Simple and nesting polymerase chain reaction, both readily identify Mycobacterium avium subspecies paratuberculosis (Map) DNA in blotted tissue impressions done on bovine tissue of cows with documented Johne's disease.

Fourteen cows, primarily derived from a Holstein herd located in Alachua County (Florida, USA) were subjected to necropsy. Each animal had been documented to have clinical Johne's disease confirmed by ELISA titer and nesting PCR done on one or more of its biological fluids. Tissue samples from ileum and mesenteric and ileocecal lymph nodes were tested by P90P91 and J1J2 primers (simple and nested PCR respectively).

The objective of this note is to report the results when blotted tissue impressions of bovine tissue obtained from Map diseased dairy cows are probed with primers P90P91 for IS900 versus primers P90P91 which are followed by a second set of primers J1J2.

Samples were first probed with primers P90P91 which recognized a 413 bp sequence of Map insertion sequence IS900. A second set of primers, J1J2 which overlapped and spanned a 333 bp region within the insertion sequence was then used as nested pcr.. Sections were stained with hematoxylin-eosin stain. At least one representative section was stained with acid-fast stain to determine the density of bacilli present.

The J1J2 primers tested positive in 11 instances in which P90P91 primers had tested negative, and P90P91 primers tested positive in one instance not identifiedby the J1J2 primers. The addition of the J1J2 set of primers appears to extend the sensitivity of PCR analysis for Map DNA in bovine tissue. Simple PCR using primers P90P91 identified Map in 64% of the ileocecal lymph nodes, 69% of the mesenteric lymph nodes and 57% of the ileal tissue samples from cows with documented Map enteritis. The addition of the J1J2 set of primers identified 100% of ileocecal lymph nodes, 100% of the mesenteric lymph nodes and 86% the ileal tissue. The blotted tissue impression method offers an additional means of rapid identification of Map within diseased tissue.