An immunocapture assay for the detection of Mycobacterium avium subsp. paratuberculosis for the diagnosis of Johne's disease.

摘要

Paratuberculosis or Johne's disease caused by Mycobacterium avium subsp. paratuberculosis (Map) is an infectious, progressive chronic digestive disorder of both wild and domestic ruminants and has great economic impact especially in the cattle industry. Due to the absence of a sensitive detection assay for identifying the infected animal at an early phase, the infection can spread silently throughout a herd. Antibiotic treatment is not recommended and culling is the only solution to the problem. In this study, we developed a diagnostic assay that captured and concentrated Map using antibody followed by amplification using polymerase chain reaction (PCR) to detect the organisms. Purified antibodies IgY were produced from eggs by injecting Map into breast muscles of Single Comb White Leghorn chickens followed by direct coating of the surface of cellulose/iron oxide beads (MagaCell(TM)-IgY beads) or indirect coating via MagaBeads(TM) rabbit anti-chicken IgG linker for capturing Map from bovine feces. Optimization for the immunocapture was performed with respect to holding time, temperature, volume and types of immunocapture beads. DNA extracted from the captured bacteria by both methods was amplified by PCR to determine the sensitivity and specificity of the assay. MagaCell(TM)-IgY beads proved to be better than indirect capture and were selected for the field study using bovine feces from positive and negative Johne's disease farms. There was 100% specificity and the sensitivity by real-time and conventional PCR after immunocapture and immunocapture culture were 88.6%, 77.3% and 61.4% respectively. In the category of heavy to moderate growth of Map colonies on culture slants, the sensitivities of real-time PCR and conventional PCR after immunocapture and immunocapture culture were 94.4%, 88.9%, 94.4% but the sensitivities dropped to 84.6%, 69.2% and 38.5% in the category of scant to light growth by culture. Conventional PCR was less sensitive than real-time PCR and inhibition was observed in field samples with conventional PCR after immunocapture. By using this immunocapture assay in combination with front-end automation for DNA extraction and real-time PCR, 50 samples can be processed within an eight hour shift by one person. This rapid turn-around time for reporting is a great improvement over conventional fecal culture that requires 8 to 16 weeks. Therefore this immunocapture-PCR assay has great potential as a routine test for Map in bovine feces for Johne's disease in a diagnostic laboratory.