Unconventional Sites for Diagnosis of Leptospirosis in Bovine Anicteric Fetuses

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.

The Role of Transplacental Infection in Leptospira spp. Epidemiology in Cattle in Caatinga Biome, Brazil

“…This explains the statistical differences between the spleen and urine and kidney and urine, in relation to positivity. Although the investigation of multiple organs is ideal for the molecular diagnosis of leptospirosis in bovine fetuses [ 37 ], our results indicate that, if it is impossible to collect many samples, the spleen and kidney may be the biological material of choice.…”

Bovine Genital Leptospirosis: An Update of This Important Reproductive Disease

“…Abortions have been widely associated with bovine leptospirosis, perhaps the most visible sign, being linked to incidental strains when occur as an outbreak, or to adapted strains when occur endemically [44]. It was suggested that abortions by leptospiral infection occur in the final third of pregnancy; however, fetal death has been reported in all stages [45]. The pathogeny of fetal death is also unclear, but the direct infection of the fetuses is the most probable cause.…”

“…The abortions infected by incidental strains such as Pomona or Icterohaemorrhagiae usually present icteric and leptospires may be demonstrated in the kidneys and liver [46]. In contrast, anicteric fetuses with hemorrhagic or even presenting no visible lesions are often associated with infection by Sejroe, which may be demonstrated in several organs, such as cardiopulmonary tissue, thymus, subcapsular kidney content, and abomasal liquid [45].…”

“…Leptospires are fastidious and very nutrient-demanding bacteria; therefore, their culture takes too long and presents low sensitivity [61]. On the other hand, molecular techniques such as PCR and qPCR present high sensitivity and specificity [62], are fast to conduct [63], and can be performed in various types of clinical samples such as urine, cervical-genital mucus, endometrial biopsy, semen, fetal tissues, and placenta [17,22,45]. It is noteworthy that the second step for diagnosis, i.e., the molecular investigation in genital samples, is indispensable for a reliable diagnosis since chronic reproductive disease due to genital leptospiral colonization is very often not detected by serology [13].…”