|Sung, K - UGA|
Submitted to: American Association of Avian Pathologists
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 24, 2003
Publication Date: January 30, 2004
Citation: Sung, K.D., Stern, N.J., Hiett, K.L. Relation of mrna reverse transcriptase polymerase chain reaction signal with campylobacter spp colonization of chicks. Avian Diseases 48:254-262. 2004. Interpretive Summary: Campylobacter, the most frequent cause of human bacterial diarrhea in the United States, is commonly associated with poultry. Discrimination of viable and dead cells is of importance in identifying sources involved in transmission. A positive molecular signal [RT-PCR] has been considered to indicate the presence of live cells because this molecular signal has an extremely short half-life. Chicken colonization by cells with positive signal, but which are not cultivable, would provide a measure to correlate cell viability. In addition, the role of a viable but not cultivable (VBNC) form of Campylobacter for infection of poultry could be verified. We observed levels of four Campylobacter poultry isolates held in the refrigerator. These levels declined progressively over time and could not be detected after 6 to 7 weeks. Cold-stored, not cultivable and heat-inactivated Campylobacter produced inconsistent signal from RT-PCR assay. These organisms producing RNA signal but which were not cultivable did not establish colonization in the intestine of chicks after challenge. These results call to question the relationship of RT-PCR, cell viability and the significance of the VBNC cells in environmental transmission of Campylobacter spp
Technical Abstract: Discrimination of viable from dead cells is of importance in the development of bacterial detection methods. A positive RT-PCR amplification signal has been considered to indicate the presence of viable cells because mRNA has an extremely short half-life. However, some researchers have suggested that the presence of mRNA is not well correlated with cell viability following heat inactivation as mRNA might also be amplified by Reverse Transcriptase-PCR (RT-PCR) after cells are dead. Chicken colonization by cells having positive mRNA signal, but which are nonculturable, would provide a measure to characterize the correlation between cell viability and persistence of mRNA. In addition, the role of a viable but nonculturable (VBNC) form of Campylobacter spp. for infection of poultry could be verified. The levels of four Campylobacter spp., previously isolated from poultry feces, declined progressively over time and loss of culturability occurred after 6 to 7 weeks incubation in phosphate buffed saline (PBS) at 4oC. Cold-stored, nonculturable and heat-inactivated (60oC for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay depending on the target genes and strains used, though all fresh cultures showed mRNA signals. Mostly, signals of mRNA species from VBNC and heat-killed Campylobacter spp. AH-1, AH-2 and CH-3 persisted. RT-PCR amplification of tkt, porA, and a 256 bp amplicon from a previously described putative haem-copper oxidase provided consistent signal while flaA did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not culturable by conventional culture-based methods, did not establish colonization in the intestine of chicks seven days after challenge. These results question the correlationship between mRNA durability as assayed by RT-PCR and cell viability, as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.