A retrospective analysis (2021-2024) of leptospirosis among suspected samples in different animal species from Tamil Nadu

Introduction

Leptospirosis is a worldwide zoonotic disease that affects humans, domestic and wild animals (Genovez, 2016) [16] . It is caused by members of pathogenic Leptospira spp. which comprises 66 different species that include more than 300 serovars (Caimi and Ruybal, 2020) [6] . It affects more than 160 mammalian species, such as dogs, cattle and humans; despite that, it is considered a neglected disease due to the lack of epidemiological data on the disease occurrence (Karpagam and Ganesh, 2020, Harren et al., 2022) [13,14] . Leptospirosis is most common in tropical and subtropical areas with high rainfall. Leptospirosis is becoming an increasingly significant public health problem, particularly in tropical developing countries. The disease is found mainly in places where humans come into contact with the urine of infected animals or a urine polluted environment (Padma Kumari et al., 2016) [18] . Transmission of leptospirosis to humans and animals is mainly by contact with urine of rodents, domestic and wild animal reservoirs (Balakrishnan et al., 2006) [3] . Numerous mammalian species act as maintenance hosts (natural carriers) including feral, farm and pet animals (Levett, 2001; Balakrishnan and Manicavasaka Dinakaran, 2014) [1,2] . Dogs may act as reservoirs and may succumb to severe illness similar to humans (Larson et al., 2017) [16] . Humans act as incidental hosts (Haake and Levett, 2015; Bonhomme and Werts, 2022) [5,12] . It is estimated that wild animals are the source of >70% of all emerging infections (Chomel and Arzt, 2013) [7] , as there is a close phylogenetic relationship between humans and other primates, creating a high potential for pathogen exchange. Humans are most commonly infected through occupational, recreational or domestic contact with the urine of carrier animals, either directly or through contaminated water or soil. Recent advances in diagnostic approaches have led to a growing awareness of the broad distribution and diversity of leptospiral pathogens both in infection and in the environment (Costa et al., 2015) [9] . Antigen detection by dark field microscopy (DFM), antibody detection by Microscopic Agglutination Test (MAT) and molecular techniques such as Polymerase Chain Reaction (PCR) are the widely accepted techniques in diagnosis of leptospirosis (WOAH, 2021; Senthilkumar et al., 2022) [22] . Prevalence of leptospirosis is more among humans and animals with occupational exposure or in the area of agricultural activities. It is inevitable that indirect exposure by the human population to the environment contaminated with urine of cattle, sheep, goats, dogs, pigs, rodents and wild animals which are infected clinically and sub clinically by leptospirosis (Balakrishnan and Manicavasaka Dinakaran, 2014) [2] . Dogs generally contract infection through direct contact, soil, food and water bodies contaminated with urine of domestic animals, rodents and humans. In developing countries including India, leptospirosis can be considered as one of the emerging public health problem. The present study was carried out to determine the sero-prevalance of leptospirosis in livestock, companion animals and wild animals in Tamil Nadu.

Materials and Methods

Study area and sample collection

A study was conducted for the serological screening for leptospirosis in livestock, companion animals (n=2649) and wild animals (n=134) to analyse the Leptospira serovar positivity in Tamil Nadu state. Serum samples were collected randomly from livestock, companion animals and wild animals with no history of vaccination to leptospirosis (Table 1). Blood/serum samples were collected randomly from animals with clinical signs of anorexia, lethargy, vomiting, diarrhoea, polyuria, polydypsia, jaundice, hematuria etc which were presented to the Zoonoses Research laboratory, Centre for Animal Health Studies, TANUVAS, Madhavaram Milk Colony, Chennai in the study area. Using serum vacutainer tubes with clot activator, 3-4 ml of blood was drawn aseptically. After allowing the blood to clot, the serum was separated by centrifuging at 2,000 rpm for 15 min and then the serum samples were used for serological study (Senthilkumar et al., 2022) [22] .

