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ABSTRACT: Leishmaniosis is a great public health problem affecting both humans and animals. The disease is caused by the protozoan Leishmania spp., which has a complex cycle involving a phlebotomine vector. The ELISA test (Enzyme-Linked Immunosorbent Assay) along with a chromatographic immunoassay was defined by the Brazil Health Ministry as the confirmatory screening protocol in 2011. Uruguaiana city is 630 km away from Porto Alegre, which makes it difficult to send samples and diagnose leishmaniasis, as well as receive quick results. In view of this, the present study evaluated an in-house indirect ELISA method compared to indirect immunofluorescence assay (IFA) and dual-path platform chromatographic immunoassay (DPP-BioManguinhos®) for the detection of an immune response to Leishmania spp. in canine species. The serological evaluation included 48 canines from the western border of Brazil (Uruguaiana and Barra do Quaraí city). Among the 48 canine samples tested, 18 were positive when using the ELISA technique, 19 were positive with IFA, and 17 were positive with rapid test DPP®. The ELISA technique showed a sensitivity/specificity of 83.3%/86.7% when compared to IFA and 100%/96.8% compared to DPP®. The present study showed a prevalence of 37.5%, demonstrating that the infection circulates in the studied population. It can be concluded that the ELISA technique was valuable for use in field conditions when performing screening tests in endemic areas.

RESUMO: A leishmaniose é um grande problema de saúde pública que afeta tanto humanos quanto animais. A doença é causada pelo protozoário Leishmania spp., que possui um ciclo complexo envolvendo um vetor flebotomíneo. O teste ELISA (Enzyme-Linked Immunosorbent Assay), juntamente com um imunoensaio cromatográfico, foi definido pelo Ministério da Saúde do Brasil como protocolo de triagem confirmatória em 2011. A cidade de Uruguaiana fica a 630 km de Porto Alegre, o que dificulta o envio de amostras e o diagnóstico da leishmaniose, além da demora para obter os resultados. Diante disso, o presente estudo teve como objetivo avaliar um método de ELISA indireto in-house comparado ao ensaio de imunofluorescência indireta (IFA) e imunoensaio cromatográfico de plataforma de dupla via (DPP-BioManguinhos®) para a detecção de uma resposta imune contra Leishmania spp. na espécie canina. A avaliação sorológica incluiu 48 caninos da fronteira oeste do Brasil (cidade de Uruguaiana e Barra do Quaraí). Das 48 amostras caninas testadas, 18 foram positivas na técnica de ELISA, 19 foram positivas com IFA e 17 foram positivas com o teste rápido DPP®. A técnica de ELISA apresentou sensibilidade/especificidade de 83,3%/86,7% quando comparada ao IFA e 100%/96,8% em relação ao DPP®. O presente estudo apresentou prevalência de 37,5%, demonstrando que a infecção circula na população estudada. Pode-se concluir que a técnica de ELISA foi valiosa para uso em condições de campo na realização de testes de triagem em áreas endêmicas.

RESUMO

Leishmaniosis is a great public health problem affecting both humans and animals. The disease is caused by the protozoan Leishmania spp., which has a complex cycle involving a phlebotomine vector. The ELISA test (Enzyme-Linked Immunosorbent Assay) along with a chromatographic immunoassay was defined by the Brazil Health Ministry as the confirmatory screening protocol in 2011. Uruguaiana city is 630 km away from Porto Alegre, which makes it difficult to send samples and diagnose leishmaniasis, as well as receive quick results. In view of this, the present study evaluated an in-house indirect ELISA method compared to indirect immunofluorescence assay (IFA) and dual-path platform chromatographic immunoassay (DPP-BioManguinhos®) for the detection of an immune response to Leishmania spp. in canine species. The serological evaluation included 48 canines from the western border of Brazil (Uruguaiana and Barra do Quaraí city). Among the 48 canine samples tested, 18 were positive when using the ELISA technique, 19 were positive with IFA, and 17 were positive with rapid test DPP®. The ELISA technique showed a sensitivity/specificity of 83.3%/86.7% when compared to IFA and 100%/96.8% compared to DPP®. The present study showed a prevalence of 37.5%, demonstrating that the infection circulates in the studied population. It can be concluded that the ELISA technique was valuable for use in field conditions when performing screening tests in endemic areas.

