masson's Trichrome to histopathological examination, and Grocotts methenamine silver to exclude fungal infections, as well immunoperoxidase to detection of Leishmania. Histopathological examination of the lungs showed a focal thickening of the alveolar septa due to congestion, edema and cellular infiltrate. Moreover, presence of fibrosis, bronchiolar exudate and flaking of the bronchiolar epithelium, presence of hemosiderin and hyperplasia of goblet cell were observed. The pattern fibrotic-cellular lesions predominated in most infected dogs but mainly in infected dogs with five or more signs clinical. However, fibrosis was higher in infected dogs with four signs clinical. Immunoperoxidase staining showed amastigotes and/or antigen of Leishmania in all infected dogs but there was an increase significant in infected dogs with five or more signs clinical. Semi-quantitative analysis of intensities antigen reveled a positive correlation between fibrosis in infected dogs. In conclusion, the presence of the parasite and/or antigens modifies the lung architecture due for fibrotic-cellular pattern in infected dogs with severe manifestation clinical.(AU)

O objetivo desse estudo foi identificar e classificar as alterações pulmonares de cães naturalmente infectados por Leishmania (Leishmania) infantum conforme as manifestações clínicas. Após o diagnóstico para LV pelo exame parasitológico, 24 cães infectados foram subdivididos em dois grupos: 10 cães com até quatro sinais clínicos; e 14 cães com cinco ou mais sinais clínicos. Como controle foram avaliados 5 cães sadios. Amostras de tecido pulmonar foram coradas com hematoxilina-eosina (H-E) e Tricrômico de Masson para avaliação histopatológica, e prata-metenamina de Grocott para excluir infecções fúngicas, bem como imunoperoxidase para detecção de Leishmania. A análise microscópica do pulmão revelou espessamento focal do septo interalveolar devido à congestão, edema e infiltrado celular. Além disso, presença de fibrose, exsudato bronquiolar e ainda descamação do epitélio bronquiolar com perda de cílios, presença de hemossiderina e hiperplasia de células caliciformes. O padrão de lesão fibrótico-celular predominou nos cães infectados com cinco ou mais sinais clínicos. Em contrapartida, fibrose estava presente em intensidade maior no grupo nos cães infectados com até quatro sinais clínicos. A análise imunoistoquímica mostrou maior intensidade de antígeno e amastigotas de Leishmania nos cães infectados com cinco ou mais sinais clínicos. A análise semi-quantitativa do antígeno mostrou correlação positiva de acordo com a intensidade da fibrose. Concluiu-se que a presença do parasita e seus antígenos modificam a arquitetura pulmonar evoluindo, em sua maioria, para o padrão fibrótico-celular em associação à intensidade das manifestações clínicas.(AU)

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RESUMEN

O objetivo deste estudo foi identificar e classificar as alterações pulmonares de cães naturalmente infectados por Leishmania (Leishmania) infantum conforme as manifestações clínicas. Após o diagnóstico para LV pelo exame parasitológico, 24 cães infectados foram subdivididos em dois grupos: 10 cães com até quatro sinais clínicos; e 14 cães com cinco ou mais sinais clínicos. Como controle foram avaliados cinco cães sadios. Amostras de tecido pulmonar foram coradas com hematoxilina-eosina (H-E) e Tricrômico de Masson para avaliação histopatológica, e prata-metenamina de Grocott para excluir infecções fúngicas, bem como imunoperoxidase para detecção de Leishmania. A análise microscópica do pulmão revelou espessamento focal do septo interalveolar devido à congestão, edema e infiltrado celular. Além disso, presença de fibrose, exsudato bronquiolar e ainda descamação do epitélio bronquiolar com perda de cílios, presença de hemossiderina e hiperplasia de células caliciformes. O padrão de lesão fibrótico-celular predominou nos cães infectados com cinco ou mais sinais clínicos. Em contrapartida, fibrose estava presente em intensidade maior no grupo nos cães infectados com até quatro sinais clínicos. A análise imunoistoquímica mostrou maior intensidade de antígeno e amastigotas de Leishmania nos cães infectados com cinco ou mais sinais clínicos. A análise semi-quantitativa do antígeno mostrou correlação positiva de acordo com a intensidade da fibrose. Concluiu-se que a presença do parasita e seus antígenos modificam a arquitetura pulmonar evoluindo, em sua maioria, para o padrão fibrótico-celular em associação à intensidade das manifestações clínicas.