Serological screening by microscopic agglutination test

Serological screening for leptospirosis was done by MAT using a panel of 12 Leptospira serovars (Table 2) representing the serogroups of Australis, Autumnalis, Ballum, Canicola, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Javanica, Pomona, Pyrogenes and Tarassovi (Senthilkumar et al., 2022) [22] obtained from Zoonoses Research Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Chennai and maintained in EMJH medium. The concentration of growth of Leptospira serovars in EMJH liquid leptospiral medium at 30±2 °C was assessed using the Petroff-Hauser chamber (0.02 mm) and estimating the number of leptospires per field by dark field microscopy. The growth of the live cultures at least 4 days old, but not more than 10 days and densities of approximately 2x10 8 leptospires per ml of culture were used as antigen for MAT (Faine et al., 1999; WOAH, 2021) [10] . Around 500 μl of culture from each of these 12 Leptospira serovars was dispensed into a sterile 10 ml screwcapped tube, which was then utilized for further processing for MAT. All the serum samples were subjected to MAT (WOAH, 2021; Senthilkumar et al., 2022) [22] . Briefly, 1:50 dilution of the sera was prepared and then the diluted sera (30μl) were mixed with pooled antigen (30 μl) in 96well Ubottom microtitre plate and kept for incubation at 37 ºC for 2 hrs. Antigen control was kept by diluting 30 μl of pooled antigen in 30 μl PBS. After incubation, 10 μl of serum sample was placed on a clean glass slide alongside the respective antigen control. The slide was then examined under DFM (20X). The serum that showed 50% agglutination, leaving 50% free cells compared with a control culture was considered as positive (Titre of 1:100 and above) for leptospirosis (Figure 1).

Result and Discussion

Leptospirosis is a zoonotic disease greatly influenced by ecological factors like rainfall, temperature and humidity. A one health approach to the epidemiology, management, surveillance and control of leptospirosis relies on accessible and early diagnostics that can be applied to companion animals, livestock and wild animals. [8] revealed that in animals, the highest prevalence of leptospirosis was found to be in buffalo (45.75%), followed by rodents (40%), dogs (26%), cows (24.26%), goats (19.42%), and pigs (15.8%) which was in accordance with our findings. Senthil Kumar et al., 2023 screened 1466 serum samples, from which 528 (36.0%) samples were seroreactive to Leptospira on the MAT, a standard serological test referred by OIE which is correlating with this present study. Arumugam et al., 2011 [1] study showed higher prevalence in Chennai (35.4%), followed by Thiruvallur (9.6%), Thiruvannamalai (5.5%), Thiruvarur (4.3%) and Kanniyakumari (3.9%). Seroprevalence of leptospirosis in animals and wild animals were reported in different areas of Tamil Nadu. Seroprevalence of leptospirosis in canines in Chennai was found to be 15.21 per cent (Kumar et al., 2009) [15] and 47.2 per cent (Bojiraj et al., 2017) [4] . Prabhavathy and Cecilia Joseph (2018) [19] who reported that 95.0 per cent in dogs, out of which 52.63 per cent samples were positive to one serovar and 47.36 per cent samples were positive to several serovars. Leptospirosis in dogs was reported in Namakkal by Sathiymoorthy et al., 2017 showing an overall prevalence of canine leptospirosis at 28.40%. Serovar prevalence indicated that grippotyphosa was the predominant serovar for about 35.21% followed by 28.62% of autumnalis, 22.53% of australis, 16.90% of pomona, 15.49% of canicola, 12.67% of icterohaemorrhagiae, 12.67% of pyrogenes and 1.40% of tarassovi. Out of 71 MAT positive serum samples, 18 (25.35%) were cross-reacted with more than one serovars. Chennai always has a higher prevalence than other parts of the state which was correlated with our present findings. Leptospirosis occurs in Chennai throughout the year. The data in this study shows that the cases are highly prevalent during monsoon (August -January) and increased after heavy rainfall and floods which implies that Leptospirosis is highly influenced by climate and environment. A notably high positivity in dogs (54%) and small ruminants (50.6%) suggests that these species act as important sentinel hosts reflecting environmental exposure to Leptospira. Australis emerged as the most predominant serogroup across all animal species and years, indicating its wide circulation in Tamil Nadu. Autumnalis, Ballum, and Hardjo were the other frequently detected serogroups, showing host adaptation tendencies. Year-wise variation, with a peak seropositivity in 2023, may be attributed to seasonal rainfall and flooding events that favour Leptospira persistence and transmission through stagnant water. The relatively lower positivity in cats compared to other species could be due to their solitary behaviour and limited environmental exposure and based on the limited available data. The detection of multiple serogroups in the same host species also points to co-exposure or co-infection scenarios, reflecting complex transmission dynamics involving both maintenance and incidental hosts. These findings underscore the zoonotic risk associated with domestic animals, particularly dogs and cattle, which frequently interact with humans. Regular serological surveillance and control measures such as vaccination, rodent management and improved farm sanitation are essential to mitigate infection risk. Furthermore, integrating One Health approaches involving veterinary, public health and environmental sectors would enhance the understanding and management of leptospirosis transmission in endemic areas.

Acknowledgement

The authors express their sincere gratitude to the Director of the Centre for Animal Health Studies, Madhavaram Milk Colony, Chennai -600051, for granting permission to undertake this study.

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