A leishmaniose é um grande problema de saúde pública que afeta tanto humanos quanto animais. A doença é causada pelo protozoário Leishmania spp., que possui um ciclo complexo envolvendo um vetor flebotomíneo. O teste ELISA (Enzyme-Linked Immunosorbent Assay), juntamente com um imunoensaio cromatográfico, foi definido pelo Ministério da Saúde do Brasil como protocolo de triagem confirmatória em 2011. A cidade de Uruguaiana fica a 630 km de Porto Alegre, o que dificulta o envio de amostras e o diagnóstico da leishmaniose, além da demora para obter os resultados. Diante disso, o presente estudo teve como objetivo avaliar um método de ELISA indireto in-house comparado ao ensaio de imunofluorescência indireta (IFA) e imunoensaio cromatográfico de plataforma de dupla via (DPP-BioManguinhos®) para a detecção de uma resposta imune contra Leishmania spp. na espécie canina. A avaliação sorológica incluiu 48 caninos da fronteira oeste do Brasil (cidade de Uruguaiana e Barra do Quaraí). Das 48 amostras caninas testadas, 18 foram positivas na técnica de ELISA, 19 foram positivas com IFA e 17 foram positivas com o teste rápido DPP®. A técnica de ELISA apresentou sensibilidade/especificidade de 83,3%/86,7% quando comparada ao IFA e 100%/96,8% em relação ao DPP®. O presente estudo apresentou prevalência de 37,5%, demonstrando que a infecção circula na população estudada. Pode-se concluir que a técnica de ELISA foi valiosa para uso em condições de campo na realização de testes de triagem em áreas endêmicas.

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The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.

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The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.(AU)

O objetivo deste trabalho foi caracterizar espécies de Leishmania em amostras de caninos e felinos, utilizando-se a reação em cadeia da polimerase (PCR)- polimorfismo de comprimento de fragmento de restrição (RFLP). O estudo foi realizado na região de fronteira no sul do Brasil, divisa com Argentina e Uruguai. Amostras foram coletadas de 116 cães (Canis lupus familiaris) e 89 gatos (Felis catus). A PCR foi realizada com o gene alvo do fragmento LT1 do DNA do cinetoplasto (kDNA) para triagem e, as amostras positivas foram submetidas a uma segunda PCR com alvo ITS1 no DNA ribossomal (rDNA). O RFLP foi realizado com a endonuclease de restrição Haemophilus aegyptius (HAE III) (Fermentas ®). As sequências positivas no PCR-ITS1 foram depositadas no NCBI GenBank, além disso, a análise filogenética foi realizada. Foram detectadas 12,9% (15/116) amostras positivas em cães. Das 89 amostras de gatos todas foram negativas. Cepas de referência de Leishmania, com padrões diferentes na PCR-RFLP, e amostras positivas apresentaram similaridade das bandas com as espécies padrão de Leishmania infantum, o que foi comprovado no sequenciamento e análise filogenética. A técnica de RFLP, sozinha, demonstrou viabilidade para a aplicação prática e a confirmação das espécies de Leishmania spp. envolvidas.(AU)

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BACKGROUND: The Leishmaniases are on the top of the global list of tropical neglected diseases. The number of infected dogs in South America is estimated in millions and correlated to disease cases in humans, especially in Brazil. Equines may get infected too and can play a role in the epidemiological chain. Thus, the aim of the present study was to evaluate risk and protective factors of leishmaniasis in rural areas of the western border region of Rio Grande do Sul state, Brazil by Leishmania spp. protozoa molecular detection and serological evaluation (ELISA) in equine and canine blood samples. This work included nine farms around the city of Uruguaiana. Epidemiologic information regarding farm characteristics and biologic material collection of canine (22) and equine (91), totalizing 113 samples was collected. The polymerase chain reaction (PCR) technique was used to detect Leishmania spp. in biological samples. Variables related to the farm were collected and evaluated through descriptive analysis followed by chi-square test and a logistic regression analysis. RESULTS: Nineteen positive samples (19/113 - 16,81%) were detected, being 18 equines and 1 canine, in six of the nine farms included in the study. No animal showed clinical signs of the disease. According to the variables analyzed, when compared each characteristic separately, the presence of abundant vegetation and poor hygiene demonstrated to be risk factors to Leishmania infection in rural areas. The logistic regression showed that excellent general hygiene, proximity to the weir and trimmed grass were protective factors (p=0.038, p=0.001 and p=0.014, respectively). Having excellent hygiene represents a 70% lower chance of getting infected, keeping the grass cut protects the animal by more than 90% and the proximity of the weir represents a protective factor of 96%. CONCLUSIONS: The presence of Leishmania infection in the western border region of Rio Grande do Sul was 16,81% and it was influenced by farm characteristics. The role of the excellent general hygiene as a protective factor is extremely relevant in the leishmaniases prevention.

