MOLECULAR EPIDEMIOLOGY OF SALMONELLA IN CONVENTIONAL AND ANTIMICROBIAL FREE SWINE PRODUCTION SYSTEMS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0214829
• Grant No.: 2008-35201-04550
• Proposal No.: 2008-01835
• Project Start Date: Sep 1, 2008
• Project End Date: Aug 31, 2012
• Grant Year: 2008
• Program Code: [32.0B]- Food Safety and Epidemiology (B): Epidemiological Approaches for Food Safety

Project Director
Thakur, S.

Recipient Organization
NORTH CAROLINA STATE UNIV
COLLEGE OF VETERINARY MEDICINE
RALEIGH,NC 27606

Performing Department
POPULATION HEALTH AND PATHOBIOLOGY

Non Technical Summary
Foodborne outbreaks due to antimicrobial resistant Salmonella strains have been reported due to consumption of pork products. The role of unidentified selective pressures that result in persistence of antimicrobial resistant strains in different swine production environments remains to be investigated. The available information on Salmonella dynamics in the conventional system does not apply to the antimicrobial free (ABF) system due to differences in them. The need to conduct a longitudinal based study to determine and compare the risk factors that are responsible for the persistence of Salmonella in these swine production environments is crucial. The long term goal of this longitudinal study is to determine and compare the molecular epidemiology and dynamics of Salmonella in the conventional and the ABF systems. Swine sampling will be done at three stages on farm (farrowing, nursery and finishing). Salmonella isolates will be characterized at the phenotypic and genotypic levels. Pertinent management data will be collected and appropriate epidemiological and statistical analysis will be performed to determine risk factors for Salmonella dissemination in pigs at farm and in these two distinct production systems. Information generated in this study will help us to determine the establishment of Salmonella infection and its dynamics within a herd and to better implement control measures. The proposal is relevant to priorities listed under the Epidemiological Approaches to Food Safety (32.0) with focus on studies to identify new risk factors that affect the prevalence, transmission and persistence of food borne organisms across the farm-to-fork continuum.

Animal Health Component

50%

Research Effort Categories

Basic

30%

Applied

50%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1040	10%
311	3599	1100	20%
311	4010	1100	10%
315	3510	1040	10%
315	3599	1100	10%
315	4010	1100	10%
712	3599	1040	10%
712	4010	1040	10%
722	3510	1100	10%

Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 722 - Zoonotic Diseases and Parasites Affecting Humans; 315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3510 - Swine, live animal; 4010 - Bacteria; 3599 - Swine, general/other;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;

Keywords

multidrug resistant salmonella

swine

swine production systems

molecular epidemiology

multi logistic regression

risk factor analysis

Goals / Objectives
The long term goal of this longitudinal study is to determine the molecular epidemiology of multidrug resistant Salmonella in swine reared in the conventional and the antimicrobial free (ABF) productions systems. Our preliminary data clearly shows the presence of multidrug resistant (MDR) Salmonella in both the production systems. This proposal is relevant to the important priorities listed under the epidemiological approaches to food safety program and focuses on studies that seek to identify new risk factors that affect the prevalence, transmission and persistence of food borne organisms in the farm-to-fork continuum. Defining the role of these risk factors in the dissemination and persistence of antimicrobial resistant Salmonella and establish critical control points is important which will aid us in implementing intervention strategies to improve food safety. With that information, we plan to achieve our long term goal of reducing the burden of pork associated salmonellosis by pursuing these objectives: 1) Determine the sources, persistence and clonality of Salmonella in the conventional and ABF production systems at farm 2) Compare and characterize the distribution of multi-drug resistant Salmonella between and within the different stages at farm in the two systems 3) Identify farm and management level risk factors that are associated Salmonella dissemination in these two swine production systems This will be the first longitudinal study to provide valuable information to the scientific community and the stakeholders on the status of Salmonella and the risk factors involved in its dissemination in different swine production systems. The proposal is multidisciplinary and we have assembled an expert team from a wide range of fields including epidemiology, microbiology and biosciences to help us achieve our goal. The results will provide important information regarding dissemination of Salmonella in the ABF system which is currently not well defined in the United States. The data generated in this study will help to design and develop intervention methods to secure our food supply.

