Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA

The qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67？% of the reactions. It also showed 100？% specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85？% (±21？%). When tested on the field samples, HYDEqPCR showed 89？% of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19？%, 36？% and 1？%, respectively. Fisher’s exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant.This study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.Johne’s disease, or paratuberculosis, is a chronic granulomatous enteritis that predominantly affects ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (Map). Infections with Map in bovine herds result in significant economic loss in the dairy industry, through slow progressive wasting and the subsequent death of the infected animals [1]. However, although Map-infected animals might not show symptoms of Johne’s disease for two to