Indian Journal of Experimental Biology Vol. 57, December 2019, pp. 967-972 Recombinant secretory proteins based new ‘Cocktail ELISA’ as a marker assay to differentiate infected and vaccinated cows for Mycobacterium avium subspecies paratuberculosis infection Kundan Kumar Chaubey*, Shoor Vir Singh* & Ashok Kumar Bhatia Department of Biotechnology, GLA University, Chaumuhan, Mathura, Uttar Pradesh-281 406, India Received 03 January 2018; revised 03 December 2018 Johne's disease is endemic in the domestic livestock population of India. Recently, we developed highly effective 'Indigenous vaccine' to control Johne’s disease in animals. In order to gain disease free status as per World Organization for Animal Health, it is essential to have a marker assay to differentiate between infected and vaccinated animals before vaccine can be used in the field. We have developed a new marker assay ‘Cocktail ELISA’ using six ‘recombinant secretary proteins’ (MAP 1693c, MAP 2168c, MAP Mod D, MAP 85c, MAP Pep AN and MAP Pep AC) and evaluated for diagnosis of Johne’s disease along with 'Indigenous ELISA kit'. This ‘Cocktail ELISA’ successfully differentiated the infected, vaccinated and healthy (non-infected) cows and will facilitate the use of Johne’s disease vaccine to control the disease in cows at national levels. Keywords: Cows, DIVA marker assay, Johne’s disease, Livestock, MAP infection, Ns_PPA Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic granulomatous debilitating disease of domestic livestock leading to high morbidity and huge economic losses 1,2 . MAP is widely distributed in domestic and wild ruminants and also reported from rabbits, primates and human beings in India 3-7 . Due to high economic / production losses, World Organization for Animal Health (OIE) recommended restrictions in the international trade of JD infected animals. JD control and eradication program is severely hampered by limited knowledge of host factors controlling immune response to MAP 8 and unrecognized sub-clinical cases of MAP infection. ‘Test and cull’ approach has been frequently used in the developed countries 9 to stamp out the disease. In view of the failure of ‘test and cull’ methodology in local conditions here, we developed 'indigenous vaccine' against JD 10 . Inactivated/live attenuated JD vaccine are applied in many countries for a period of 5-35 years for controlling JD and were able to reduce JD incidence from 87.5 to 5% 10-12 . However, as per Office International des Epizooties (OIE), before 'indigenous vaccine' can be used in the field, it is essential to have a marker assay which can Differentiating Infected from Vaccinated Animals (DIVA). In the absence of DIVA assay, it is not possible to launch any control program using whole cell 'Indigenous vaccine'. In developing DIVA marker assay, selection of antigens is a major challenge. Antigens should be pathogen specific for sensitive and specific detection of MAP. Cho et al. 13 identified fourteen proteins with potential diagnostic value for JD. They reported that secretory proteins/cultural filtrate proteins (CFPs) of MAP showed greater reactivity with MAP positive cows sera, with respect to number of antigens detected and the reaction intensity on western blots. These MAP CFPs are good antigen candidates for the development of improved sero-diagnostic tests for bovine JD. Use of CFPs increased sensitivity of ELISA by 25% over the commercial ELISAs for low MAP shedders in cows 14 . However, selection of antigens remains major challenge, since there is no single MAP-specific antigen that is recognized by all the stages of infected animals, especially those in early and sub-clinical stages of the disease. Six recombinant secretory proteins (RSPs) of MAP were used to develop MAP based cocktail ELISA as 'marker assay', and their ability to differentiate infected and vaccinated animals. Materials and Methods Samples Profile Faecal and serum samples were driven from cow available in 4 Gaushalas of the states [Rajasthan (RJ), Haryana (HR), Uttar Pradesh (UP) and Madhya Pradesh (MP) in 3 years (2013–2016). Four gaushalas volunteered to participate in the ‘vaccine trial’. A total of 458 cows (82-Yadu dairy farm, Alwar, Rajasthan; ———— *Correspondence: Phone: +91 5662 250900-09 Ext. 756; Fax: +91 5662 241687 E-mail: shoorvir.singh@gmail.com (SVS); kundan2006chaubey@gmail.com (KKC)