Seroprevalence of Bovine Leptospirosis using Indirect ELISA

Leptospirosis is known to be endemic in India since the early 20^th century with most outbreaks reported from the coastal regions. The transmission cycle of leptospirosis involves the maintenance hosts, the carrier hosts, the environment and human beings. Cattle are known to be the carrier of leptospirosis and urinary shedding is very evident in cattle. Acute systematic infection in adult dairy cattle is reported as a cause of acute onset agalactia, called as milk drop syndrome (Ellis, 2015). In cattle, acute haemolytic syndrome of leptospirosis is characterized by fever, icterus, anaemia and haemoglobinuria including additional findings of anorexia, apathy, dehydration, dyspnoea, hyperpnea, bilirubinuria, mastitis and abortion. Chronic infections with leptospirosis can result in abortions, stillbirths and reduced fertility in ruminants (Pande et al., 2020).

Enzyme-Linked Immuno Sorbent Assay (ELISA) is one of the methods used for carrying out seroprevalence study and presumptive diagnosis and was predominantly designed to detect antibodies, referred to as indirect ELISA. It is more specific than MAT, though MAT is the gold standard serological test for leptospirosis. Proper understanding of the epidemiological features of these organisms is a critical step in designing interventions for diminishing the risk of the disease transmission Seroprevalence study contributes in understanding the disease’s epidemiology in cattle.

Place and duration of work
 
The proposed work was conducted in the Department of Veterinary Medicine, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University, Jabalpur for a period of 12 months i.e. from January 2021 to December 2021.
 
Location and features of the place
 
In Central Madhya Pradesh, Jabalpur is located at latitude 23^o10 N to longitude 79^o59 E. The city covers an area of 367sq km and lies at an altitude of 412 m above sea level. Jabalpur has a humid subtropical climate characteristic of north-central India. Summer season begins from March and last until June. May is the hottest month of the year with an average temperature exceeding 40^oC (104^oF). Summer season is followed by the southwest monsoon which extend from July to October.  The average annual precipitation is nearly 1,386 mm (54.6 in).
 
Experimental animals
 
For the present study, 6755 cattle were screened from organized and unorganized farms of Jabalpur. Out of 6755 cattle, 300 bio-samples were collected from cattle with a history of repeat breeding, abortion, haemogalactia and mastitis.

Collection of blood samples
 
Five ml of blood was collected aseptically from the jugular vein of properly restrained cattle in clot-activated vacutainers. They were kept in an upright position at room temperature for 2 hours for serum separation. The straw-coloured serum was then poured into 1.5 ml sterile cryovials and aliquoted for future use. Samples were transported to the laboratory and stored at -20°C till further use.
 
Seroprevalence-Indirect ELISA
 
The Vet Check bovine Leptospira indirect Elisa kit which detects specific antibodies (Ab) directed against Leptospira interrogans in cattle. Indirect ELISA was performed strictly as per the standard protocol.
 
Material provided in kit
 
Bovine Leptospira antigen coated micro wells (12x8 wells), positive control, negative control, wash buffer (10x), sample diluent buffer (1x), protein G HRP conjugate (12 ml), chromogen solution (12 ml), stop solution (12 ml of 1 M H2SO4). Additional materials used for the test includes accurate adjustable micro-pipettes and disposable pipette tips, deionized/distilled water, micro plate washing system, micro plate reader, timer, test tube for serum dilution, graduated cylinder and vortex.
 
Test procedure
 
Test samples were diluted in sample diluent buffer separately in 1:100 dilutions. Both serum and diluent buffer were mixed thoroughly. Then 100 µl of ready-to-use controls/diluted sera was dispensed into their respective wells. The micro titre plate was covered with aluminium foil and incubated at 37^oC for one hour. After one hour of incubation, the plates were taken out and washed four times with 300 µl diluted wash buffer provided with the kit. Wash buffer residues were completely removed by tapping the plate on a towel or tissue paper. The 100 µl of the conjugate solution was added to each well and incubated at 37^oC for one hour. The plates were taken out and washed four times with 300 µl diluted wash buffer provided with the kit. Wash buffer residues were completely removed by tapping the plate on a towel or tissue paper. Then 100 µl of chromogen solution was added to each well. The plates were incubated at room temperature for 10-15 minutes in the dark until the colour developed. The reaction was stopped after the colour development, by adding 100 µl of stop solution provided with the kit in each well. The OD values of the micro titre plate was taken with an ELISA reader at 450 nm and results were interpreted.
 
