Comparison of PCR versus Culture for Detection of Mycobacterium bovis after Experimental Inoculation of Various Matrices Held under Environmental Conditions for Extended Periods

The purpose of this study was to compare the performance of a molecular detection technique (nested PCR) with that of mycobacterial culture in the detection of Mycobacterium bovis DNA in a set of 687 samples of experimentally inoculated environmental substrates (hay, soil, corn, water) exposed to natural weather conditions in Michigan. Four replicates of each substrate were used; half were autoclaved for sterilization, all were inoculated with 50,000 CFU ofM. bovis isolated from Michigan livestock, and all were placed in outdoor enclosures, with half under shade and the other half exposed to direct sunlight. Samples were tested for the presence ofM. bovis during one 12-month period, with monthly sample testing and during three 12-week periods (winter, spring, summer) with weekly sample testing. Samples were subjected to mycobacterial culture for isolation ofM. bovis and a nested PCR with two primer sets targeting IS6110 to detectM. bovis DNA. In 128 samples tested during the 12-month period, M. bovis was not detectable by culture after 2 months butM. bovis DNA was detectable by PCR for at least 7 months. Of the 559 samples tested during the 12-week periods, PCR detectedM. bovis DNA for up to 88 days in all of the sample types. There were no significant differences in the detection ofM. bovis between shade and sun samples or between sterile and unsterilized samples, regardless of the detection method (PCR or culture). For use in epidemiologic investigations, the PCR assay was more rapid than mycobacterial culture, was not hindered by contaminating organisms, and detectedM. bovis DNA in environment samples much longer after initial contamination than mycobacterial culture did.