Evaluation of a homemade ELISA kit for serological diagnosis of cattle paratuberculosis

Paratuberculosis is a bacterial infection caused by Mycobacterium avium subsp. paratuberculosis. It is characterized by chronic gastroenteritis, diarrhea, weight loss, reduced milk production and occasionally death. A long subclinical stage can be occurred in infected cattle. Subclinically infected animals transmit the infection to healthy animals by shedding the agent in their faeces, milk and colostrum. Paratuberculosis is a zoonotic disease which is seen all over the world and causes significant economic losses in dairy cattle farms. The aim of this study is to investigate the prevalence of paratuberculosis in dairy cattle over 2 years of age in the Elazig province by using a homemade Enzyme-Linked Immunosorbent Assay (ELISA) kit. For this purpose, blood serum samples were collected from 150 dairy cattle without showing any clinical signs. Antigen was prepared from Mycobacterium avium subsp. paratuberculosis strain (DSMZ 44133) grown on Herrold's Egg Yolk Agar medium with Mycobactin J and ANV (amphotericin, nalidixic acid and vancomycin). After optimization, the dilution of 0.125 µg/well of the antigen, 1/60 of the serum and 1/20000 of the conjugate were determined to be used in the ELISA. Each of serum samples was tested in dublicate. The arithmetic averege optical density (OD) value of each serum sample was transformed to sample-to-positive (S/P) ratio. Of the total 150 serum samples, 15 (10%) were positive, 4 (2.66%) were low positive, 3 (2%) were suspicious and 128 (85. 33%) were negative. In comparison with a commercial ELISA kit, the sensitivity and specificity of optimized ELISA kit were 95% and 96%, respectively. Although the difference between the positivity rates according to the age of the animals was statistically significant (p˂0.05), the difference between the positivity rates according to animal breed was not significant (p> 0.05). The results of this study indicate that paratuberculosis is an important problem in Elazığ province and that protection and control programs should be applied carefully. Keywords: Paratuberculosis, Mycobacterium avium subsp. paratuberculosis, Cattle, ELISA, Diagnosis