Liquid culture has been used to identify Mycobacteria in liquid culture for several decades, only recently have non-radiometric liquid culture methods been available for M. paratuberculosis. Our laboratory has evaluated a number of variables in both sample processing and culture media to enhance MAP growth and reduce time to detection (TTD).

Objectives

1) To develop an enhanced sample processing method and improved media formulation to reduce TTD. 2) To determine the relationship between MAP cfu in the sample to TTD.

Materials and methods

Sample processing components evaluated included: fecal sample size; inoculum size; HPC percentage; antibiotic concentrations and types: (ANV, PANTA and Nisin). Media component variables included: egg yolk (EY) volume; EY sources; species of EY, OACC, and OADC, among others.

Results

Increased EY volume up to 2,000 ul per tube reduces TTD by the equivalent of nearly two logs of MAP compared to 60 ul EY. Both ANV and PANTA reduce TTD compared to no antibiotics, but vancomycin is more inhibitory than naladixic acid or amphotericin B. Nisin has promise since it has minimal inhibitory effect on MAP growth. Different sources of EY have little effect on MAP growth in liquid culture. Reduced HPC percentage (0.3%) is well tolerated with little increase in contamination, and on occasion does enhance MAP recovery, but not consistently. Increased inoculum size reduces TTD up to a limit of 1,000 ul but does result in a higher contamination rate. Increased MAP concentrations reduce TTD proportionately. Repeated experiments with MAP alone, MAP spiked fecal samples and field samples positive on HEYM showed a high degree of correlation between TTD and MAP cfu in the original sample.

Conclusion

Liquid culture system with MGIT has proven to be a robust diagnostic culture system that has the potential to replace traditional culture using solid media (HEYM).