Development and Standardization of Visual Loop Mediated Isothermal Amplification (LAMP) Essay for Specific Diagnosis of Johne’s Disease

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

Comparitive study on the efficacy of ELISA and IS900 PCR for the diagnosis of Paratuberculosis in goats

Paratuberculosis, one of the chronic granulomatous enteritis that predominantly affects ruminants worldwide, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is most efficiently diagnosed by MAP from faeces by Polymerase Chain Reaction (PCR). Serological tests like Enzyme Linked Immuno Sorbent Assay (ELISA) also provides a rapid and cost-effective alternative diagnostic tool. Present study was carried out to directly evaluate the sensitivity and specificity of ELISA (ID vet innovative diagnostics; France) using IS900 PCR as a gold standard. Serum and faecal samples were collected from 180 adult goats of either sex, from Malappuram and Thrissur districts of Kerala with unknown paratuberculosis status. Faecal samples were processed for direct IS900 PCR and serum samples were tested for MAP antibodies using Indirect ELISA kit. IS900 PCR detected 38 out of 180 confirmed to be shedding MAP. ELISA detected 22 out of 180 animals as positive. Overall, ELISA was 50 % sensitive and 97.9 % specific in comparison to IS900 PCR. The IS900 PCR outperformed ELISA in detecting animals potentially infected with MAP and is more sensitive than ELISA at detecting animals suspected of paratuberculosis. But, for early diagnosis of paratuberculosis in goats, ELISA can be done as easy and rapid farm level identification and IS900 PCR as individual confirmatory test.