Relationship of messenger RNA reverse transcriptase-polymerase chain reaction signal to Campylobacter spp. viability.

SUMMARY. Discriminating viable from dead cells is of importance in the development of bacterial detection methods. A positive reverse transcriptase-polymerase chain reaction (RT-PCR) amplification signal was tested as a potential predictor of chick colonization. Some researchers have suggested that the presence of messenger RNA (mRNA) may not correlate with cell viability. Chicken colonization by cells that have positive mRNA signal but that are noncultivable would provide a correlation in cell viability and persistence of mRNA. The role of a viable but noncultivable (VBNC) form of Campylobacter spp. for colonization of poultry could be verified by such an mRNA signal. The levels of four strains of Campylobacter spp., previously isolated from poultry feces, declined progressively over time, and loss of cultivability occurred after 6 to 7 wk incubation in phosphate-buffered saline (PBS) at 4 C. Cold-stored, noncultivable and heat-inactivated (60 C for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay, depending on the target transcripts and strains used, although all fresh cultures showed mRNA signals. For the most part, signals of mRNA species from VBNC and heat-killed Campylobacter spp. AH-1, AH-2, and CH-3 persisted. RT-PCR amplification of transcripts originating from the tkt and cmp genes and a 256-base pair amplicon (from a previously described putative haem-copper oxidase) provided consistent signals, whereas transcripts from the flaA gene did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not cultivable by conventional culture-based methods, did not establish colonization in the intestine of chicks 7 days after challenge. These results lead us to question the correlation between mRNA durability with cell viability as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.

The optimization of serum-free medium for the production of the scu-PA by the addition of algal extracts.

The addition of 2.8 µg/ml algal extracts enhanced both scu-PA production and cell growth in a serum-free medium, compared to a conventional serum-free medium for the cultivation of recombinant CHO cells. The growth rate and scu-PA production were relatively lower in the serum-free medium than 5% serum containing medium: however, specific scu-PA production rate was higher in the serum-free medium due to the long-term period of cultivation (3.66×10(-4) vs. 2.48×10(-4) IU/cell/day). Overall scu-PA production rate was also greater in an enforced serum-free medium as 25,000 IU/day over 50 d of perfusion cultivation. The conversion ratio of scu-PA to tcu-PA was greatly reduced in the serum-free medium during perfusion cultivation (10% compared to 20% conversion in a serum containing medium).