Source: NORTH CAROLINA STATE UNIV submitted to
AN INTEGRATED APPROACH TO DETERMINE SALMONELLA DYNAMICS IN THE NICHE-MARKET ANTIMICROBIAL-FREE SWINE PRODUCTION SYSTEM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
0214304
Grant No.
2008-51110-04341
Cumulative Award Amt.
(N/A)
Proposal No.
2008-01580
Multistate No.
(N/A)
Project Start Date
Sep 1, 2008
Project End Date
Feb 28, 2013
Grant Year
2008
Program Code
[111]- National Integrated Food Safety Initiative
Project Director
Thakur, S.
Recipient Organization
NORTH CAROLINA STATE UNIV
COLLEGE OF VETERINARY MEDICINE
RALEIGH,NC 27606
Performing Department
(N/A)
Non Technical Summary
Non-typhoidal Salmonella serovars have been known to be among the most common bacterial foodborne pathogens worldwide, and are known to be important reservoirs of antimicrobial resistance. There is an increasing emphasis on eliminating Salmonella in swine during pre-harvest production, and in swine-products during post-harvest processing in order to ensure the safest pork for human consumption. Recent increases in demand for niche-market antimicrobial-free (ABF) pork highlight the importance to determine Salmonella dynamics in these production systems. The goal of our integrated longitudinal study is to determine the epidemiology and risk factors that aid in the dissemination and persistence of antimicrobial-resistant Salmonella among swine reared in both intensive (indoor) and the extensive (outdoor) ABF production systems. Our central hypothesis is that unique sets of conditions (e.g., exposure to variable management and environment) in the two major ABF systems favors the dissemination and persistence of antimicrobial-resistant Salmonella strains that colonize and persist in the host and their environment. This longitudinal study is the first study of its kind in the US and will focus on defining the role played by ecological and management risk factors in the emergence, dissemination and persistence of antimicrobial resistant Salmonella in ABF swine production systems. Integral to this project is the preparation and evaluation of extension and education materials for swine-industry stakeholders that will provide them pertinent information on the best practices to be undertaken to maximize pre-harvest food safety.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113510110010%
3113510117010%
3114010110010%
3114010117020%
7123510110010%
7123510117010%
7124010110010%
7124010117020%
Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3510 - Swine, live animal; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1170 - Epidemiology;
Goals / Objectives
The goal of the proposed integrated study is to determine the epidemiology and risk factors that aid in the dissemination and persistence of antimicrobial resistant Salmonella among swine reared in the intensive (indoor) and the extensive (outdoor) antimicrobial free (ABF) production system. An important component of this proposal is the preparation and evaluation of extension and education material for the stakeholders that will provide them pertinent information on the best practices to be undertaken to maximize pre-harvest food safety. The proposal will address two important priority areas of the National Integrated Food Safety Initiative priority areas including: 1) Filling knowledge gap about sources and persistence of microbial pathogens in meat and applying control measures to reduce these pathogens (111.D), and 2) Providing food safety education and training for farmers, industry and small farm vendors and processors (111.B) This will be the first longitudinal study conducted in the US to determine the risk factors that are responsible for the dissemination and persistence of antimicrobial resistant Salmonella in the ABF production system. With this information, we plan to achieve our long term goal of reducing the burden of pork associated human cases of salmonellosis by pursuing these objectives: 1. Research Objectives: 1.1) To determine the sources of Salmonella in the intensive and extensive ABF production systems on farm and at slaughter. 1.2) To compare the distribution of antimicrobial resistant Salmonella among ABF pigs and their environment at the phenotypic and genotypic levels. 