Correlation between Herrold’s egg yolk medium culture results and quantitative real-time PCR for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental slurry samples

Authors: ALY, SHARIF - University Of California; MANGOLD, BEVERLY - Tetracore, Inc; ANDERSON, RANDALL - California Department Of Food And Agriculture; JIANG, JIMING - University Of California; SHUKKEN, YNTE - Cornell University; HOVINGH, ERNEST - Pennsylvania State University; WOLFGANG, DAVID - Pennsylvania State University; Van Kessel, Jo Ann; Karns, Jeffrey; LOMBARD, JASON - US Department Of Agriculture (USDA); SMITH, JULIA - University Of Vermont; WHITLOCK, ROBERT - University Of Pennsylvania; GARDNER, IAN - University Of California

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2010
Publication Date: 9/1/2010
Citation: Aly, S.S., Mangold, B., Anderson, R.J., Jiang, J., Shukken, Y., Hovingh, E., Wolfgang, D., Van Kessel, J.S., Karns, J.S., Lombard, J., Smith, J.M., Whitlock, R.H., Gardner, I.A. 2010. Correlation between Herrold’s egg yolk medium culture results and quantitative real-time PCR for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental slurry samples. Journal of Veterinary Diagnostic Investigation. 22:677-683.

Interpretive Summary: Johne’s disease is an infectious disease of cattle caused by infection with the bacterium, Mycobacterium avium subsp. paratuberculosis (MAP). A large number of US dairy herds are infected with MAP and the dairy industry incurs large economic losses as a result of Johne’s disease. Diagnostic testing for MAP is critical for management and successful control of this disease. The ‘gold standard’ of testing for the presence of MAP in feces or environmental samples uses traditional bacterial culture methods. However, MAP is a very slow growing microorganism and test results can take as long as 4 months to obtain. This four month lag from sample collection to result makes it very difficult for producers to make effective use of the test results. A method of testing for the presence of MAP DNA has been developed and is commercially available. This method is a real-time PCR method that can be completed in less than 2 days after the sample is collected. The objective of the present study was to estimate the relationship between the quantitative results of the PCR method and the culture results in fresh and frozen pooled fecal and environmental slurry samples and model the association between results of both assays. Nearly 2000 pooled fecal samples from cows in 14 herds in 4 states (CA, NY, PA, VT), and 802 environmental samples from 113 dairies nationwide were tested for the level of MAP contamination using both methods. Sometimes samples must be frozen between collection and analysis so frozen samples were also tested. The study showed that results of using a commercially-available PCR analysis to quantify MAP in fresh and frozen pooled samples, such as environmental slurry and pooled fecal samples of different size pools, were strongly correlated to quantitative results of traditional culture methods. This further validates this new, rapid method of analysis as a useful tool in the management of Johne’s disease.

Technical Abstract: Quantitative real-time PCR (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold’s egg yolk medium (HEYM), the traditional ante-mortem reference test for MAP. Although the sensitivity and specificity of these two tests has been described using binary results (test-negative and test-positive), the correlation between qPCR cycle (Ct) values and colony-forming units (CFU) on HEYM has not been evaluated. The objective of the present study was to estimate the correlation of qPCR results to HEYM culture results in fresh and frozen pooled fecal and environmental slurry samples and model the association between results of both assays. Quantitative HEYM culture results for 1997 pooled fecal samples from cows in 14 herds in 4 states (CA, NY, PA, VT), and 802 environmental samples from 113 dairies nationwide were correlated with their respective qPCR results. The correlation between assays was good (-0.66) in both fresh and frozen pooled fecal samples and excellent (-0.76) and good (-0.61) in fresh and frozen environmental slurry samples, respectively. Furthermore, the correlation varied from good (-0.53) to excellent (-0.90) with different size fecal pools. Truncated linear regression models indicated a significant negative association between CFU and Ct in fecal pools of all sizes and in both individual and pooled environmental slurry samples. The use of qPCR instead of HEYM can yield more timely quantitative MAP detection on a herd basis and allow for incorporation of qPCR in dairy herd testing strategies to reduce the risk of MAP transmission.