Introduction

Leptospirosis is a zoonotic disease with ubiquitous distribution, caused by infection with pathogenic Leptospira species. It is a rapidly emerging disease affecting the health of domestic animals and humans in most countries with tropical and sub-tropical climates (Vijayachari et al. 2008). Despite being so severe, this disease is neglected in most endemic countries in the world because of a lack of information and awareness about the extent of the problem. The disease affects a variety of domestic animals viz. cattle, buffalo, goats, sheep, horse and swine resulting in heavy economic losses to the farming community on account of reproductive problems (Srivastava 2008). The leptospirosis situation in India is a cause of concern, and it is endemic in all Southern states as well as in coastal states like Gujarat, Maharashtra, including Andaman and Nicobar Islands of India, where high prevalence was recorded both in animals and humans.

Leptospirosis in cattle and water buffaloes causes direct or indirect economic losses which include costs of abortion, early embryonic death, stillbirth, infertility, birth of weak calves, failure to thrive, mastitis, reduced productivity and decreased milk yield (Quinn et al. 1994), and associated veterinary costs in domestic and commercial livestock, with potential for malnutrition and impoverishment amongst individuals and communities dependent on animal sources of protein, especially in subsistence economies (Srivastava 2008). Leptospirosis can cause abortion as early as the fourth month, but abortion after 7 months is more common in cattle and buffaloes. However, in subclinical infection, cattle show asymptomatic signs with chronic renal colonization of organisms and shedding in urine, infertility due to bacterial colonization in reproductive tract and occasionally abortion, whereas in clinical infection in calves, outbreaks of clinical diseases with haematuria and death could be observed some times. The presence of the complexity in the case of identification need for early diagnosis and lack of diagnostic facilities hinders diagnosis. Hence, leptospirosis is frequently under diagnosed because of the nonspecific symptoms and the difficulty of performing both culture and the international reference serological test, microscopic agglutination test (MAT) (WHO 2011; OIE 2013).

In India, a comprehensive study covering wider geographical locations on the prevalence of leptospirosis in cattle especially in dairy farms is lacking except few isolated location-specific reports (Balamurugan et al. 2016b). Generally, in organized or unorganized farms, the abortions of animals in various stages of gestation more specifically due to leptospirosis are over looked, and the studies on level of infection in animals in the farming sector throw light on the status of the disease per se, which in turn help in early diagnosis and subsequent prompt treatment, prevention and its control including zoonotic transmission to humans.

The prevalence study is imperative in order to develop the appropriate long-term prevention and control strategies, including implementation of vaccination against diseases in the farm animals as well as to implement mitigating measures to reduce the incidence of the leptospirosis in animals and humans (Balamurugan et al. 2016b) as the number of Leptospira serovars and serogroups constantly increasing due to new strains isolation from animals, humans and the environment (Levett 2001). Generally, cattle are the maintenance host for serovar Hardjo, which consist of two serologically indistinguishable but genetically distinct species. Further, serovars causing infection in cattle have been classified into two groups: (a) those adapted to and maintained by other cattle (Hardjo) and (b) incidental infections caused by strains maintained by other domestic and free-living animals. For effective prevention of the leptospirosis in cattle and water buffalo dairy farms, vaccine should contain specific Leptospira serovars prevalent in that particular geographical area, in order to reduce or prevent the abortions and other reproductive disorders caused by Leptospires and eliminate the carrier status of animals by preventing the colonization of Leptospires in the kidney tubules, from which the spread of infection occurs through urine by contaminations. This can be achieved only through regular systematic yearly vaccination of farm animals with inactivated vaccine containing multiple major prevalent serovars to make the population immune against leptospirosis. This in turn reduces the zoonotic potential of disease in the farm workers and contact personnel besides preventing the periodical Leptospira abortions occurrence in the dairy farms. Hence, investigation was undertaken to know the status of Leptospira serogroup-specific antibody distribution and its prevalence in dairy cattle associated with reproductive disorders in endemic states of India.

Materials and methods

A total of 373 cattle serum samples from 45 dairy farms in various endemic areas of different states (Maharashtra, Gujarat, Punjab, Tamil Nadu, Haryana, Telangana, Jharkhand, Chhattisgarh and Karnataka, Andhra Pradesh, Sikkim, Himachal Pradesh and Uttarakhand) of India were collected by Veterinary Officers from animals with a history of reproductive disorders like abortion, repeat breeders, anoestrus, endometritis and also from apparently healthy animals with a status of non-vaccinated against leptospirosis during survey period of 2015–2016 (Table 1). These samples were from either randomly selected herd with history of reproductive disorders and collected by ICAR-NIVEDI teams or suspected samples from the farm submitted for either screening or diagnosis purpose with the same history. Collected serum samples were transported on ice to the ICAR-NIVEDI for testing, and upon arrival, the samples were stored at − 20 °C until further use.

