Identification and isolation of Leptospira from bovine cases of reproductive disorders in Kerala, India

It is becoming more widely acknowledged that leptospirosis, a zoonosis of global importance, is a major but underdiagnosed cause of bovine reproductive failure. The purpose of this study was to look into the role of Leptospira in causing abortions, stillbirths and recurring breeding problems in cattle in Kerala. A combination of serological, molecular and cultural techniques were used to sample and assess a total of 20 animals. Five animals tested positive for Leptospira DNA by polymerase chain reaction (PCR) targeting the LipL32 gene, while two animals tested seropositive in microscopic agglutination test (MAT). Leptospira were successfully isolated from two samples (liver and blood). The results emphasise how crucial it is to use a variety of diagnostic techniques in order to accurately identify both active infections and carrier states.

Introduction

Leptospirosis is an infectious disease that is re-emerging and affects many types of domestic and wild animals. It is a serious threat to public health as it can spread from animals to people. Leptospirosis is a major cause of reproductive problems in cattle, such as infertility, abortion, stillbirth and weak calves, but it often goes unnoticed because due to non-specific symptoms and lack of awareness. The fact that the infection is chronic and that the organism likes to live in the kidneys and reproductive organs makes it hard for herds to get rid of the pathogen, even when there are no obvious clinical signs. Bovine leptospirosis is still not diagnosed enough in many parts of India, including Kerala. Infected urine, water, feed, or fomites are the most common ways for the disease to spread. It is even harder to control because the organism is very resilient in the environment and carrier animals are common. The goal of the study was to find out the occurrence of Leptospira infection in cattle Kerala that have had problems with reproduction in the past by using a thorough diagnostic method.

Materials and Methods

The study included 20 cows that had clinical histories that suggested they had Leptospiral infection then or in the past. These included mid-to late-term abortions, stillbirths, and repeat breeding. Samples submitted to the Department of Veterinary Microbiology, i.e., blood and serum samples from 17 animals and urine samples from 3 animals were included. Also, samples of aborted foetal tissue, vaginal discharge, and post-mortem liver, kidney, and uterus were taken from cases where they were available. Serological detection was performed using MAT, against 12 reference Leptospira serovars maintained at the Department of Veterinary Microbiology at a screening dilution of 1:200, following the protocol described by Faine et al. (1999) [1] . The samples were examined under a dark field microscope for agglutination. Molecular detection using PCR assay targeting LipL32 gene, which is specific to pathogenic Leptospira was performed. DNA was extracted using a Himedia Multisamples DNA extraction kit and PCR was performed using primers described by Amutha et al. (2007) [2] . For bacterial isolation, samples were inoculated into EMJH semi-solid medium and incubated at 28 to 30°C for up to 3 months. Cultures were monitored for Dinger's ring formation and confirmed using dark field microscopy. Confirmatory identification of isolates was done using LipL32 PCR.

Results

Out of the 20 cattle examined, two animals tested positive for Leptospira antibodies via MAT, showing reactivity to serovars Hebdomadis and Pomona. PCR targeting the LipL32 gene revealed Leptospira DNA in five animals across blood, urine, and liver samples. Leptospira was successfully cultured in two cases (from blood and liver samples). Isolates showed typical morphology and were PCR confirmed. These findings underscore the value of multi-sample diagnostics over sole reliance on serum.

Animal no. (sample)

Animal no. (sample)

Discussion

Soman et al. (2014) [3] tested 100 bovine samples from central and northern Kerala using MAT and found a higher seropositivity rate of 47 per cent. Sreekutty et al. (2020) [4] reported an overall seropositivity of 52.2 per cent from 90 bovine serum samples collected from Alappuzha district. Divya (2021) [5] performed MAT on 29 bovine serum samples and identified a seropositivity of 20.68 per cent. Murigavelu et al. (2022) [6] examined 165 blood samples from slaughtered cattle in Thrissur district, revealing an overall seropositivity of 44.24 per cent. Cheema et al. (2007) [7] collected 50 bovine serum samples across various states in India and found that four of the samples (eight per cent) tested positive in a lipl32 genespecific real-time PCR. Senthilkumar et al. (2022) [8] identified Leptospira DNA in 9.2 per cent of urine samples from a total of 305 samples collected from cattle from Tamil Nadu. Patel et al. (2017) [9] detected positive amplicons in 9 out of 120 blood (7.5 per cent) and 33 out of 304 urine (10.85 per cent) samples from cattle, screened using lipl32 gene specific PCR in Gujarat. The findings demonstrate a strong link between Leptospiral infection and reproductive issues in cattle. Antibody detection using MAT detected only two positives, while PCR identified five, and cultures yielded two viable isolates. The discrepancy between MAT and PCR outcomes highlights the limitation of serological tests alone for confirmatory diagnosis of leptospirosis. The low detection rates could be ascribed to the intermittent shedding of leptospires in ruminants (WOAH, 2021) [10] , with negative PCR results potentially reflecting a lack of detectable Leptospira excretion at the time of sampling.

In the present study, samples were collected from suspected cases of bovine leptospirosis as well as animals suffering from chronic reproductive disorders. This underlines the importance of repeated sampling over time to accurately determine carrier status, as suggested by Denipitiya et al.

(2017) [11] . Furthermore, the comparative analysis by Nally et al. (2020) [12] found that no single assay was optimal, and there was a need for combining multiple diagnostic methods, such as real-time PCR alongside culture and FAT techniques, to enhance the accuracy of Leptospira detection in cattle. Moreover, the study reinforces the importance of considering the sample type viz., blood, urine or tissue, when diagnosing leptospirosis, as different sample types may yield varying detection rates in different species, with urine often showing higher positivity in cattle and pigs, due to the prolonged presence of leptospires in the urinary system. Additonally, while prior studies in cattle from Kerala employed a lower threshold of 1:50 for bovine samples (Sreekutty et [4,6,13] , a higher cutoff titre of 1:200 was used in this study as majority of bovine samples were collected from animals with chronic reproductive disorders. Isolation of Leptospira from internal organs affirms systemic infection and raises public health concerns. PCRpositive but culture-negative urine samples may indicate low bacterial load or intermittent shedding. These findings support previous reports by Ellis et al. (1982) [14] . Under the circumstances, comprehensive diagnostic protocol is essential in endemic areas like Kerala, where environmental factors favour transmission. Asymptomatic carriers may continue to spread the infection, underscoring the need for regular herd screening using all available tools. reinforces the need for a tiered diagnostic approach. Regular screening, vaccination, hygiene and biosecurity are critical in disease management and in reducing the zoonotic risk.

Conclusion

Conclusion

Acknowledgement

AcknowledgmentsThe authors thank Kerala Veterinary and Animal Sciences Univeristy for the facilities and Indian Council of Medical Research for funding the present work.

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