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Title: Real-time PCR testing of pooled (1:5) fecal samples comparison to HEYM culture

Author
item WHITLOCK, R - U. OF PENNSYLVANIA
item MANGOLD, B - TETRACORE INC.,MARYLAND
item MCADAMS, S - U. OF PENNSYLVANIA
item FYOCK, T - U. OF PENNSYLVANIA
item SWEENEY, R - U. OF PENNSYLVANIA
item SCHUKKEN, Y - CORNELL UNIVERSITY,NY
item SMITH, J - UNIVERSITY OF VERMONT
item Van Kessel, Jo Ann
item HOVINGH, E - PENN STATE UNIVERSITY
item Karns, Jeffrey
item WOLFGANG, D - PENN STATE UNIVERSITY
item JOHNSON, T - VET.SRVCS.USDA,VERMONT

Submitted to: International Colloquium on Paratuberculosis
Publication Type: Proceedings
Publication Acceptance Date: 9/24/2007
Publication Date: 10/30/2007
Citation: Whitlock, R.H., Mangold, B.L., Mcadams, S., Fyock, T., Sweeney, R., Schukken, Y., Smith, J., Van Kessel, J.S., Hovingh, E., Karns, J.S., Wolfgang, D., Johnson, T. 2007. Real-time PCR testing of pooled (1:5) fecal samples comparison to HEYM culture. International Colloquium on Paratuberculosis, Tsukuba, Japan. No.75b. http://www.paratuberculosis.org/pubs/proc9/abst75b_o1.htm.

Interpretive Summary:

Technical Abstract: Increased sample submission for laboratory testing for the detection of Mycobacterium subsp avium paratuberculosis (MAP) in bovine fecal has necessitated utilization of techniques to enhance efficiency of MAP detection. Pooling of fecal samples offers a method to enhance diagnostic efficiency when coupled with real-time PCR (RT-PCR) with minimal loss of test sensitivity. Most importantly, the action cut-point for the identification of individually infected cattle can be adjusted to account for herd prevalence and for owner management decision making. Individual fecal samples from 736 cows in four dairy herds were processed by standard techniques for the detection of MAP and 1:5 pools were created concurrently for both culture and for RT-PCR with the Tetracore Vet AlertTM Johne’s Real-Time PCR assay. For the purposes of this investigation all individual and pooled fecal samples were cultured using the standard three day culture protocol with four tubes of HEYM. The 1:5 pools were created by transfer of five ml of each standard fecal water tube-step to a 50 ml conical tube. From the 25 ml of pooled fecal water tube, 5 ml was transferred to 25 ml of BHI and incubated overnight; centrifuged at 900 X G for 30 minutes and the pellet was re-suspended in 1 ml of antibiotic brew on day 2 and incubated overnight. On the third day the sample was vortexed and approximately 200 ul inoculated on the surface of each of four tubes of HEYM with mycobactin J. The remaining 20 mls of the pooled fecal water tube was centrifuged at 900 X G for 30 minutes, decanted and the pellet re-suspended in water and processed according to manufacturer’s recommendations for RT-PCR. Samples tested by RT-PCR were assayed in duplicate wells. Of the 148 pooled fecal samples representing 736 individual cows, 34 pools were RT-PCR positive on both wells. Of the 34 RT-PCR positive pools, 19 had all individual samples within the pools tested where 18/19 (95%) contained at least one RT-PCR positive individual sample. Culture identified MAP in 14/34 (41%) of the RT-PCR positive pools and in 26/170 (15.2%) of the individual samples from those 34 pools. Only one pool was RT-PCR positive where all individual samples were both RT-PCR negative and culture negative. Of the 31 culture positive fecal samples among the 736 tested, only one individual fecal sample (1, 0, 0, 0) was not detected by either RT-PCR or by culture in the 1:5 pool. An additional 24 pools had one of two wells positive on RT-PCR. These represented lower concentrations of MAP with Ct values between 37 and 42, the cut-off value for a negative sample. Of the 9 positive pools with one positive well and all individual samples tested by RT-PCR, 8/9 (89%) had at least one positive individual sample. Only four (3.5%) individual fecal samples within the 115 fecal samples represented by the 24 pooled samples were culture positive, all were low shedders and ¾ in one pooled sample. The use of a commercially available RT-PCR with pooled fecal samples offers a very economical, flexible, rapid and exquisitely sensitive method to identify those MAP infected cattle at the greatest risk to spread MAP infection to herd-mates. Only individual samples in pools with the highest concentration of MAP (lowest Ct values) need to be tested for MAP, thus significantly reducing the testing cost for the herd.