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The physiological measurements of collateral ligaments of distal interphalangeal joint (CL-DIPJ) differ in the literature. The factors that influence these differences are not well described. The aims of this study are to compare CL-DIPJ sizes in equines with different withers height, as well as to correlate body weight and hoof size to the size of these ligaments. In total, 52 horses were used in the study. They were divided into two groups according to wither height: Group 1 (G1) - 21 animals with up to 147 cm - and group 2 (G2) - 21 animals with greater than 148 cm. CL-DIPJ was ultrasonographically measured in order to find the mean of dorso-palmar (DPD) and latero-medial (LMD) diameters and the cross-sectional area (CSA). Hoof width and length were measured, and the results were used to calculate the hoof surface area. Withers height and body weight were also measured. Groups were statistically compared by Student's t test and Pearson's correlation application to each group. Groups were different in body size, HS and CL-DIPJ size when p < .05, except for the LMD of the medial collateral ligament of left thoracic limb. Withers height and body weight did not show significant correlations to CL-DIPJ size in G1, but they had little influence on the size of CL-DIPJ on G2. No correlation between the hoof size and the CL-DIPJ was noted in any of the two groups. In conclusion, the CL-DIPJ were larger in taller horses and their sizes were correlated to their height and weight, but they were not correlated to hoof size.

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Com o propósito de estabelecer valores de hematócrito, proteínas plasmáticas totais, fibrinogênio, creatina quinase , aspartato transferase e lactato em potros da raça Crioula, do nascimento até os dois anos, utilizaram-se amostras sanguíneas de 85 animais, divididos pela estratificação etária: Grupo 1 (G1) Até 15 dias de vida (n=70); grupo 2 (G2), entre 16 dias até um mês (n=67); grupo 3 (G3), entre 1 e 3 meses (n=75); grupo 4 (G4), entre 3 e 6 meses (n=64); grupo 5 (G5), entre 6 e 9 meses (n=59); grupo 6 (G6), entre 9 e 18 meses (n=39); e grupo 7 (G7), entre 18 meses até 2 anos (n=17). Foi realizado estudo estatístico entre os grupos pela análise de variância unidirecional (one-wayANOVA), complementada pelo teste de Tukey. Para comparação das médias entre os sexos utilizou-se o teste t de Student. O hematócrito foi significativamente mais elevado até os 90 dias e nas fêmeas do G7. Para proteínas plasmáticas totais, notou-se aumento significativo nos grupos 3, 4, 6 e 7. Os valores de fibrinogênio foram maiores no G1. A CK apresentou maior concentração no G5 e a AST no G7. A AST assumiu valores semelhantes dos 30 dias até os 2 anos. A concentração de lactato foi mais elevada no G3. Conclui-se que na interpretação dos exames laboratoriais de potros da raça crioula, o gênero não interfere significativamente nos resultados, porém a idade deve ser considerada devido à ocorrência de variações relevantes. Recomenda-se que para interpretação sejam consultadas tabelas específicas para cada análise.

Plasma levels of hematocrit, total plasma protein, fibrinogen, creatine phosphokinase, aspartate transferase, and lactate were analyzed in blood samples of 85 Crioula breed foals, from birth to two years of age. The animals were divided into age groups: G1 (up to 15 days of age; n=70), G2 (from 16 days to one month of age; n=67), G3 (between one and three months of age; n=75), G4 (between three and six months of age; n=64), G5 (between six and nine months of age; n=59), G6 (between nine and 18 months of age; n=39), and G7 (between 18 months and two years of age; n=17). These groups were statistically analyzed by one-way variance analysis (ANOVA) and Tukey’s test. Male and female means were compared by Student’s t-test. Hematocrit levels were significantly higher up to 90 days of age and in G7 females. Total plasma proteins increased significantly in groups 3, 4, 6, and 7. The highest fibrinogen levels were found in G1. Yet for creatine phosphokinase, the highest concentrations were detected in G5, whereas those of aspartate aminotransferase in G7. The levels of this enzyme remained similar from 30 days to two years of age. Lactate concentrations were higher in G3. We concluded that the sex of the animal had no significant effect on laboratory test interpretations. By contrast, the age of the animal should be considered since relevant variations were observed with time. Nevertheless, specific tables for each analysis should be consulted for interpretation of results.

