Diagnostic accuracy of an in-house ELISA using the intermediate species Leptospira fainei as antigen for diagnosis of acute leptospirosis in dogs.

Diagnosis of animal leptospirosis is still challenging. The microscopic agglutination test, is the current method for diagnosing leptospirosis. However, this technique requires specific equipment, highly trained staff and the maintenance of live cultures of several reference strains of Leptospira for use as antigens. Recently, an ELISA (enzyme-linked immunosorbent assay) employing a Leptospira fainei serovar Hurstbridge based antigen for the early diagnostic of human leptospirosis was developed. In this study we estimate the diagnostic sensitivity and specificity of this test in identifying acute canine leptospirosis. A total of 271 serum samples divided into five panels and tested by MAT as a reference test, were used to evaluate the ELISA. Comparing acutely and non-acutely infected dogs, ELISA-Hb showed 95.6% sensitivity and 93% specificity. L. fainei-based ELISA is adequate for diagnosing acute canine leptospirosis, with high sensitivity and specificity and presenting practical advantages when compared to current techniques.

Molecular demonstration of intermittent shedding of Leptospira in cattle and sheep and its implications on control / Demonstração molecular da intermitência eliminando Leptospira em vacas e ovelhas e suas implicações no controle

ABSTRACT: For a long time, it has been stated that urine leptospiral shedding is intermittent, which was observed primarily by culturing. However, culturing presents serious limitations, mainly low sensitivity, and failure on detection of leptospires cannot be neglected. PCR presents several advantages, mainly higher sensitivity. The present study aimed to analyze the occurrence of intermittency on leptospiral shedding by PCR in naturally and experimentally infected animals. In this study two experiments were conducted, the first with 60 cows naturally infected from an endemic herd. The second one was conducted in three sheep experimentally infected, each one with a different strain of Leptospira (strains Copenhageni L1-130, Canicola LO-4 and Pomona Fromm). Considering cattle, 43.3% presented negative in all tests, the remaining (56.7%) were positive at least once. From these, only one (1.6%) was positive in all samples, and seven (11.8%) were positive only in the last sampling, making it impossible to evaluate the intermittency. Noteworthy, 26 cows (43.3%) presented the typical intermittent pattern of leptospiral shedding in urine. In sheep, all experimentally infected animals presented the typical intermittent shedding patterns, independently of the inoculated leptospiral strain. We considered that a careful serial analysis of urine samples for a more definitive and reliable individual diagnosis would be required for a successful antimicrobial therapy and control of leptospirosis on a herd.

RESUMO: Durante muito tempo, foi afirmado que a eliminação de leptospiras na urina era intermitente, o que havia sido demonstrado principalmente por meio do cultivo microbiano. No entanto, a cultura apresenta graves limitações, principalmente com relação à baixa sensibilidade. Em contraste, a PCR apresenta várias vantagens em relação ao cultivo bacteriológico para leptospiras, sendo esta ferramenta cada vez mais utilizada para o diagnóstico de animais eliminadores da bactéria em diversos sítios. Assim, o presente estudo teve como objetivo analisar a ocorrência de intermitência na eliminação de leptospiras por meio de PCR em animais natural e experimentalmente infectados. Para este estudo foram realizados dois experimentos, sendo o primeiro com 60 vacas naturalmente infectadas de um rebanho sabidamente endêmico e o segundo em três ovelhas experimentalmente infectadas, cada uma com uma estirpe diferente de Leptospira (estirpes Copenhageni L1-130, Canicola LO-4 e Pomona Fromm). Considerando-se os bovinos, 43,3% apresentaram negatividade em todos os testes, sendo os demais 56,7% positivos ao menos uma vez. Destes, apenas um (1,6%) foi positivo em todas as amostras, e sete (11,8%) foram positivos somente na última coleta, o que impossibilitou a avaliação da intermitência. Não obstante, 26 vacas naturalmente infectadas (43,3%) apresentaram o padrão de eliminação tipicamente intermitente de leptospiras na urina. Das três ovelhas experimentalmente infectadas, todas apresentaram eliminação intermitente da bactéria na urina, independentemente da estirpe inoculada. Consideramos que seria necessária uma cuidadosa análise seriada de amostras de urina para um diagnóstico individual mais definitivo e confiável para uma terapia antimicrobiana bem-sucedida e o controle da leptospirose em um rebanho.

Detection of wild animals as carriers of Leptospira by PCR in the Pantanal biome, Brazil.

Leptospiral infection is widespread in wildlife. In this context, wild ecosystems in tropical countries hold a vast biodiversity, including several species that may act as potential reservoirs of leptospires. The Pantanal biome presents highly favorable environmental conditions for the occurrence of leptospirosis, such as high temperatures, constant flooding, and high biodiversity. The purpose of this study was to detect wild animals as carriers of Leptospira sp. using direct methods (PCR and culture) in the Pantanal biome, Brazil. A total of 35 animals were studied, namely Cerdocyon thous, Nasua nasua, Ozotoceros bezoarticus, and Sus scrofa species. Blood for serology (MAT) and urine for bacteriological culturing and PCR was sampled. The most prevalent serogroups were Javanica and Djasiman. Additionally, 40.6% of these animals presented PCR positive reactions. Seroreactivity associated with the high frequency of leptospiral carriers among the different studied species suggests a high level of exposure of the studied animals to pathogenic Leptospira strains. Our results are still limited and the actual role of the studied animals in the epidemiology of leptospirosis in the Pantanal region remains to be elucidated.

Recovery of Leptospires in Short- and Medium-Term Cryopreservation Using Different Glycerol and Dimethyl Sulfoxide Concentrations.

Cryopreservation is a recognized method for the maintenance of Leptospira collections. Although cryoprotectants are commonly used in order to prevent or reduce the adverse effects of freezing, there is no consensus regarding the protocols of cryopreservation. This study aimed to compare cryopreservation protocols for Leptospira using different glycerol and dimethyl sulfoxide (DMSO) concentrations. Leptospira interrogans serovar Icterohaemorrhagiae, L. interrogans serovar Bratislava, and L. borgpetersenii serovar Hardjo were used as the experimental strains. For each strain, three protocols were tested using 5% and 10% glycerol and 2.5% DMSO. For each protocol, 12 tubes containing 1.5 mL of serovar were frozen at -70°C on the same day. An aliquot of each serovar/protocol was thawed once a month throughout 1 year. The viability of leptospires was evaluated by the recovery of those at days 7, 14, and 21 after thawing. Although no significant difference was found among the leptospiral recovery rates for the 9 serovar/protocols tested, DMSO (2.5%) was shown to be slightly better than glycerol, and its use should be encouraged as a cryoprotectant for leptospires.