Use of Direct LAMP Screening of Broiler Fecal Samples for Campylobacter jejuni and Campylobacter coli in the Positive Flock Identification Strategy

Abstract

Rapid identification of Campylobacter-positive flocks before slaughter, following freezing and heat treatment for the Campylobacter-positive carcasses at the slaughterhouses is an effective control strategy against foodborne campylobacteriosis. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the direct screening of naturally contaminated chicken cloacal swabs for C. jejuni/C. coli to compare this assay with conventional quantitative culture methods. In a comparison study of 165 broilers, the LAMP assay showed 82.8% (48/58 by conventional culture) sensitivity, 100% (107/107) specificity, 100% (48/48) positive predictive value (PPV), and 91.5% (107/117) negative predictive value (NPV). In a comparison of 55 flocks, LAMP showed 90.5% (19/21) sensitivity, 100% (34/34) specificity, 100% (19/19) PPV, and 94.4% (34/36) NPV. In the cumulative total of 28 farm-level comparisons, LAMP showed 100% (12/12) sensitivity, 100% (16/16) specificity, 100% (12/12) PPV, and 100% (16/16) NPV. The LAMP assay required less than 90 min from the arrival of the fecal samples to final results in the laboratory. This suggests that the LAMP assay will facilitate the identification of C. jejuni/C. coli-positive broiler flocks at the farm level or in slaughterhouses before slaughtering, which would make it an effective tool in preventing the spread of Campylobacter contamination.

A Rapid LAMP-Based Method for Screening Poultry Samples for Campylobacter Without Enrichment

“…A recent publication ( Sabike et al, 2016 ) reported the use of Campylobacter LAMP assay for direct detection in chicken fecal samples. They achieved detection of 3.89 and 3.60 log cfu × g −1 of fecal sample for C. jejuni and C. coli , respectively, in artificially contaminated fecal samples, and a three-step DNA precipitation protocol was used to improve sensitivity.…”

“… Sabike et al (2016) reported that their analysis of naturally contaminated specimens from one particular farm showed a high frequency of false negative results for Campylobacter loadings ranging from <3 to 5.81 log cfu × g −1 . The authors discussed that this high rate of false negatives may be explained by inhibitors of DNA amplification, possibly derived from substances in the poultry feeding stuffs.…”

“…Rapid and robust testing systems are needed for implementation on-site to inform rapid action. Other methods for detection and identification of Campylobacter have been published in recent years ( Velusamy et al, 2010 ), including antibody-based detection ( Wadl et al, 2009 ), PCR ( Josefsen et al, 2004 ; Leblanc-Maridor et al, 2011 ; de Boer et al, 2015 ), DNA microarrays ( Quinones et al, 2007 ; Donatin et al, 2013 ), and loop-mediated isothermal DNA amplification (LAMP) ( Yamazaki et al, 2009 ; Dong et al, 2014 ; Sabike et al, 2016 ). Nevertheless, most procedures are still not fully adequate to be deployed on-site, as they involve specialized skills or facilities to perform certain steps such as DNA extraction or enrichment culture.…”

A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production

“…LAMP was developed for the detection of Campylobacter spp. in poultry samples such as meat, carcass swabs, and fecal samples (Yamazaki et al, 2008, 2009b; Sabike et al, 2016; Romero and Cook, 2018; Sabike and Yamazaki, 2019). To study the epidemiology of Campylobacter spp.…”

“…However, to detect the presence of the Campylobacter spp. in cloacal swabs, ceca, meat and environmental cleaning samples, an enrichment step of 22 to 24 h is needed (Yamazaki et al, 2009b; Sabike et al, 2016; Sabike and Yamazaki, 2019). Consequently, it takes a total of at least 24–26 h for sample enrichment, preparation, amplification, and detection.…”