1.3) To identify the risk factors at the ecological and management levels that are associated with the dissemination and persistence of Salmonella among ABF pigs. 2. Education Objectives: To develop and implement new curricula aimed at providing education and training of veterinary, animal and food science students for generating a safe and wholesome ABF pork. 3. Extension Objectives: To convey the results of the study to stakeholders including swine producers, swine veterinarians and production companies via seminars and gatherings and address the crucial aspects that will make the final pork product safe for human consumption. Expected Results. The results will provide important information to stakeholders regarding dissemination and persistence of antimicrobial resistant Salmonella in the different ABF system which is currently not well defined in the United States. We expect the outcome of our education component will result in a better trained and educated student in veterinary, animal or food sciences focus. We believe the student will be more aware of the challenges posed by the foodborne pathogens in the pre-harvest food safety area and ways to counteract them. The information generated in this study using a scientific approach will greatly benefit the ABF swine industry by providing them options to mitigate Salmonella challenge at farm and slaughter. We strongly believe in that this study will provide the groundwork to conduct quantitative risk assessments in various ABF swine production systems.
Project Methods
Sample Collection and Processing: A total of 20 farms (30 pigs per farm) will be included in the study including 10 farms each from the intensive and extensive ABF farms with pigs sampled twice at nursery and finishing stages. Sample collection at the slaughter plant will include post-evisceration and post-chill samples stages and the mesenteric lymph nodes. Environmental samples will include five samples each of the barn and truck floor drag swabs. We will also collect feed and soil samples (5 samples weighing approximately 10 gm each in sterile whirl pack bags). Salmonella isolation will be done following standard bacteriological methods. Pertinent management and herd data that could play a role in dissemination and persistence of Salmonella in pigs will be collected. Salmonella Characterization: We will follow the Kauffman-White scheme for serotyping Salmonella. Antimicrobial susceptibility testing will be done using the broth microdilution method to determine the minimum inhibitory concentration (MIC) against a panel of 15 antimicrobials following the manufacturer recommendations. Salmonella isolates will be genotyped using Pulsed Field Gel Electrophoresis as recommended by CDC. We will use polymerase chain reaction for characterizing the genes, integrons and transposons coding for antimicrobial resistance. We will sequence the resistance coding regions to determine how similar or dissimilar they are from different sources. Data Analysis: Statistical analysis will be done for two outcome variables including the 1) presence of Salmonella in the fecal and environmental samples based on microbiological culture in the two ABF production systems at farm and slaughter and, 2) The occurrence of antimicrobial resistant Salmonella; exhibiting resistance to a single antimicrobial or multiple antimicrobials in the two production systems. The data for sample type including Salmonella positive samples, MIC values, serotype, antimicrobial resistance profile and patterns will be entered into a Microsoft Access database. All the data analyses will be run on SAS v.9.2. Education: Information generated in the study will be developed into educational materials and disseminated among veterinary, animal and food science students. We will combine the information generated in this study with pertinent pre- and post-harvest food safety issues that will be discussed with students in a didactic setting. Formal in class-evaluations of the material and information disseminated among the students will be collected. Extension: Information will be disseminated to the swine producers in the Healthy Hog Seminar series which is held every fall in North Carolina and attended by swine veterinarians, hog producers and stakeholders to exchange information pertaining to swine production. Data generated will also be presented at the North Carolina Veterinary Association meetings that are held six times a year. We will host four workshops in collaboration with NC A&T, Texas A&M and The Ohio State University. Evaluation of extension efforts will be performed using questionnaire survey at meetings as well as through links on the web site.

