Assessment and genetic analysis of Salmonella and Campylobacter from various stages of an integrated No-Antibiotics-Ever (NAE) broiler complex

Authors: ADHIKARI, YAGYA - Auburn University; BAILEY, MATTHEW - Auburn University; KREHLING, JAMES - Auburn University; KITCHENS, STEVEN5 - Auburn University; GAONKAR, PANKAJ - Auburn University; MUNOZ, LUIS - Auburn University; ESCOBAR, CESAR - Pulseforge Inc; Buhr, Richard; LAURA, HUBER - Auburn University; PRICE, STUARD - Auburn University; BOURASSA, DIANNA - Auburn University; MACKLIN, KENNETH - Auburn University

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/8/2024
Publication Date: 8/14/2024
Citation: Adhikari, Y., Bailey, M.A., Krehling, J.T., Kitchens, S., Gaonkar, P., Munoz, L.R., Escobar, C., Buhr, R.J., Laura, H., Price, S.B., Bourassa, D.V., Macklin, K.S. 2024. Assessment and genetic analysis of Salmonella and Campylobacter from various stages of an integrated No-Antibiotics-Ever (NAE) broiler complex. Poultry Science. 103(11):104212. https://doi.org/10.1016/j.psj.2024.104212.
DOI: https://doi.org/10.1016/j.psj.2024.104212

Interpretive Summary: Salmonella and Campylobacter are the leading bacterial foodborne pathogens that are frequently associated with consumption or mishandling of raw or undercooked poultry meat and meat products. The objectives of the research were to isolate these two pathogens from a wide range of sample sites in the production of broiler chickens and determine potential risk factors from 840 Salmonella and 531 Campylobacter on farm environmental samples. The 11 different sample types were collected from two pullet rearing farms, four breeder egg-production farms, a hatchery, nine broiler growout farms, transport coops, and a processing plant, all from an integrated No-Antibiotics-Ever (NAE) broiler complex located in south-east region of the United States. All samples were analyzed utilizing the 3M-Molecular Detection System (MDS) for rapid screening of these pathogens and suspect positive samples were further processed for confirmation of result and identification. Based on MDS results, 4.5% (38/840) of samples were positive for Salmonella while 18% (97/531) of total samples were positive for Campylobacter. The pathogens were present in almost all the production stages (except the breeder farms where no Salmonella was recovered). The odds of occurrence of Salmonella were not significantly different among various stages of broiler production. However, the odds of occurrence of Campylobacter in breeder farms (34%) and processing plant (35%) was significantly higher than pullet (17%) and broiler farms (12.6%) followed by the hatchery (2%) and transport coops (0%). Among the 11 different sample types, boot swabs were best useful for predicting the occurrence of Salmonella as compared to other sample types, while boot swabs, soil samples and carcass rinses were equally useful for predicting the occurrence of Campylobacter among other sample types. Altogether 16 Salmonella and 17 Campylobacter isolates were positive on culture and were further processed for Illumina sequencing. The Salmonella serotypes identified in this study were S. Typhimurium (25%), S. Barranquilla (25%), S. Liverpool (19%), S. Kentucky (12%), S. Enteritidis (6%), S. Luciana (6%), and S. Rough O:r:1,5 (6%). The species identified for Campylobacter were C. jejuni (10%) and C. coli (90%). From core genome-based phylogeny results, the Salmonella isolates recovered from water puddles/drainage near the entrance of broiler farmhouses had a close genetic relation with isolates recovered from swabs of transport coop floors as well as from fly papers placed outside the processing plant near the live birds unloading area. Similarly, the C. coli isolates recovered from boot swabs of broiler farmhouses, cow feces outside the breeder and broiler farmhouses and carcass rinses in the processing plant had close genetic relation based on phylogeny results. With these findings, it can be inferred that the surroundings of poultry-houses and processing facilities include various risk factors that could transfer these foodborne pathogens into the poultry complex, and hence, potentially to the consumers.

Technical Abstract: Salmonella and Campylobacter are the leading bacterial foodborne pathogens that are frequently associated with consumption or handling of raw poultry meat and meat products. With the objectives of isolating these pathogens from a wide range of samples and detecting potential risk factors, a total of 840 Salmonella and 531 Campylobacter farm environmental samples were collected for isolating Salmonella and Campylobacter respectively, from two pullet farms, four breeder farms, a hatchery, nine broiler farms, transport, and a processing plant of an integrated No-Antibiotics-Ever (NAE) broiler complex located in south-east region of the United States. All the samples were analyzed utilizing the 3M-Molecular Detection System (MDS) for rapid screening of these pathogens and suspect positive samples were further processed for confirmation of result and identification. The core genome-based phylogenies were built for both Salmonella and Campylobacter isolated from this study along with the genomes downloaded from NCBI based on environmental sources. Data were analyzed with R version 4.3.1 using Generalized Linear Model for binomial distribution. The odds ratio and confidence limits were calculated to separate groups for the stages of broiler complex and 11 different sample types. The level of significance was measured at a = 0.05 and 95% C. I. Based on MDS results, 4.52% (38/840) of samples were positive for Salmonella while 18.27% (97/531) of total samples were positive for Campylobacter. The pathogens were present in almost all the stages (except the breeder farms for Salmonella). The odds of occurrence of Salmonella wereas not significantly higher among various stages. However, the odds of occurrence of Campylobacter in breeder farms and processing plant was significantly higher (??.??%. P < 0.05) than pullet and broiler farms followed by hatchery and transport. Among 11 different sample types, boot swabs were best useful for predicting the occurrence of Salmonella as compared to other sample types, while boot swabs, soil samples and carcass rinses were equaluseful for predeitecting the occurrence of Campylobacter among other sample types. Altogether 16 Salmonella and 17 Campylobacter isolates were positive on culture and were further processed for Illumina sequencing. The Salmonella serotypes identified in this study were S. Typhimurium (25%), S. Barranquilla (25%), S. Liverpool (198.75%), S. Kentucky (12.5%), S. Enteritidis (6.25%), S. Luciana (6.25%), and S. Rough_O: r: 1,5 (6.25%). The species identified for Campylobacter were C. jejuni (10.53%) and C. coli (89.47%). From core genome-based phylogeny results, the Salmonella isolates recovered from water puddles/drainage near the entrance of broiler farmhouses had a close genetic relation with isolates recovered from swabs of transport truck/ cages’ floors as well as from fly papers placed outside the processing plant near the live birds unloading area. Similarly, the C. coli isolates recovered from boot swabs of broiler farmhouses, cow feces outside the breeder and broiler farmhouses and carcass rinses in the processing plant had close genetic relation based on phylogeny results. With these findings, it can be inferred that the surroundings of poultry-houses and processing facilities include various risk factors that couldan transmit these foodborne pathogens into the poultry complex, and hence, potentially to the consumers. It also provides information about the genetic relatedness among the collected isolates to further determine the dynamics of these pathogens along the poultry production through processing food chain.