Project Methods
Sampling will be done over a period of two years and every conventional farm sampled will be followed by sampling of an ABF farm. A total of 20 farms will be included in the study including 10 farms each from the conventional and the ABF farms in North Carolina. We will follow a cohort of pigs (n=30) all the way from the farrowing to finishing farms. At the farrowing unit, we will sample the sows (n=10) and their piglets (n=30; 3 piglets/sow). The same cohort of pigs will be sampled twice at the nursery farm, at 6 and 10 weeks of age. At the finishing level, pigs will be sampled twice at 16 and 26 weeks of age. At every farm visit, approximately 10 grams of fresh fecal samples will collected per pig with gloved hands directly from the rectum. Samples will be transported to the laboratory on ice and processed for Salmonella on the same day upon arrival. Confirmed Salmonella isolates will be stored at -80oC till further characterization. Salmonella isolates will be serotyped and antimicrobial susceptibility testing to determine the minimum inhibitory concentration (MIC) will be done by using the broth microdilution method. Genotyping will be done using Pulsed Field Gel Electrophoresis (PFGE) and analysis will be done using the Bionumerics software. We will use polymerase chain reaction (PCR) and sequencing for molecular characterizing the antimicrobial resistance determinants. Pertinent management, facility and herd information that could possibly play a role in emergence, dissemination and persistence of antimicrobial resistant Salmonella in pigs will be collected at farm. We have considered 2 outcome variables for the study that includes a) Presence of Salmonella in the fecal samples based on microbiological culture in the two production systems at farm. This will be recorded as a dichotomous variable (absent=0 and present=1) and, b) The occurrence of antimicrobial resistant Salmonella; exhibiting resistance to a single antimicrobial or multiple antimicrobials (resistance to greater than three or more antimicrobials) in the two production systems at farm. This will be also recorded as a dichotomous variable (absent=0 and present=1). All the data analyses will be run on SAS v.9.2 (SAS Institute, Inc, 2002). A combination of different modeling techniques and statistical models will be used including multivariate analysis techniques, logistic regression and cluster analysis will be used to provide a more thorough investigation of the associations described above. In addition to between production system comparisons, comparisons within a production system will be made to investigate potential risk factors that affect the occurrence of Salmonella. Trend analysis will be undertaken to ascertain the effect of time on the proportion of Salmonella within the cohorts. Logistic regression modeling will be performed using PROC LOGISTIC, repeated measures logistic analysis will be done using PROC NLMIXED and PROC GENMOD and GEE (generalized structural equations) will be used to account for the dependency structure of the observations registered along time.

