Problems in Isolation of Campylobacter jejuni from Frozen-Stored Raw Milk and Bovine Fecal Samples: Genetic Confirmation Using Multiplex PCR

Abstract

The objectives of this study were to evaluate the use of various protocols for the isolation of Campylobacter jejuni from bulk tank milk and bovine fecal samples that were stored frozen for varying times, and to develop a rapid DNA-based protocol that distinguishes C. jejuni from other thermophilic Campylobacter spp. The pathogen was recovered from fecal samples that had been stored for 96-251 days at -20 degrees C with glycerol as the cryopreservative. In a separate study, C. jejuni-positive bovine fecal samples were stored at 5 degrees C for up to 70 days without compromising subsequent recovery of the pathogen. However, the pathogen could not be recovered from pathogenpositive fecal samples stored with or without glycerol (5 mL/11 g sample) for 21 days at -20 degrees C. Bolton broth (BB) and Bolton broth with 5% blood (BBB), containing BB supplement, were used for enrichment. Bacterial isolation was achieved by streaking from BB and BBB, and filtering from BBB onto blood-free charcoal cefoperazone deoxycholate agar (CCDA). The use of BB for the recovery of Campylobacter was more sensitive than BBB, and streaking achieved better isolation rates than filtration. Multiplex PCR incorporating thermophilic Campylobacter-specific 23S rRNA and C. jejuni-specific hippuricase gene sequences was used to confirm C. jejuni. All 265 bulk tank milk samples analyzed were negative for C. jejuni, whereas five of 411 (1.2%) fecal samples tested positive. This is the first report that has used a combination of sequences of the two genes in a multiplex format to identify C. jejuni to the species level. The method described has potential for routine use in the detection of thermophilic Campylobacter in farm environmental samples as well as other samples.

Tetracycline Resistance Genes in Campylobacter jejuni and C. coli Isolated From Poultry Carcasses

“…The melted samples were cultured in an enriched broth media containing tryptic soy broth (TSB) (30 g/L), dextrose (2.5 g/L), sodium thioglycolate (0.5 g/L), rifampicin (10 mg/L), trimethoprim (10 mg/L), vancomycin (10 mg/L), ceftriaxone (10 mg/L), amphotericin-B (10 mg/L), incubated in a microaerophilic atmosphere (Anaerocult C, Merck) and at 37°C for hours, followed by incubation at 42°C for 44 hours ( 8 ). Unfortunately, due to the sensitive nature of this bacterium in frozen conditions ( 9 ), we could not retrieve the live bacteria. The DNA extraction was carried out directly on the culture media, using phenol-chloroform extraction ( 10 ).…”

Applications of Biotechnology in Today's Dairy Industry

“…We have also used a multiplex PCR format to confirm and identify Campylobacter jejuni isolated from the dairy farm environment and from dairy cows. 25 Campylobacter is a leading cause of bacterial foodborne illness in the US and in many other industrialized countries. This organism is widespread in nature and can be isolated from the gastrointestinal tracts of many animal species, including poultry, freshwater and bulk tank milk.…”