Molecular Identification of Bovine Brucellosis Causing Organisms at Selected Private Farms in Pothohar Plateau, Pakistan

Pothohar Plateau is located in north east of Pakistan consisting of four districts; Rawalpindi, Chakwal, Jhelum, Attock and Islamabad Capital Territory (ICT). It is surrounded by Jhelum River in the east, Indus River in the west, Margalla hills in north and the Salt range in south. This area falls under arid climate zone of Pakistan (Khan, 2004; Kazmi and Rasul, 2012). Livestock are mainly fed by grazing on range lands (Chaudary et al., 2007). Both indigenous and exotic cattle are prevalent with exotic animals raised primarily for milk production. However, intensive husbandry practices are only seen by exotic cattle.

A total of 399 blood samples (250 from indigenous and 149 from exotic cattle) were collected from three different livestock farms in the region. Blood samples (3 ml) were collected directly from jugular vein with a 5 ml syringe. Each blood sample was transferred into a non-EDTA coated vacutainer, placed at room temperature for 30 min, and centrifuged at 4,000 rpm for 15 min. Serum was discharged into a 1.5 ml Eppendorf tubes and stored at

-20 °C until further analysis.

Serum samples were screened initially by Rose Bengal Plate Test (RBPT) and then confirmed by Serum Agglutination Test (SAT) as described by Alton et al. (1988). Standard reagents were provided by Veterinary Research Institute (VRI), Lahore, Pakistan. A sample of 1:160 titers or higher was considered positive for SAT.

For molecular detection DNA was extracted from seropositive serum samples by using High Pure PCR Template preparation kit (Roche Diagnostic, Germany) as per manufacturer’s recommendations. DNA samples were stored at -20ºC till further analysis. Multiplex real-time PCR technique was used for molecular detection of the etiology as described by Probert et al. (2004).

Table I shows primers used for amplification of Brucella genus (bcsp31) and species (IS711 for B. abortus and B. melitensis). The cut-off cycle threshold (Ct) value for a positive sample was set as ≤ 40.

A total of 20 (5.01%) serum samples were found seropositive by RBPT and 19 (4.76%) by SAT (Table II). Out of these sero-positive samples, 13 (overall 3.25%) were found positive only for both Brucella genus (BCSP31) and species Brucella abortus (IS711) by real time-PCR (Table III). None of these seropositive samples were found positive for Brucella melitensis. Only one sample, found positive by RBPT but negative by both SAT and real-time PCR, could be attributed to the low positive predictive value of RBPT (Gul and Khan, 2007).

Brucella abortus is a common etiology of brucellosis in bovines as well as in camels and small ruminants in the country (Ali et al., 2014, 2015; Fatima et al., 2016). Although open grazing, mixed farming and nomadic rearing pose brucellosis risk to animals, intensive farming also does not guarantee absolute protection (Gul and Khan, 2007; Abubakar et al., 2012; Ullah et al., 2015; Munir et al., 2011). Infected semen, breach in biosecurity and latent infection could be the main reasons. Recently, felines and canines have been reported to disseminate the infection at dairy farms (Wareth et al., 2016). Our study revealed the exotic animals tending to be more seropositive (6.04%) than indigenous animals (4.4%). Same results have been observed elsewhere in the country (Ali et al., 2013; Mangi et al., 2015; Kaleem et al., 2016) but one study has shown opposing results (Khan et al., 2016). These observations however, need to be studied further through intense inter-breed comparison of seropositive animals.

The authors declare no conflict of interest regarding this paper.