Progress 09/01/08 to 08/31/12

Outputs
OUTPUTS: The aim of this longitudinal study was to determine the dynamics of Salmonella in 30 conventional and 8 ABF cohorts at farm, slaughter and in their environment. Overall, a total of 2889 fecal and 2122 environmental samples were collected at farm. In addition, 1363 slaughter and 205 lairage and truck samples each were collected at slaughter. The overall prevalence of Salmonella in conventional and ABF pigs was 4.0% (n=66) and 0.2% (n=2), respectively. At the environment level, 11.8% (n=156) of conventional and 0.6% (n=5) ABF samples tested positive. Salmonella was isolated from 8.3% (conventional) and 5.5% (ABF) slaughter samples. In this study we identified 25 different serotypes among pigs and their environment. In the conventional system, S. Typhimurium (33.7%) was predominant among pig and their environment at farm and slaughter, followed by S. Infantis (16.4%), S.Derby (11.4%), S. Anatum (9.1%) and S. Ouakam (7.8%). In the ABF system, S. Infantis (42.8%) was the predominant serotype followed by S. Anatum (17.8%) and S. Inverness (7.7%). In addition, our study highlights the identification of S. Rissen in pigs and environment, which is rarely found in the US. The isolates were tested as per the CLSI recommended protocol against a panel of 15 antimicrobials. Isolates from conventional pigs were showed highest frequency of resistance to tetracycline (82%) followed by sulfisoxazole (48%), and ampicillin (17%). Whereas isolates from conventional environment showed highest frequency of resistance to tetracycline (88%) followed by sulfisoxazole (49%). ABF isolates from pigs and environment both showed highest frequency of resistance to tetracycline (23%). Multi drug resistant (MDR) (resistance to ≥ 3 antimicrobials) Salmonella were detected in 23% (n=257) of the isolates. Salmonella were characterized for their antimicrobial resistance profile, resistance genes, class I integrons. We detected blaTEM, blaPSE, cmlA, strA, strB, aadA1, aadA2, tetG and blaCMY-2 resistance genes by PCR. Genotypic relationships among Salmonella isolated from the pigs and their environment were detected by Pulsed Field Gel Electrophoresis (PFGE). PARTICIPANTS: Dr. Morgan Morrow: He is a swine extension veterinarian in the Department of Animal Science at NCSU. His primary role was to talk to the swine companies and producers and get them to agree to participate in this USDA project. Dr. Morgan Scott: He is an epidemiologist and his primary role has been in helping with the study design, conduct and oversight, as well as analysis and reporting of these studies as we progress in this project. Dr. Wondwossen Gebreyes: His role in the project is to genotype the Salmonella isolates in years two and three. At this stage, Dr. Gebreyes has provided us input with sample collection and technical information. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This study highlights the prevalence of antimicrobial resistant Salmonella from the conventional and ABF systems at farm and slaughter. The phenotypic and genotypic results of our study indicate that the environment plays an important role in transmission of antimicrobial resistant Salmonella to the conventional and ABF pigs at different stages of production. The prevalence of AR Salmonella in ABF pigs in the absence of selection pressure is concerning.

Publications

• Keelara, S., Gebreyes, W. A., Morrow, W. A., Scott, H. M. Correa, M., and Thakur, S. (2012). Role of environment in the persistence of antimicrobial resistant Salmonella in antimicrobial free (ABF) and conventional pigs at farm and slaughter. Conference of Research Workers in Animal Diseases (CRWAD). December 2-4, Chicago, Illinois.

Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: The aim of this longitudinal study was to determine the dynamics of Salmonella in 30 conventional and 8 ABF pigs at farms and slaughter and in their environment. Overall, a total of 2889 fecal and 2122 environmental samples were collected at farm. In addition, 1363 slaughter and 205 lairage and truck samples each were collected at slaughter. The overall prevalence of Salmonella in conventional and ABF pigs was 4.7% (n=78) and 0.4% (n=5), respectively. At the environment level, 12% (n=159) of conventional and 0.75% (n=6) ABF samples tested positive. Salmonella was isolated from 8% (conventional) and 6% (ABF) slaughter samples. The isolates were tested as per the CLSI recommended protocol against a panel of 15 antimicrobials. Isolates from conventional pigs were showed highest frequency of resistance to tetracycline (83%) followed by sulfisoxazole (52%), streptomycin (26%) and ampicillin (13%). Whereas isolates from conventional environment showed highest frequency of resistance to tetracycline (78%) followed by sulfisoxazole (32%). ABF isolates from pig and environment showed highest frequency of resistance to tetracycline (29%). Salmonella was characterized for their antimicrobial resistance profile, resistance genes, class I integrons. We detected blaTEM, blaPSE, cmlA, strA, strB, aadA1, aadA2, tetG and blaCMY-2 resistance genes by PCR. Genotypic relationships among Salmonella isolated from the pigs and their environment were detected by Pulsed Field Gel Electrophoresis (PFGE). PARTICIPANTS: 1. Dr. Morgan Morrow: He is a swine extension veterinarian in the Department of Animal Science at NCSU. His primary role was to talk to the swine companies and producers and get them to agree to participate in this USDA project. 2. Dr. Morgan Scott: He is an epidemiologist and his primary role has been in helping with the study design, conduct and oversight, as well as analysis and reporting of these studies as we progress in this project. 3. Dr. Wondwossen Gebreyes: His role in the project is to genotype the Salmonella isolates in years two and three. At this stage, Dr. Gebreyes has provided us input with sample collection and technical information. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This study highlights the prevalence of antimicrobial resistant Salmonella from the conventional and ABF at farm and slaughter. The phenotypic and genotypic results of our study indicate that the environment plays an important role in transmission of antimicrobial Salmonella to the conventional and ABF pigs at different stages of production.

