Introduction.
Increased sample submission for laboratory testing for the
detection of Mycobacterium avium subsp. 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
for owner management decision making.
Materials and
methods. 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 Alert™ 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.
Results: 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.
Conclusion. 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.
Acknowledgements:
Financial support for this work was provided by the USDA
Agricultural Research Service Cooperative Grant (58-1265-3-115) and
by USDA-APHIS-VS field studies funding.