Table 1 Details of the cattle samples screened and its test results for leptospirosis
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All these samples were screened for Leptospira serogroup-specific antibodies by MAT at 1:100 dilution. Ellinghausen McCullough Johnson and Harris (EMJH) liquid medium was prepared and used for the propagation of Leptospira reference serovar cultures (Balamurugan et al. 2016a, 2017). MAT was performed as reported earlier (OIE 2013) using a panel of 18 live reference Leptospira serovar antigens (Australis, Bankinang, Canicola, Hardjo, Hebdomadis, Pyrogenes, Tarassovi, Icterohaemorrhagiae, Pomona, Shermani, Kaup, Grippotyphosa, Hurstbridge, Javanica, Panama, Djasiman, Copenhageni and Bataviae) obtained from WHO National Reference Laboratory, Indian Council of Medical Research (ICMR)-Regional Medical Research Centre (RMRC), Port Blair, and being maintained in Leptospira Laboratory, ICAR-NIVEDI, Bengaluru, for the presence of specific Leptospira antibodies as described earlier (Balamurugan et al. 2017), and a MAT titre of 1:100 is taken as positive reactor as per WHO/OIE manual for leptospirosis (WHO 2011; OIE 2013).

Chi-square test was used for testing the independence of seropositivity of leptospirosis across age groups and across the sampled regions (Snedecor and Cochran 1989). The null hypothesis (H0) is independent of seropositivity with specific disease history, age groups and study regions (vs H1 dependent). Further, odds (risk-relation), risk of disease history status and apparently healthy status associated with seropositivity was compared statistically with β estimate, at 95% confidence interval (CI), with p value (< 0.05). The estimation of apparent prevalence with 95% CI and statistical data analysis were carried out using Microsoft Office Excel 2013 and SPSS version 22 (IBM), India.

Results

Out of total 373 sera tested, 263 samples found to be reactive in MAT representing 70.51% seropositivity (263/373, 95% CI 0.65 to 0.75) in dairy cattle, of which 20 samples from apparently healthy animal and 243 samples from cattle associated with reproductive problems showed reactivity with different serovars. The details of samples screened and its test results are presented in Table 1. It is evident from the pooled chi-square analysis that the seroprevalence of leptospirosis in cattle across the disease history is significantly dependent (χ2 = 55.71, p < 0.01) and sampled states (χ2 = 32.99, p < 0.01). However, seropositivity is independent across the age groups of cattle (χ2 = 0.91, p > 0.10) (Table 2) and apparently healthy animals (χ2 = 15.6, p > 0.10) (Table 1). Out of total 263 reactors in MAT, 186 samples showed reaction with multiple serovars representing 70.72% prevalence. The cross-reactivity observed between the different serovars tested is shown in Tables 3 and 4. Further, the odds (risk-relation) of reproductive disorders was 5.29 compared to apparently healthy animals (0.25 odds) (Table 5). The frequency distribution of predominant serogroup-specific Leptospira antibodies was determined against the serovars—Hardjo (27.76%), Pyrogenes (18.63%), Canicola and Javanica (17.49%), Hebdomadis (17.11%), Shermani and Panama (16.73%), Djasiman (16.35%), Tarassovi, Grippotyphosa and Pomona (15.97%), Icterohaemorrhagiae (15.59%), Copenhageni (14.83%), Australis (13.69%), Kaup and Hurstbridge (10.65%), Bankinang (10.27%) and Bataviae (9.51%)—and are summarized in Table 6. Distribution of reactive pathogenic serovars in different states is presented in Table 7. Further, the number of seropositive samples to intermediate and other Leptospira spp. serovars tested in MAT is summarized in Table 8. State-wise coverage of the positive reactive samples with selected panel of serovars was tabulated (Table 9).

Table 2 Age-wise prevalence of Leptospira serogroup-specific antibodies in cattle
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Table 3 Cross-reactivity amongst different Leptospira serovars in cattle
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Table 4 Major selected reacted serovars versus cross-reacted serovars
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Table 5 Odds ratio test of seropositive reaction versus history of disease status
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Table 6 Frequency distribution of reacted pathogenic serovars (descending order)
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Table 7 State-wise distribution of reacted pathogenic serovars in cattle
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Table 8 Details of the samples reacted with the Leptospira intermediate and other species serovars
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Table 9 State-wise coverage of the positive reacted samples with selected panel of the serovars
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Discussion

