Investigação sobre a soroprevalência, leptospiremia e leptospirúria em cães contactantes de pessoas acometidas por leptospirose

Orientadora: Professora Dra. Simone Tostes de Oliveira StedileCoorientador: Professor Dr. Rafael Felipe da Costa VieiraDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa : Curitiba, 22/03/2017Inclui referênciasResumo: A presente dissertação apresenta uma pesquisa sobre a eliminação urinária de leptospiras por cães que tiveram contato com pessoas que foram acometidas por leptospirose. Sabe-se que cães podem atuar como reservatório para esta importante zoonose disseminada pelo mundo, porém considera-se que seu papel como portadores assintomáticos seja subestimado. Desta forma, o objetivo deste estudo foi investigar uma possível relação entre a presença de seres humanos com leptospirose e de cães portadores assintomáticos que possam servir como fonte de infecção. Amostras de sangue e urina foram coletadas de cães contactantes de pessoas com leptospirose para realização de exames de PCR e sorologia. Nenhum resultado foi positivo nos exames realizados, o que leva a crer que os cães testados não serviram, naquele momento, como fonte de infecção de leptospirose para seus proprietários. Palavras chave: PCR; portador assintomático; reservatório; saúde pública; zoonose.Abstract: This thesis presents a study about urinary shedding of leptospires by dogs who had contact with people affected by leptospirosis. Dogs may play like a reservoir to this important zoonosis widespread throughout the world, but is considered that their asymptomatic carrier role be underestimated. Therefore, the aim of this study was to investigate a possible relation between humans with leptospirosis and asymptomatic carrier dogs who can be an infection source. Blood and urine samples were collected from contactant dogs of people with leptospirosis to performing PCR and serology. None positive result was found, what suggests that the dogs, in that moment, didn't serve like source of infection of leptospirosis to their owners. Key words: asymptomatic; PCR; public health; reservoir; zoonosi

Comparison of Three Protocols to Preserve Leptospira spp. in Cat Urine for Efficient DNA Extraction and PCR Amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recent studies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine may inhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processing to optimize the detection of pathogenic leptospires in cat urine.Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested. Aliquots of standard concentration of L interrogans serovar Canicola culture were added to urine samples to achieve concentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by the addition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS was added to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B, PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24 and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Samples were then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets were discarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and the pellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20 min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºC for 10 min. DNA was stored at -20ºC until the molecular analysis. The PCR detection limit was evaluated. In samples from protocol A, leptospires were detected in concentrations up to 1×103 (4 h) and 1×104 (24 and 48 h). In protocol C, leptospires were detected in concentrations up to 1×104 (4 h) and 1×105 (24 and 48 h). No leptospiral DNA was detected in samples from protocol B.Discussion:   Leptospires are sensitive to acid conditions, at pH 6.8 or lower and the urine pH of cats may vary from 5 to 7. In the present study, we found best results for DNA amplification with the addition of PBS immediately after urine collection (protocol A). Previous studies have shown the importance of neutralizing urine samples immediately after collection to avoid loss of bacterial DNA during the extraction process. However, protocols B and C may be an alternative in clinical practice, when PBS cannot be added immediately after collection. The delay after urine collection before DNA extraction is one more factor that may interfere with the PCR sensitivity. This was observed in the samples from protocol A, because although these samples were neutralized immediately, there was a 10-fold decrease in the detection limit of the test at 24 and 48 h. Leptospires rapidly lose their integrity in urine and the detection limit declines considerably over time, so prompt extraction is essential. These results show that the in-house method of preserving cat urine is useful to maintain the viability of leptospiral DNA extraction. In addition, this study highlights the importance of neutralizing urine samples immediately after collection and the need for prompt DNA extraction to improve PCR detection limit. However, if PBS cannot be added to the collected sample immediately, it is better to process the sample without PBS and extract DNA as soon as possible to minimize the risk of false-negative results

INVESTIGAÇÃO DO CONHECIMENTO DE PESSOAS INFECTADAS POR Leptospira spp. E DA SOROPREVALÊNCIA, LEPTOSPIRÚRIA E LEPTOSPIREMIA DE SEUS CÃES CONTACTANTES

A leptospirose é uma zoonose, endêmica no Brasil, que afeta diversos mamíferos, inclusive o homem. Os cães portadores assintomáticos podem transmiti-la e é provável que o número de animais nesta condição seja subestimado. Apesar de a maioria dos estudos ser baseada em resultados sorológicos, este teste não prediz se o animal apresenta leptospirúria. Para a detecção de leptospiras na urina é indicado o diagnóstico molecular por meio da reação em cadeia da polimerase (PCR). Devido à escassez de dados para avaliar o quão presentes estes cães reservatórios são e o quanto estão envolvidos na transmissão para seres humanos, este estudo teve como objetivo investigar o estado de portador renal para Leptospira spp. em cães contactantes de pessoas acometidas por leptospirose no município de Curitiba/Paraná. Sangue e urina foram coletados de 15 cães, pertencentes a oito tutores com histórico recente de leptospirose para realização de sorologia (soro-aglutinação microscópica) e PCR destas amostras. Todos os testes sorológicos dos cães foram negativos, bem como os resultados das PCR do sangue e da urina. Este resultado sugere que os cães submetidos à pesquisa não serviram como fonte de infecção, não havendo, portanto, relação entre portadores assintomáticos e tutores infectados neste estudo

Investigação sobre a soroprevalência, leptospiremia e leptospirúria em cães contactantes de pessoas acometidas por leptospirose

Orientadora: Professora Dra. Simone Tostes de Oliveira StedileCoorientador: Professor Dr. Rafael Felipe da Costa VieiraDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciências Veterinárias. Defesa : Curitiba, 22/03/2017Inclui referênciasResumo: A presente dissertação apresenta uma pesquisa sobre a eliminação urinária de leptospiras por cães que tiveram contato com pessoas que foram acometidas por leptospirose. Sabe-se que cães podem atuar como reservatório para esta importante zoonose disseminada pelo mundo, porém considera-se que seu papel como portadores assintomáticos seja subestimado. Desta forma, o objetivo deste estudo foi investigar uma possível relação entre a presença de seres humanos com leptospirose e de cães portadores assintomáticos que possam servir como fonte de infecção. Amostras de sangue e urina foram coletadas de cães contactantes de pessoas com leptospirose para realização de exames de PCR e sorologia. Nenhum resultado foi positivo nos exames realizados, o que leva a crer que os cães testados não serviram, naquele momento, como fonte de infecção de leptospirose para seus proprietários. Palavras chave: PCR; portador assintomático; reservatório; saúde pública; zoonose.Abstract: This thesis presents a study about urinary shedding of leptospires by dogs who had contact with people affected by leptospirosis. Dogs may play like a reservoir to this important zoonosis widespread throughout the world, but is considered that their asymptomatic carrier role be underestimated. Therefore, the aim of this study was to investigate a possible relation between humans with leptospirosis and asymptomatic carrier dogs who can be an infection source. Blood and urine samples were collected from contactant dogs of people with leptospirosis to performing PCR and serology. None positive result was found, what suggests that the dogs, in that moment, didn't serve like source of infection of leptospirosis to their owners. Key words: asymptomatic; PCR; public health; reservoir; zoonosi