Dipstick immunoassay for rapid diagnosis of paratuberculosis in small ruminants

Highly sensitive and specific gold nanoparticle based dipstick immunoassay using protoplasmic antigen of Mycobacterium avium subspecies paratuberculosis (MAP) for diagnosis of paratuberculosis was introduced. In this method the colloidal gold nanoparticles (GNPs) were coated with MAP protoplasmic antigen using alkanethiols derivatives and anti-MAP rabbit antibodies. These antigen coated GNPs acts as a detector reagent in this assay. The antibody (test sera) immobilized onto nitrocellulose (NC) membrane binds with antigen coated GNPs and this was detected visually by development of red color (due to gold nanoparticles) on the nitrocellulose membrane (NC). This immunoassay was specific to MAP when compared with other common mycobacterial species. In addition, the dipstick immunoassay was able to detect an antibody dilution of 1:50 of polyclonal anti-MAP antibody raised in rabbit. Further the efficacy of this dipstick immunoassay was evaluated by comparing this test to other serological tests like agar gel immunodiffusion (AGID) and absorbed ELISA in detecting MAP antibodies in sheep and goats. Out of 536“531” changed to 536 and percentage was also changed sera samples (271 sheep and 265 goats) collected from different parts of India, positive results recorded were; AGID 79 (14.74%), dipstick immunoassay 83 (15.49%), and absorbed ELISA 88 (16.42%). Though dipstick immunoassay was less sensitive compared to absorbed ELISA, but it was simple to perform in field conditions and requires less time. This dipstick immunoassay was also compared in live animals using intradermal Johnin test and nested PCR (detecting mycobacterial DNA in feces) in 65 animals (35 sheep and 30 goats) of our institutional animal houses. Of which, positive results recorded in animals were; Johnin test 21 (32%), dipstick immunoassay 26 (40%) and fecal PCR detected mycobacterial DNA in 28 (43%) animals. Though fecal nested PCR for mycobacterial DNA gave best results, dipstick immunoassay might be considered for large scale use in field condition for on-site screening of paratuberculosis.