Recombinant secretory proteins based new ‘Cocktail ELISA’ as a marker assay to differentiate infected and vaccinated cows for Mycobacterium avium subspecies paratuberculosis infection

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.

‘Indigenous’ and ‘Ethanol Vortex’ ELISA kits for diagnosis of Mycobacterium avium subsp. paratuberculosis infection in cattle: Is there a ‘globally relevant kit’ in the ‘Reverse Ice-burg’ environment?

Highly versatile and robust 'Indigenous ELISA kit' for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in cattle herds was compared with 'Ethanol Vortex (EV) ELISA kit' of USA. Of 160 (118 vaccinated and 42 non-vaccinated) cattle screened, 129 and 35 were positive in 'Indigenous' and 'Ethanol Vortex' ELISA kits, respectively. 'I-ELISA', using 'semi-purified protoplasmic antigen' from native highly prevalent biotype ('Indian Bison type') of MAP of goat origin, was highly sensitivitive (91.4%) as compared to the 'EV-ELISA. 'I-ELISA kit using whole cell sonicate from native 'S 5' (‘Indian Bison type’) strain of MAP as 'antigen source' was significantly superior than EV-ELISA kit using surface antigens from 'Linda' strain ('cattle type') of cattle origin in USA. Therefore, 'i-ELISA kit' may be recommended for the screening of domestic cattle herds against MAP infection in India. The present study has demonstrated that in the diagnosis of chronic infections and diseases, such as Johne's disease, 'Indigenous kits' are significantly superior to kits made in other countries, EV-ELISA, in the present case, particularly screening of native cattle herds endemically infected with MAP.

Evaluation of goat based ‘Indigenous vaccine’ against Bovine Johne’s Disease in endemically infected native cattle herds.

‘Indigenous vaccine’ prepared from ‘Indian Bison Type’ a native bio-type of Mycobacterium avium subspecies paratuberculosis strain ‘S5’ of goat origin (goat based) was evaluated in indigenous cattle herds located in gaushalas (cow shelters), endemic for Bovine Johne’s disease. Cows (893) were randomly divided into vaccinated (702 = 626 adults + 76 calves) and control (191 = 173 adults + 18 calves) groups. Response to vaccination was evaluated on the basis of health (mortality, morbidity), productivity (growth rate, reproductive performance, total milk yield), immunological parameters (LTT, ELISA titer), survivability of animals naturally infected with MAP, bacterimia (by specific blood PCR), sero-conversion (by indigenous ELISA) and status of shedding of MAP in feces (by microscopy) in the two groups before and after vaccination. Reduction in MAP shedding [to the extent of 100% in Herd A; and from 82.1% (0 DPV) to 10.7% (270 DPV) in Herd C] was the major finding in vaccinated cows. Whereas, the control group cows have shown no improvement. As the first indicator of vaccine efficacy, MAP bacilli disappeared from the blood circulation as early as 15 days post vaccination, however, peak titers were achieved around 90 DPV. Peak titers initially declined slightly but were maintained later throughout the study period. Control animals did not show any pattern in antibody titers. Mortality was low in vaccinated as compared to the control groups. Vaccination of endemically infected native cattle herds with inactivated whole-cell bacterin of novel ‘Indian Bison Type’ bio-type of goat origin strain ‘S5’ effectively restored health and productivity and reduced clinical BJD. Application of goat based ‘indigenous vaccine’ for therapeutic management of BJD in native cattle herds (gaushalas) is the first of its kind.

Evaluation of ‘cattle’ and ‘Indian Bison’ type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of Bovine Johne’s Disease using ‘indigenous ELISA’ and AGPT .

Two antigens (‘cattle’ type and ‘Indian Bison’ type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne’s disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows.