Leptospirosis is a highly endemic disease in Kerala affecting man and animals. In cattle the disease causes abortions, stillbirths, reduced milk production and haemagalactia. The present study reports seroprevalence of leptospirosis in cattle in and around Thrissur district. One hundred and eighty eight serum samples collected randomly from adult cattle of various organized farms and households in and around Thrissur district of Kerala during 2013-2014 were screened for antibodies to pathogenic leptospires. Enzyme linked immunosorbent assay based on recombinant outer membrane protein rLipL32 was used. Among 188 samples tested 12 were positive for antibodies. Eventhough the seroprevalence is low i.e 6.38 per cent it has to be viewed seriously as it forms one of the major zoonosis in this area.
Leptospirosis is a worldwide zoonoses caused by pathogenic leptospires. In cattle the disease is associated with abortions, stillbirths, reduced milk production and mastitis with blood tinged milk (Quinn et al., 1994). The disease in cattle is mainly caused by serovar Hardjo and Grippotyphosa (Ellis, 1994). The commonly used methods for diagnosis of Leptospirosis include isolation and identification of leptospires or detection of leptospiral antibodies. The isolation and identification is time consuming and can be done only in specialized reference laboratories. Serological testing is the most widely used diagnostic test and Microscopic Agglutination Test (MAT) is considered as the gold standard test (OIE, 2008). But MAT involves handling of live antigen and poses a risk to laboratory personnel.
Enzyme linked immunoassays (ELISA) are used nowadays as screening test for leptospirosis (Ribotta et al., 2000). ELISA has several advantages over MAT, including use of killed antigen. But one of the disadvantage of conventional ELISA is that it requires a separate test for each serovar (Bercovich et al., 1990). This can be overcome by using outer membrane proteins (OMPs) as antigen by which antibodies to all pathogenic serovars can be detected. Among various OMPs studied lipL21 and lipL32 were found to be the most abundant and immunodominant proteins conserved among the pathogenic species and OMP based ELISA was used for diagnosis of bovine leptospirosis (Srivastava et al., 2006; Joseph et al., 2012). The present paper describes seroprevalence of leptospirosis among cattle in and around Thrissur district of Kerala detected using recombinant antigen lipL32 based ELISA.
Materials and Methods
A total of 188 cattle sera were collected from different organized farms and households in and around Thrissur district. Sera were kept in sterile micro centrifuge tubes and stored at -20°C until further use. The recombinant lipL32 antigen was supplied from department of Veterinary Microbiology, CVAS, Mannuthy and ELISA was performed as per Vishak (2015). The ELISA plates were coated with 150ng/well of recombinant lipL32 proteins suspended in 0.05M carbonate bicarbonate buffer (pH 9.6) and incubated overnight at 40C.The plates were then washed 5 times with phosphate buffer saline (PBS) containing 0.05 per cent Tween-20 (PBST). Wells were blocked with five per cent skim milk in PBS (SM-PBS) for one hour at 370C and again washing process was repeated. Then 100µl of diluted serum samples (1:50 dilution in 5% SM-PBS) were added to each well and incubated at 370C for 45 minutes to allow binding of antibodies to antigen present in each well. The plates were then washed five times with PBST. Then Anti-Bovine IgG peroxide conjugate (100 µl of 1:2500) was added and kept it for 30 minutes which bound to any available primary antibody. The plates were then washed five times in PBST and added 100 µl of enzyme substrate (O-phenylenediamine (OPD), and incubated at room temperature for ten minutes in the dark for colour development. The reaction was stopped by adding 100µl of 1.25M sulphuric acid. The absorbance was measured at 492nm in an ELISA reader.
Results and Discussion
Out of 188 samples screened 12 samples were positive for antibodies to pathogenic leptospires making 6.38 percent seroprevalence. In recent years, recombinant based ELISA has become the preferred test compared to whole cell based ELISA as a serodiagnostic tool of leptospirosis due to better sensitivity and specificity (Dey et al., 2004; Bomfim et al., 2005). Bahera et al., 2014 also suggested that indirect ELISA using rLipL32 as antigen may be of higher sensitivity as well as specificity than MAT for detection of leptospiral antibodies in cattle. Even though a higher rate of seroprevalence was reported for leptospirosis in various states of India such as Gujarat, Odisha, Tamilnadu and Andaman Nicobar islands, (Sharma et al., 2003; Patel et al., 2014; Balmurugan et al., 2013), the present study showed a low prevalence. This may be due to the fact that the majority of the samples were collected from apparently healthy animals in various organized farms. Leptospirosis in cattle is a worldwide problem associated with abortion, agalactia, still birth and infertility resulting in major economic losses to dairy industry (Lilenbaum and Souza, 2003). Carrier state is reported in cows in which the organism persists in the kidneys and genitals of animals without showing clinical signs of disease (Ellis et al., 1986). They often excrete leptospires in their urine making important sources of infection for other cows and people (Waitkins, 1986). The disease is widely prevalent in different parts of India (Mariya et al., 2007; Balakrishnan et al., 2011). It has been recognized as a re-emerging infectious disease which has the potential to become widely prevalent with anticipated global warming ((Kamath and Joshi, 2003; Yang, 2007).
Several factors such as herd size, co-grazing with infected cattle, access to contaminated water sources, use of infected bulls, inadequate husbandry practices and replacement with animals from other farms have been found to be associated with leptospiral infection in cattle (Guitian et al.,2001). Control of leptospirosis in cattle herds relies upon a combination of management decisions to reduce risk of infection, strategic antibiotic treatment, and vaccination. Results of the present study revealed a low prevalence of leptospirosis among cattle in this region. However further studies should be performed to understand the epidemiology of leptospirosis in farm animals and its association with human leptospirosis. Future studies utilizing more number of samples would assist in determining the seroprevalence and variations of disease pattern and impact of leptospirosis in cattle in this region.
The authors are grateful to the Kerala Government for the funding through the plan schemes 2014-2015 and to Department of Veterinary Microbiology CVAS, Mannuthy for supply of the recombinant lipL32 antigen.