Pennsylvania Animal Diagnostic Laboratory System
PADLS Conferences Spring 2001 7. Detection M. paratuberculosis
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Objectives:

1. To determine the differential sensitivity of pooling fecal samples from cows with known M. paratuberculosis infection
2. To evaluate two different pooling methods
3. To estimate the homogeneity of M. paratuberculosis in bovine fecal samples.

Procedures:

Ten known positive fecal samples were selected from the University of Pennsylvania repository (6 light shedders with less than 10 colonies per tube and 3 moderate shedders with 1- to 30 colonies per tube and one heavy shedder with more than 50 colonies per tube). One known uninfected cow, based on previous history of no life time exposure to infected cattle and repeated negative fecal cultures was selected as the source of the uninfected samples.

Pools of 5 and 10 fecal samples, each with one positive fecal sample and either 4 or 9 uninfected fecal samples were compared. Two methods on fecal pooling were evaluated, in method A the resuspended fecal pellets (just prior to inoculation of the culture tubes) were mixed together and for method B, the fecal samples were mixed together in a small glass beaker with a sterile wooden stick for approximately one minute, then a two grams of the resultant fecal mixture was used for the sample processing with the double incubation centrifugation method.

Results:

Combining fecal samples prior to processing provided similar culture sensitivity as did combining the fecal pellets just prior to tube inoculation. Pools containing known positive fecal samples with higher colony counts were more likely to be detected than those with a lower colony counts.

Pooled culture detected M. paratuberculosis in 60% of pools as compared to 90% and 80% for individual cow samples. Within the low fecal shedding group, pooled culture detected M. paratuberculosis in 43% of pools compared to 83% and 67% for individual samples. Within the moderate to high shedding group, pooled culture detected 85% of the pools compared to 100% of the eight individual cow cultures at each replicate.

Discussion:

Based on the results of these experiments, pooled fecal cultures offers an alternative to ELISA testing to detect herd infections with M. paratuberculosis. Pools of ten fecal samples combined together prior to fecal sample processing for culture provided similar sensitivity of detection as did mixing the fecal pellets, thus reducing the technician time to process the samples for culture. Additional experiments need to be completed in infected herds to compare pooled fecal cultures to ELISA for the detection of M. paratuberculosis in herds of cattle.

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