Molecular Diagnosis and Prevalence of Brucellosis in N.W.F.P (Pakistan)

Abstract

Brucellosis is a disease with major social and financial implications in many countries, particularly in the developing world. In Pakistan, although there are systems for the detection of brucellosis based on serum agglutination testing, more sophisticated methods such as Polymerase Chain Reaction (PCR), Real-Time PCR, Enzyme Linked Immunosorbent Assay (ELISA) and Fluorescence Polarization Assay (FPA) are not applied routinely. Similarly, the classical and particularly molecular characterization approaches to understand the epidemiological features of Brucella are in infancy. In the present study, serum was used as clinical specimen for serological and molecular diagnosis of brucellosis whereas field culture isolates were used for conventional and molecular characterization purposes. Milk samples were also tested where possible. In total, 277 serum samples of both human and animal origin were collected from 6 districts of Khyber Pakhtunkhwa Province of Pakistan. These samples were then tested at the Animal Health and Veterinary Laboratories Agency, United Kingdom for further validation. From 167 animal sera, 4%, 6%, 13%, 21% and 31% animals were positive by RBPT, cELISA, PCR BCSP31, PCR IS711 and Real-Time PCR, respectively. Out of total 110 human sera belonging to various occupational groups, 5%, 9%, 31%, 38%, 53% and 60% were detected positive by RBPT, cELISA, FPA, PCR BCSP31, PCR IS711 and Real-Time PCR, respectively. A total of 51 milk samples were tested which revealed the prevalence of brucellosis as 12% with Indirect ELISA and none of the sample produced positive results by Milk Ring Test (MRT). In human, positive male showed clinical complaints of arthritis and hepatitis while in female abortion in 4-5 month of pregnancy, headache and arthritis were the main clinical signs. Amongst the positive detected animals, abortion in last trimester, retention of placenta and mastitis were the key clinical features. Cultured field isolates when subjected to phenotypic and molecular characterization techniques found to be in resemblance with Brucella melitensis biovar1. These results give an indication of the prevalence of brucellosis in Pakistan and show that the disease is not only confined to livestock but is also present in the human population. In Pakistan, where there are few centres for brucellosis testing and large areas to cover, serum might provide the best specimen for the determination of infection, based on its stability over time.