The aim of this study was to identify and classify the pulmonary alterations in dogs naturally infected by Leishmania (Leishmania) infantum according to clinical manifestation. After diagnosis to VL by parasitological exam, 24 infected dogs were subdivided in two groups: 10 dogs with until four clinical signs; and 14 dogs with five or more clinical signs. As control were evaluated 5 healthy dogs. Pulmonary tissue samples were stained by hematoxylin-eosin (H-E) and Masson´s Trichrome to histopathological examination, and Grocott's methenamine silver to exclude fungal infections, as well immunoperoxidase to detection of Leishmania. Histopathological examination of the lungs showed a focal thickening of the alveolar septa due to congestion, edema and cellular infiltrate. Moreover, presence of fibrosis, bronchiolar exudate and flaking of the bronchiolar epithelium, presence of hemosiderin and hyperplasia of goblet cell were observed. The pattern fibrotic-cellular lesions predominated in most infected dogs but mainly in infected dogs with five or more signs clinical. However, fibrosis was higher in infected dogs with four signs clinical. Immunoperoxidase staining showed amastigotes and/or antigen of Leishmania in all infected dogs but there was an increase significant in infected dogs with five or more signs clinical. Semi-quantitative analysis of intensities antigen reveled a positive correlation between fibrosis in infected dogs. In conclusion, the presence of the parasite and/or antigens modifies the lung architecture due for fibrotic-cellular pattern in infected dogs with severe manifestation clinical.

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INTRODUÇÃO: A Leishmaniose Visceral (LV) é uma zoonose causada pelo protozoário Leishmania infantum, e tem o cão como reservatório doméstico. Nessa espécie, a leishmaniose visceral canina (LVC) é caracterizada por um amplo espectro de sinais clínicos, variando de acordo com a resposta imune do animal parasitado, o que dificulta o seu diagnóstico. Na busca de um tratamento eficaz, os cães são submetidos a vários agentes terapêuticos e protocolos. Embora alguns protocolos resultem em melhora dos sinais clínicos, as recidivas são frequentes. Além disso, a detecção do parasito na pele dos cães tratados faz com que sua participação no ciclo de transmissão continue, inclusive com risco de infecção de humanos e seleção de cepas resistentes às drogas de referência. Um dos fármacos que já demonstraram ação contra a Leismania é o Dietilditiocarbamato (DETC), um potente inibidor de Superóxido Dismutase (SOD), enzima que atua facilitando a sobrevivência de parasitos intracelulares. OBJETIVO: Os objetivos deste estudo foram testar o DETC, in vitro e in vivo, para o tratamento contra LVC e desenvolver um algoritmo que auxilie no diagnóstico clínico da doença. MÉTODOS: Nos ensaios in vitro foram utilizadas células mononucleares de sangue periférico de cães saudáveis. Nos ensaios in vivo foram utilizados 10 cães experimentalmente infectados e 16 naturalmente infectados com Leishmania infantum...

INTRODUCTION: Visceral Leishmaniasis (VL) is a zoonosis caused by Leishmania infantum, and has the dog as domestic reservoir. In this species, canine visceral leishmaniasis (CVL) is characterized by a broad spectrum of clinical signs, varying with the immune response of parasitized animals, which complicates the diagnosis. In the search for an effective treatment, the dogs are subjected to various therapeutic agents and protocols. Although some protocols result in improvement of clinical signs, recurrences are frequent. Furthermore, the detection of the parasite in the skin of treated dogs makes participation in the transmission cycle to continue, including the risk of human infection and selection of resistant strains to the reference drugs. One of drugs that have shown activity against Leishmania is diethyldithiocarbamate (DETC), a potent inhibitor of superoxide dismutase (SOD), an enzyme that acts facilitating survival of intracellular parasites. AIM: This study aimed to test the DETC, in vitro and in vivo for the treatment against LVC and develop an algorithm to assist in clinical diagnosis of disease. METHODS: In vitro tests Mononuclear cells from peripheral blood of healthy dogs were used. To in vivo assays were used 10 experimentally infected dogs and 16 naturally infected with Leishmania infantum...

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An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

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An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

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