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 for 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 degrees C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42 degrees C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37 degrees C followed by overnight enrichment in RV10 at 42 degrees C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42 degrees C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viable Salmonella bacteria. Presumptive Salmonella isolates were biochemically and serologically 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) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invA gene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement between Salmonella detection using cultivation with preenrichment and detection 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 positive samples, while Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detecting Salmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study.
Feder
I
I
Microbial Food Safety Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, Wyndmoor, Pennsylvania 19038, USA. ifeder@arserrc.gov
Nietfeld
J C
JC
Galland
J
J
Yeary
T
T
Sargeant
J M
JM
Oberst
R
R
Tamplin
M L
ML
Luchansky
J B
JB
D000822:Animal Husbandry; D000818:Animals; D015139:Blotting, Southern; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D004587:Electrophoresis, Agar Gel; D005243:Feces / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012481:Salmonella Infections, Animal / Q000382:microbiology*; D019779:Salmonella enterica / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*; D014871:Water Microbiology
D003470:Culture Media; D004269:DNA, Bacterial