Unconventional Sites for Diagnosis of Leptospirosis in Bovine Anicteric Fetuses

SIMPLE SUMMARY: Abortion is one of the main signs of bovine leptospirosis. The necropsy of the fetuses followed by molecular analysis of the tissues is recommended for the diagnosis. In general, the target sites for diagnosis are the kidneys and liver. However, due to the high diversity of leptospiral strains and anatomic lesions caused by those, this may not be valid for the major agents of bovine leptospirosis. In this context, this study aimed to analyze unconventional sites for the presence of leptospiral DNA in bovine anicteric aborted fetuses. Five anicteric fetuses of a dairy herd with seroreactive cows were submitted for necropsy. After conventional PCR of the lipL32 gene of multiple organs, leptospiral DNA was identified in the lungs, heart, subcapsular kidney contents, thymus, liver, kidneys, and the abomasal liquid. Only one fetus was positive in the liver and kidney. Leptospirosis would have been misdiagnosed if only kidney/liver samples had been tested. Therefore, we recommend the investigation of multiple organs, beyond the liver and kidneys, especially in fetuses with anicteric lesions. ABSTRACT: Background: Bovine leptospirosis is an important reproductive disease and abortion is a major sign, leading to economic impacts. Due to its multifactorial etiology, the proper diagnosis of the cause of the abortion is crucial. Necropsy of the fetuses followed by molecular analysis is recommended for diagnosis, and the investigation mainly occurs in the kidneys and liver. This study aimed to analyze unconventional sites for the presence of leptospiral DNA in bovine anicteric aborted fetuses. Methods: Five fetuses of the same herd were received for necropsy and diagnosis. Conventional lipL32-PCR was performed in the fetuses’ kidneys, livers, lungs, hearts, spleens, subcapsular kidney content, abomasal fluid, and in the cavity’s hemorrhagic contents. To complete the investigation, the sera of 30 cows of the herd were collected to perform the serologic screening by Microscopic Agglutination Test. In addition, six subfertile non-pregnant cows from the same herd were selected due to their low reproductive performance, and genital samples (uterine fragment and cervicovaginal mucus) and urine were collected for lipL32-PCR. PCR-positive samples were submitted to a nested PCR of the secY gene and intended for sequencing. Results: The herd presented seroreactive animals (11/30, 36.6%), all against the Sejroe serogroup, with titers between 200 and 1600. In necropsy, four fetuses showed hemorrhagic and anicteric lesions, while one fetus had no macroscopic lesions. Regarding molecular analysis, all the fetuses were positive in lipL32-PCR and the positive sites were the heart, lungs, subcapsular kidney content, thymus, kidneys, liver, and abomasal fluid. Only one fetus presented positive results in the kidney and liver, while three fetuses were positive in the abomasal fluid. Five of six cows were positive for lipL32-PCR, all being positive only in genital samples. Of the fetuses and the cows, seven sequences were obtained and all were identified as Leptospira interrogans serogroup Sejroe serovar Hardjoprajitno. Conclusions: In order to improve the diagnosis of leptospirosis in cows, it is recommended to perform a comprehensive analysis of the samples, beyond the kidneys and liver. Thus, we highly encourage testing multiple organs by PCR to investigate abortions suspected of bovine leptospirosis, particularly in anicteric fetuses.