Statistical analysis
 
Significance of prevalence was calculated by chi square test

The screening of cattle for the presence of clinical signs pertaining to leptospirosis was done in present study.  Out of 6755 cattle screened, a total of 300 samples were suspected on the basis of clinical signs and 146 cattle were found positive for leptospirosis. Thus the overall seroprevalence was found to be significantly high i.e. 2.16 per cent (Table 1).



The seroprevalence of leptospirosis among suspected cases was 48.67 per cent. Among 300 samples, 177 were from organized farming systems and 123 were from unorganized farms. The seroprevalence of organized sector was significantly higher (53.67 per cent) than unorganized sector (41.46 per cent). In clinically ailing cattle, the significantly higher sero-prevalence (53.30 per cent) was reported in cattle with the history of abortion followed by repeat breeding (46.48 per cent). In the cases of hemogalactia/mastitis and still death the seroprevalence was 26.92 per cent and 16.67 per cent, respectively. The seropositivity was significantly higher i.e. 55.41 per cent (87 out of 157 cattle) in the cattle of age above 6 years followed by 47.22 per cent (51 out of 108) in 4-6 years of age and least i.e.  22.85 per cent (8 out of 35) in the cattle below 4 years of age. The breed wise seroprevalence study of leptospirosis in cattle revealed a significantly higher prevalence in exotic and crossbred cattle as compared to indigenous breeds (Table 1).

Leptospirosis is an apparently emerging as a significant public health problem in developing countries which remains under diagnosed due to non-specific symptoms in the majority infection cases and also absence of advanced laboratory facilities.  The most frequent sources of infection are urine contaminated water through which leptospires enter into the host body is through intact skin. Humid, wet and damp environment favours the transmission of leptospires. Till date there is no published data of seroprevalence of leptospirosis in Madhya Pradesh since most of the outbreaks are recorded in coastal states of India. Thus the study was planned to know the sero-epidemiological status of leptospirosis. A high seroprevalence in cattle (47.06 per cent, 24/51) was recorded from Valsad, Gujrat by Patel (2014) and (42.15 per cent, 180/427) from Andaman and Nicobar by Jai Sunder et al., (2018) are in agreement with the present study. 

In buffaloes high seroprevalence i.e. 54.14 per cent was reported in Gujarat (Balakrishnan et al., 2011), 26.66 per cent in Andaman and Nicobar Islands (Varma et al., 2001) and 88.8 per cent (125/111) in Chennai (Selvaraj et al., 2010). A sero-survey among animal populations of Andaman and Nicobar Islands (Sharma et al., 2006) showed that about 40 per cent of the cows and 26 per cent of the bulls were seropositive. In contrary to this, less prevalence in cow was reported by Mandal et al., (2008) in West Bengal which was 16.84 percent. The high sero-prevalence in Jabalpur might be due to the location of organized dairy farms which are situated nearby to water bodies leading to favourable conditions for growth of leptospires and easy transmission. 

Reproductive signs seen in cattle could be due to the localization of leptospires in reproductive tract/uterus. Role of leptospira infection in abortions and reproductive disorders is well established by earlier workers including our earlier studies (Poonacha et al., 1993; Gangadhar et al., 2008). Similar findings in clinically ailing seropositive cattle history of mastitis/ agalactia was recorded in maximum number of cases (24.00 per cent, 6/25) followed by abortion (20.00 per cent, 3/15) and repeat breeding (11.76 per cent, 2/17) by Patel (2014).

Balakrishnan et al. (2011) reported the similar findings that the risk of leptospirosis was more in the cattle of age group above seven years (55.88 per cent), followed by four to seven years (51.28 per cent) and below seven years (28.42 per cent). Thus, it was concluded that higher age groups (above 4 years) in cattle favour the occurrence of leptospirosis and supported the observations of earlier workers (Agrawal et al., 2005; Balakrishnan et al., 2011). The older cattle are maximum prone for diseases due to senility and immune exhaustion along with the lodging of leptospira in kidney and reproductive system.

The high seropositivity in exotic and cross bred cattle may be attributed to their higher susceptibility to disease and poor disease resistance The findings of present study are similar to those reported by Balakrishnan et al., (2011) who reported the risk of leptospirosis was highest in exotic pure breed, followed by indigenous pure breeds and cross bred cattle. Varma et al., (2001) and Nagarajan (2005), also reported more seropositivity among cross bred cattle than indigenous cattle.

It can be concluded from the present study that the cattle showing the reproductive signs were found to be seropositive for leptospirosis. Since it is a disease of public health importance, a comprehensive and good understanding of the prevalence of the disease and its epidemiological characteristics is an essential prerequisite for evolving an effective control measures.

Authors would like to acknowledge Environment Planning and Coordination Organization (EPCO), Bhopal for Chief Minister’s Scholarship on Climate Change.

The authors declare that they have no conflict of interest.