Effects of shipping and storage conditions of fecal samples on viability of Mycobacterium paratuberculosis.

Johne’s disease is characterized by chronic granulomatous enteritis of ruminants caused by Mycobacterium paratuberculosis infection. Early diagnosis of infected animals is necessary to reduce contamination of the environment and to control disease spread through feces (3, 6). About 75% of positive animals are either low or very low shedders (7, 8). Diagnostic samples for M. paratuberculosis identification are generally collected, shipped, and stored under various conditions that may influence the viability of M. paratuberculosis (2, 4, 5), thus changing the true status of the infected animal; in fact, the culture status especially of individual low or very low shedder animals could change from positive to negative. The aim of the present study was to mimic the most relevant conditions of shipment and storage of clinical samples obtained from naturally infected cattle and to study the effects of these conditions on the detection of M. paratuberculosis by standard bacteriological culture and real-time PCR. Fecal samples were collected from 11 cows confirmed to have Johne’s disease; samples were transported on ice and stored as described in Table 1. Fecal samples were tested for colony counts on Herrold’s egg yolk agar slant (three tubes each for three dilutions with mycobactin J and one without mycobactin J) (9) and by immunomagnetic bead capture of M. paratuberculosis from feces and extraction of genomic DNA from captured M. paratuberculosis, followed by PCR (1). Several of the firstand second-dilution tubes had an M. tuberculosis organism too-numerous-to-count status; thus, data analyses were restricted to dilution 3. The mean colony counts at week 16 were compared across treatments, using analysis of variance (ANOVA) (SPSS version 13.0 software for Windows; SPSS Inc., Chicago, IL). Pairwise comparisons of treatment means between treatment groups were performed with the Tukey correction for multiple comparisons. Results for each tube were dichotomized into positive or negative, based on the presence of at least one colony. Fisher’s exact test (intercooled STATA version 9.2; STATA, College Station, TX) was used to compare the proportions for positive tubes and cattle. Significance was defined as a P value of 0.05 for all analyses. Pairwise comparisons (ANOVA) between treatment means identified a significantly higher number of colonies with treatment 5 than with treatment 3 (P 0.04). Treatments 1, 2, 4, 5, and 6 had a significantly higher proportion of positive tubes than treatment 3. At the individual animal level, significantly larger proportions of cattle were classified as positive for treat-