Publications

• Keelara, S., Zou, M., Pierce, H., Whitsell, A., Susick, E., and Thakur, S. (2011). Prevalence, molecular characterization and genotypic analysis of Campylobacter and Salmonella from commercial poultry farms. Sigma Xi Annual Meeting and International research Conference. November 10-13, Raleigh, North Carolina.
• Veerappa, S. K., and Thakur, S. (2011). Genotypic and molecular characterization of multidrug resistant (MDR) Salmonella Typhimurium of swine and human origin. Annual CVM Research Forum, Raleigh, North Carolina.
• Veerappa, S. K., and Thakur, S. (2010). Molecular characterization and Genotypic analysis of Multidrug Resistant (MDR) Salmonella Typhimurium of swine and human origin. Sigma Xi Symposium on Food Safety and Security: Science and Policy, November 11-13, Raleigh, North Carolina.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: The aim of this longitudinal study was to determine the dynamics of Salmonella in conventional and ABF pigs at 10 different farms and slaughter and in their environment. Overall, a total of 2672 fecal and 1942 environmental samples were collected at farm. In addition, 1026 slaughter and 175 lairage and truck samples each were collected at slaughter. The overall prevalence of Salmonella in conventional and ABF pigs was 4% (n=63) and 0.5% (n=5), respectively. At the environment level, 11% (n=141) of conventional and 0.7% (5) ABF samples tested positive. Salmonella was isolated from 8% (conventional) and 7% (ABF) slaughter samples. The isolates were tested as per the CLSI recommended protocol against a panel of 15 antimicrobials and the highest frequency of resistance was detected to tetracycline in pigs and environment. Salmonella was characterized for their antimicrobial resistance profile, resistance genes, class I integrons. We detected blaTEM, blaPSE, cmlA, strA, strB, aadA1, aadA2, tetG and blaCMY-2 resistance genes by PCR. Genotypic relationships among Salmonella isolated from the pigs and their environment were detected by Pulsed Field Gel Electrophoresis (PFGE). PARTICIPANTS: Dr. Morgan Morrow: He is a swine extension veterinarian in the Department of Animal Science at NCSU. His primary role was to talk to the swine companies and producers and get them to agree to participate in this USDA project. Dr. Morgan Scott: He is an epidemiologist and his primary role has been in helping with the study design, conduct and oversight, as well as analysis and reporting of these studies as we progress in this project. Dr. Wondwossen Gebreyes: His role in the project is to genotype the Salmonella isolates in years two and three. At this stage, Dr. Gebreyes has provided us input with sample collection and technical information. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This study highlights the prevalence of antimicrobial resistant Salmonella from the conventional and ABF at farm and slaughter. The phenotypic and genotypic results of our study indicate that the environment plays an important role in transmission of antimicrobial Salmonella to the conventional and ABF pigs at different stages of production.

Publications

• Veerappa, S. K., and Thakur, S. 2010. Environmental role in the transmission of multidrug resistant (MDR) Salmonella to conventional pigs at different stage of production at farm and slaughter. Conference of Research Workers in Animal Diseases (CRWAD). December 5-7, Chicago, Illinois.
• Thakur, S., Correa, M., Morrow, W. M., and Scott, H. M. 2010. Molecular Epidemiology of Salmonella in Conventional and Antimicrobial Free Swine Production (ABF) Systems. USDA National Research Initiative Principal Investigator Annual Meeting and Institute of Food Technologists Annual Meeting. July, Chicago, Illinois.
• Veerappa., S. K., Morrow, W. E., Gebreyes, W. A., Correa, M., Scott, H. M., and Thakur, S. 2010. Role of environment in transmission of antimicrobial resistant salmonella in antimicrobial free (ABF) pigs at farm and slaughter. 110th General ASM Meeting in San Diego, California.

Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: Sample Collection at commercial and antimicrobial free (ABF) swine production systems in North Carolina Sample collection is currently underway in North Carolina. Every pig selected in this study will be sampled five times as it moves from farrowing to the finishing farms. The pigs will again be sampled at the slaughter plant at post-evisceration and post-chill stages. In addition, environmental samples will be collected at every stage at farm and slaughter. Multiple Salmonella isolates (up to three) will be collected from every positive sample to determine the diversity of the pathogen within individual pigs. To date, we have completed sample collection at five ABF farms including the farm and slaughter stages. In addition, two commercial farms are currently being sampled in their final sampling stages at the finishing farms. Questionnaires and other related information at farm and slaughter is being collected from individual farm owners and currently being coded and analyzed. Salmonella prevalence among pigs and carcasses in North Carolina: Two pigs out of 706 pigs in ABF farm (0.28%) and four pigs out of 197 pigs in Commercial farm (1.93%) were positive. Salmonella prevalence at slaughter level is 15.6% (35 positive out of 223 samples). At post chill we isolated the pathogen from five carcass of pigs reared in the ABF production system. A total of 183 isolates were isolated from both ABF and Commercial farms (farm: 83 and slaughter: 100). Antimicrobial susceptibility testing of Salmonella isolates: We are in the process of testing the Salmonella isolates for their antimicrobial susceptibility profile. The isolates were tested as per the CLSI recommended protocol. The isolates were tested for their susceptibility against 15 antimicrobials namely: Amikacin, ampicilin, amoxicillin/clavulanic acid, ceftriaxone, chloramphenicol, ciprofloxacin, trimethoprim/sulfamethaxazole, cefoxitin, gentamycin, kanamycin, nalidixic acid, sulfisoxazole, streptomycin, tetracycline and ceftiofur. So far we have tested 55 isolates out of 183 isolates. Salmonella isolates from slaughter exhibited maximum frequency of resistance against tetracycline (69.09%, n=38) where as only two out of 16 from farm were resistant to tetracycline, streptomycin and kanamycin. PARTICIPANTS: Individuals 1. Dr. Morgan Morrow: He is a swine extension veterinarian in the Department of Animal Science at NCSU. His primary role is to talk to the swine companies and producers and get them to agree to participate in this USDA project. All the farms that we have enrolled and sampled in this project so far have been arranged through the extension work done by him. Dr. Morrow will also be responsible for the extension and education aspects of this proposal as the project enters in years two and three. 2. Dr. Morgan Scott: He is an epidemiologist and his primary role has been in helping with the study design, conduct and oversight, as well as analysis and reporting of these studies as we progress in this project. The specific tasks that he will contribute to in the proposed research relate to initial design and implementation of the study, oversight of conduct and compliance and sample size issues, descriptive and analytical approaches for data and reporting. He will assist with presenting and discussing the findings at key scientific meetings. So far, we have utilized his expertise in designing the questionnaires. 3. Dr. Wondwossen Gebreyes: His role in the project is to genotype the Salmonella isolates in years two and three. At this stage, Dr. Gebreyes has provided us input with sample collection and technical information. 4. Deena Bermudez: Deena has been hired through this USDA grant to work as a field technician. She has been responsible for scheduling, arranging and packing for the farm trip. She has also aided in sample processing and Salmonella isolation in this project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Impact This study highlights the prevalence of antimicrobial resistant Salmonella from both the commercial and the ABF at farm and slaughter. Even though only a few pigs were positive at the farm level, a higher number of carcass swabs came out positive at the slaughter plant indicating that the slaughter plant environment is possibly playing an important role in Salmonella transmission.

Publications

• No publications reported this period