Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples

A total of 150 fecal and water samples from four swine farms were tested fo r the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37 de greesC followed by overnight enrichment in Rappaport-Vassiliadis 10 broth ( RV10) at 42 degreesC; (ii) 24 water samples from the third farm were preenr iched overnight in 3MC broth at 37 degreesC followed by overnight enrichmen t in RV10 at 42 degreesC; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42 degreesC with no addi tional enrichment, Following each of the enrichment techniques, samples wer e subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar pri or to transfer to Hektoen Enteric agar plates for the recovery of viable Sa lmonella bacteria. Presumptive Salmonella isolates were biochemically and s erologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) d erived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridizatio n with a 20-mer oligonucleotide probe specific for the Salmonella invA gene . Neither the standard microbiological method nor the molecular method dete cted all of the 65 samples that tested positive by both methods or either m ethod alone. Salmonella bacteria were detected by both cultivation and PCR- hybridization in 68% (17 of 25) of the positive samples that were preenrich ed in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC bro th, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreemen t between Salmonella detection using cultivation with preenrichment and det ection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 posit ive samples, while Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approac h is equivalent to or better than cultivation for detecting Salmonella in s wine feces or water samples from swine farms when using the medium combinat ions evaluated in this study.