It is evident from the analysis that the seroprevalence across the disease history is significantly dependent with odds, indicating that animal with history of reproductive disorders has more probability of having leptospirosis than in apparently healthy animals. Besides being an important cause of cattle abortion, reduced fertility and agalactia, serovar Hardjo also poses a potential zoonotic threat to humans who are exposed to infected cattle. Significant percentage of animals that are actively infected with Leptospira spp. serovar are shedding Leptospires and have antibody titres ≥ 100 against serovar Hardjo that are considered to be seropositive (Balamurugan et al. 2016a, 2017). Infection with host-adapted serovars has been reported to produce subclinical infection with apparently healthy animals serving as chronic carriers and persistent shedders of the organism through their urine, body fluid or tissue (Balamurugan et al. 2013, 2016a), which may act as source and pose a potential risk to livestock owners, farm workers, occupational workers, other domestic species in the farm, etc. (Balamurugan et al. 2013). It was found that seroprevalence was higher in some farms which might be due to exposure of infection from newly introduced unscreened animals into the farm or intensive farm management practices, or apparently healthy seropositive status animals may be shedding Leptospirae and that may serve as source of infection to others, since vaccination against leptospirosis in cattle is not regularly practiced in India and higher percentage positivity in all age groups indicates natural circulation of Leptospira in the farms.

Further, the results are dependent with the history of disease status for the sampled regions, and the studies of cattle leptospirosis in different parts of the globe also indicated that serovars responsible for reproductive losses vary depending on which serovars are locally endemic. Seropositivity in apparently healthy animals also indicates that animals were exposed or harbouring Leptospira organism, irrespective of the history. Further, seropositivity is independent across the age group of cattle indicating that leptospirosis affects all the age group of animals. The most prevalent predominant Leptospira antibodies amongst the reacted samples were against serovars Hardjo, Pyrogenes, Canicola, Javanica, Hebdomadis, Pomona, Icterohaemorrhagiae, etc. The observed majority of the cross-reactivity was between these aforesaid major reacted serovars with other serovars Australis, Grippotyphosa, Shermani, Tarassovi, Panama, Copenhageni, Hurstbridge, Djasiman, Kaup, etc. Further, on analysis of frequency distribution of major serovars, Hardjo showed the highest positive reactions followed by Pyrogenes; Canicola and Javanica; Hebdomadis; Shermani and Panama; Djasiman, Tarassovi, Grippotyphosa and Pomona; Icterohaemorrhagiae; Copenhageni; Australis; Kaup and Hurstbridge; Bankinang; and Bataviae. Out of 186 multiple serovars reacted sera, 168 samples have reacted with aforesaid determined seven major serovars. These serovars may be considered as highly infective serovars in different regions of surveyed farms. These results on seroprevalence from this study correspond with earlier reports. It is a well-known fact that Hardjo serovar is the common one and dairy cattle have a role as a natural host of serovars Hardjo, Pomona and Grippotyphosa (Leonard et al. 2004). Moreover, different serogroup-specific antibodies have been reported from different parts of India by various researchers viz. from Karnataka (4.6%), Andaman (24.2%), Tamil Nadu (51.4%) (Natarajaseenivasan et al. 2011) and Andhra Pradesh (10.5%) (Srivastava 2008), Maharashtra (7.3%) and Uttar Pradesh (4 to 8%) (Srivastava and Kumar 2003), Odisha (37.74%) (Balamurugan et al. 2017), Konkan Region of Maharashtra (41%) (Balamurugan et al. 2016c), Gujarat (12.8%) (Patel et al. 2014) and organized cattle farm (12%) (Balamurugan et al. 2016b) during different surveys.

The reacted serovars (Kaup and Hurstbridge) representing Leptospira intermediate species may be of significance, as the prevalence of this species in India has been reported (Balamurugan et al. 2013, 2017). However, most of the reacted samples also showed multiple reaction with either of the aforesaid major serovars, and only a few samples showed reaction with serovars representing Leptospira intermediate species. It is to be noted that reacted samples to Kaup, Hustbridge serovar representing the Tarassovi serogroup, did not react with the serovar Tarassovi (Perepelicin strain) belonging to species L. borgpetersenii. Further, on inclusion of the L. santarosai, L. noguchii and L. kirschneri serovars, it is possible to detect additional reacted positive samples, which helped in knowing or identifying the prevalence of emerging or re-emerging serovars in various geographical regions as reported earlier (Balamurugan et al. 2016a, 2017). The local abundance of several species of pathogenic Leptospires may be a useful indicator to assess the potential for transmission to humans and livestock, and it may help to take appropriate control measures to reduce the impact of leptospirosis (Chadsuthi et al. 2017).

In overall, with the selected major reacted serovars in the panel, it is possible to provide the diagnostic screening more than 80% in MAT with coverage ranges from 90 to 100% in some states. However, in the present study, as the samples were limited to few farms in some states, it has to be further tested and verified with large samples size. In other way, initially, dairy cattle farm samples are to be screened by using these selected major serovars panel in MAT followed by screening of negative samples with remaining panels of Leptospira serovars antigens, which will avoid tedious effort and time and funds associated with handling of more number of reference Leptospira serovars in the panel for providing specific diagnosis. Further, study supports that dairy cattle maintaining important several serovars in endemic states of India warrants mitigating measures to reduce the disease burden by an intensive surveillance programme, preventive vaccination and control strategies.