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Plasma levels of hematocrit, total plasma protein, fibrinogen, creatine phosphokinase, aspartate transferase, and lactate were analyzed in blood samples of 85 Crioula breed foals, from birth to two years of age. The animals were divided into age groups: G1 (up to 15 days of age; n=70), G2 (from 16 days to one month of age; n=67), G3 (between one and three months of age; n=75), G4 (between three and six months of age; n=64), G5 (between six and nine months of age; n=59), G6 (between nine and 18 months of age; n=39), and G7 (between 18 months and two years of age; n=17). These groups were statistically analyzed by one-way variance analysis (ANOVA) and Tukey's test. Male and female means were compared by Student's t-test. Hematocrit levels were significantly higher up to 90 days of age and in G7 females. Total plasma proteins increased significantly in groups 3, 4, 6, and 7. The highest fibrinogen levels were found in G1. Yet for creatine phosphokinase, the highest concentrations were detected in G5, whereas those of aspartate aminotransferase in G7. The levels of this enzyme remained similar from 30 days to two years of age. Lactate concentrations were higher in G3. We concluded that the sex of the animal had no significant effect on laboratory test interpretations. By contrast, the age of the animal should be considered since relevant variations were observed with time. Nevertheless, specific tables for each analysis should be consulted for interpretation of results.

Com o propósito de estabelecer valores de hematócrito, proteínas plasmáticas totais, fibrinogênio, creatina quinase , aspartato transferase e lactato em potros da raça Crioula, do nascimento até os dois anos, utilizaram-se amostras sanguíneas de 85 animais, divididos pela estratificação etária: Grupo 1 (G1) Até 15 dias de vida (n=70); grupo 2 (G2), entre 16 dias até um mês (n=67); grupo 3 (G3), entre 1 e 3 meses (n=75); grupo 4 (G4), entre 3 e 6 meses (n=64); grupo 5 (G5), entre 6 e 9 meses (n=59); grupo 6 (G6), entre 9 e 18 meses (n=39); e grupo 7 (G7), entre 18 meses até 2 anos (n=17). Foi realizado estudo estatístico entre os grupos pela análise de variância unidirecional (one-wayANOVA), complementada pelo teste de Tukey. Para comparação das médias entre os sexos utilizou-se o teste t de Student. O hematócrito foi significativamente mais elevado até os 90 dias e nas fêmeas do G7. Para proteínas plasmáticas totais, notou-se aumento significativo nos grupos 3, 4, 6 e 7. Os valores de fibrinogênio foram maiores no G1. A CK apresentou maior concentração no G5 e a AST no G7. A AST assumiu valores semelhantes dos 30 dias até os 2 anos. A concentração de lactato foi mais elevada no G3. Conclui-se que na interpretação dos exames laboratoriais de potros da raça crioula, o gênero não interfere significativamente nos resultados, porém a idade deve ser considerada devido à ocorrência de variações relevantes. Recomenda-se que para interpretação sejam consultadas tabelas específicas para cada análise.

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Com o propósito de estabelecer valores de hematócrito, proteínas plasmáticas totais, fibrinogênio, creatina quinase , aspartato transferase e lactato em potros da raça Crioula, do nascimento até os dois anos, utilizaram-se amostras sanguíneas de 85 animais, divididos pela estratificação etária: Grupo 1 (G1) Até 15 dias de vida (n=70); grupo 2 (G2), entre 16 dias até um mês (n=67); grupo 3 (G3), entre 1 e 3 meses (n=75); grupo 4 (G4), entre 3 e 6 meses (n=64); grupo 5 (G5), entre 6 e 9 meses (n=59); grupo 6 (G6), entre 9 e 18 meses (n=39); e grupo 7 (G7), entre 18 meses até 2 anos (n=17). Foi realizado estudo estatístico entre os grupos pela análise de variância unidirecional (one-wayANOVA), complementada pelo teste de Tukey. Para comparação das médias entre os sexos utilizou-se o teste t de Student. O hematócrito foi significativamente mais elevado até os 90 dias e nas fêmeas do G7. Para proteínas plasmáticas totais, notou-se aumento significativo nos grupos 3, 4, 6 e 7. Os valores de fibrinogênio foram maiores no G1. A CK apresentou maior concentração no G5 e a AST no G7. A AST assumiu valores semelhantes dos 30 dias até os 2 anos. A concentração de lactato foi mais elevada no G3. Conclui-se que na interpretação dos exames laboratoriais de potros da raça crioula, o gênero não interfere significativamente nos resultados, porém a idade deve ser considerada devido à ocorrência de variações relevantes. Recomenda-se que para interpretação sejam consultadas tabelas específicas para cada análise.(AU)

