Field study on bovine paratuberculosis using real-time PCR and liquid culture for testing environmental and individual fecal samples implemented in dairy cow management

摘要

Bovine paratuberculosis (Johne’s disease) is a bacterial,chronic, and wasting intestinal disease causedby Mycobacterium avium ssp. paratuberculosis (MAP).Johne’s disease causes severe losses in dairy farmproductivity and is also suspected to be a potentialtrigger for Crohn’s disease in humans. The fecal-oralinfection of MAP to neonates is recognized as an importantwithin-herd transmission route. Our objectivewas to recommend diagnostic methods for herds withsuspected paratuberculosis requiring fast results, aswell as for herds with breeding programs or others thataim at being nonsuspected of paratuberculosis infection.We determined a period of 8 wk from sampling todiagnostic findings suitable for testing of cows duringthe dry period. We therefore tested environmental andindividual fecal samples with one rapid and one highlysensitive diagnostic method. Environmental samples(boot swabs) were taken as a first step in 3 herds andtested using a DNA extraction protocol for feces andsubsequent real-time PCR (referred to as fecal PCR).Additionally, cultivation in liquid medium for 6 wk wasperformed and verified with real-time PCR (referredto as liquid culture). Automation of DNA extractionbased on magnetic beads and the PCR setup wasperformed with pipetting robots. As a result, we successfullydetected MAP in boot swabs of all herds byboth methods. In a second step, 245 individual fecalsamples from the 3 herds were examined using alsofecal PCR and liquid culture. The results obtainedby fecal PCR were compared with detection of MAPusing cultivation in liquid medium for 6 wk. Testingindividual cows, we identified MAP-specific DNA in 53fecal samples using the liquid culture. Using fecal PCR,we revealed 43 positive samples of which 39 also testedpositive in the liquid culture, revealing MAP-positivecows in all 3 herds. The fecal PCR procedure allowsrapid detection of MAP-specific DNA with 74% of thesensitivity of liquid culture. For the purpose of testingwith maximal sensitivity, cultivation in liquid mediumis recommended. Cultivation of MAP in liquid mediumM7H9C means a significant time gain in comparison tocultivation on solid media, which requires twice as muchtime. Thus, this testing fits within the 6- to 8-wk dryperiod of gravid cows and provides test results beforecalving, a prerequisite to prevent fecal-oral transmissionto newborn calves.