Plasma levels of hematocrit, total plasma protein, fibrinogen, creatine phosphokinase, aspartate transferase, and lactate were analyzed in blood samples of 85 Crioula breed foals, from birth to two years of age. The animals were divided into age groups: G1 (up to 15 days of age; n=70), G2 (from 16 days to one month of age; n=67), G3 (between one and three months of age; n=75), G4 (between three and six months of age; n=64), G5 (between six and nine months of age; n=59), G6 (between nine and 18 months of age; n=39), and G7 (between 18 months and two years of age; n=17). These groups were statistically analyzed by one-way variance analysis (ANOVA) and Tukeys test. Male and female means were compared by Students t-test. Hematocrit levels were significantly higher up to 90 days of age and in G7 females. Total plasma proteins increased significantly in groups 3, 4, 6, and 7. The highest fibrinogen levels were found in G1. Yet for creatine phosphokinase, the highest concentrations were detected in G5, whereas those of aspartate aminotransferase in G7. The levels of this enzyme remained similar from 30 days to two years of age. Lactate concentrations were higher in G3. We concluded that the sex of the animal had no significant effect on laboratory test interpretations. By contrast, the age of the animal should be considered since relevant variations were observed with time. Nevertheless, specific tables for each analysis should be consulted for interpretation of results.(AU)

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Background: Dilated cardiomyopathy (DCM) is a multifactorial disease characterized by systolic dysfunction of myocardium, affecting domestic animals like dogs, cats, and ferrets. It was sporadically described in non-domestic species, generally as a necropsy observation. The hog-nosed skunk (Conepatus chinga) is a small mammal, carnivore, belonging to the Mephitidae family, found in southern South America, and considered as concerned in a conservation status of the species. The goal of this issue is report the first clinical approach of dilated cardiomyopathy in a young hog-nosed skunk, elucidating the challenging aspects of the diagnostic, therapy, and clinical outcome. Case: A newborn hog-nosed skunk (Conepatus chinga) was found in a farm of southern Brazil in poor nutritional and behavioral conditions. Thirty days of nutritional supplementation based on cow's milk, fruits and insects were necessary to recover its body weight score and activity level. However, 2 months after adoption, the skunk showed acute dyspnea and abnormal breath sound, decreased appetite, and loss of weight. Firstly, these signs were associated with a possible pulmonary infection or other respiratory disease. However, the general cardiomegaly and pulmonary edema pattern observed on the thoracic radiography, changed the clinical approach, conducting the presumptive diagnosis to congestive heart failure (pulmonary edema) caused by an unknown cardiac disease as a dilated cardiomyopathy (CDM). An adequate physical, echocardiographic, electrocardiographic, and hematological evaluation was possible only after chemical restraint by sevofluorane. Systolic heart murmur and pulmonary cracking sounds were detected on thoracic auscultation. These findings associated to the echocardiography features of four chamber dilatation, systolic (fractional shortening-FS = 9% and ejection fraction-EF= 22%) and diastolic (mitral E/A ratio = 4.93) dysfunctions, and bilateral ventriculoatrial regurgitation (mitral and tricuspid) were highly correlated with DCM. Considering the etiologies of CDM described in domestic species and the poor nutritional condition previously observed in the case, nutritional and idiopathic etiologies of DCM were considered for this case. Therapy was based on furosemide at hospital (4 mg/kg subcutaneous, single doses) and home (2 mg/kg orally, BID), enalapril maleate (0.5 mg/kg orally, every 48 h), taurine supplementation (100 mg orally, SID), and pimobendan (0.5 mg/kg orally, BID). Clinical improvement was already observed on the second day of treatment, and monitored for 5 months, when the skunk was completely revaluated. The second echocardiographic exam showed improvement in systolic (FS = 20% and EF = 43%) and diastolic functions (mitral E/A ratio = 2.05), tricuspid regurgitation was not observed, and decrease the left atrial and ventricular dimensions were seen. Due to good clinical outcome, furosemide was gradually reduced until complete withdrawal without any clinical complications or worsening. After 30 months of therapy of pimobendan, taurine, and enalapril maleate, the skunk has good quality captive life without congestive heart failure recurrence. Discussion: The lack of information about DCM in hog-nosed skunk turns the clinical diagnosis and therapy challenging. However, the radiographic and echocardiographic features seamed to lead the same domestic animal patterns. The therapy based on decrease the cardiac overload, increase the myocardial inotropic function (pimobendan and taurine) resulted on excellent clinical and echocardiographic outcome. Although the etiologies of DCM in this species are not stablished, the diagnostic and therapeutic approaches applied resulted on excellent clinical results, and therefore may provide useful information about this cardiac condition in skunk species.

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