Progress 09/01/08 to 02/28/13

Outputs
Target Audience: The target audiences for this work include the groups in the swine industry, food safety, and public health. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? 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. Thakur, S., Magestro, L., and Quintana-Hayashi, M. (2012). Molecular Epidemiology of Campylobacter coli isolated from Conventional and Antimicrobial-free (ABF) Swine and their Environment. International Association for Food Protection. July 22-25, Providence, Rhode Island. Quintana-Hayashi M., Magestro, L., Kobylanski, A., Whitesell, A., and Thakur, S. (2012). Multilocus Sequence Typing and Phylogenetic Analysis of Campylobacter Isolated from Conventional and Antimicrobial-Free (ABF) Swine and their Environment. Seventh Annual North Carolina State University Graduate Student Research Symposium. March 20th, Raleigh, North Carolina. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A total of 59 Salmonella isolates were recovered from ABF pigs, environment at farm, and slaughter. Salmonella prevalence was low in ABF pigs (0.16%; n=2) and environment (0.62%; n=5) (P < 0.01). At slaughter, Salmonella was isolated from all the stages including post chill. The isolates exhibited highest frequency of resistance to tetracycline including ABF pigs (60.0%) and its environment (21.4%). The PFGE analysis identified 100% similarity among the pig and environmental Salmonella isolates in both at farm and slaughter. Therefore, there is definite role of the environment in transmission of AR Salmonella in the two production systems. Clostridium difficile was isolated predominantly in the farrowing stage of production (23%) with a significant reduction in prevalence at the nursery (1.4%) and finishing stages (0.2%; P=0.001). At slaughter, C. difficile was isolates rarely in carcass samples, and not in the MLN. Repeated sampling of the farm environment revealed higher C. difficile prevalence in soil (17.9%) and swab (40%) samples at the farrowing stage, which decreased significantly over time. C. difficile was isolated from a single feed sample at farrowing, but not from any water provided to the pigs at any production state. All environmental samples collected at the finishing stages were negative. The majority of isolates collected were tcdA+tcdB+cdtb+ (96.3%) and Toxinotype V (81.3%). The remaining isolates were Toxinotype XIII. Campylobacter coli was the predominant species identified in this study (n=1235, 99.8%), with the remaining isolates C. jejuni (n=4, 0.2%). Overall, Campylobacter prevalence in environmental samples collected on farm mirrored the trends seen in fecal prevalence, with a higher number of isolates recovered from the nursery and finishing stages of production. At slaughter, Campylobacter was detected in 73% of PE, 11% of MLN and 1% of PC samples. In the slaughter environment, Campylobacter was recovered from 21% of lairage swabs but not from the truck transporting pigs from farm to slaughter. A total of 100 representative C. coli isolates selected from fecal, environmental, and carcass samples were typed by MLST. The majority of the C. coli isolates belonged to ST-854 (n=31), and were grouped in clonal complex ST-828 (68%). The index of association (IsA) for the ABF was IsA= 0.1513. C. coli populations were close to linkage equilibrium, indicative of a freely recombining population. A minimum spanning tree revealed the close clustering of C. coli STs that originated from swine and carcass with those from the environment. Our results highlight the role of the environment in the persistence and dissemination of AR Campylobacter, Salmonella, and C. difficile in alternative swine production systems that do not use antimicrobials. The environmental reservoirs present in ABF farms could potentially explain antimicrobial resistance in pigs that were not exposed to antimicrobials. The presence of these pathogens at slaughter, particularly the post chill carcass, is also concerning and closely relates to public health risks.

Publications


    Progress 09/01/11 to 08/31/12

    Outputs
    OUTPUTS: The objective of this study is to determine the dynamics and the risk factors associated with the transmission of Salmonella in the antibiotic free (ABF) swine production system. However, Dr. Thakur used his new faculty start-up funds and further isolated and characterized Campylobacter and Clostridium difficile from the same group of pigs and their environment. By 2011, we had completed all aspects of sampling for this project and progressed to phenotypic and molecular characterization of the Salmonella (n=59), Campylobacter (n=1,239) and C. difficile (n=107) isolates obtained during sampling. Antimicrobial susceptibility testing was performed on all confirmed isolates. Campylobacter and Salmonella isolates were tested for antimicrobial susceptibility by broth micro dilution method using the Sensititre semi-automated system. The minimum inhibitory concentration (MIC) was determined using the CVM1AGNF plate for Salmonella and the CAMPY plate for Campylobacter isolates against 15 and 9 antimicrobials, respectively, of both human and animal health importance. The minimum inhibitory concentration (MIC) levels of the C. difficile isolates were determined for six antimicrobials using E-test strips (bioMerieux, Inc., Durham, NC, USA) containing gradients of antimicrobial concentrations plated on Mueller-Hinton plates with 5% sheep blood. The protocol and breakpoints were determined according to Clinical Laboratory Standards Institute (CLSI) recommendations. All the resistant Salmonella isolates were screened for the presence of class 1 Integron and corresponding resistance genes based on their AR phenotypes. The primers used in for detection of different AR genes are as follows: ESBL genes-blaTEM and blaPSE , blaCMY-2 genes encoding tetracycline resistance tet(A), tet(B) and class I integrons, aminoglycosides-aad A1/A2 and strA/B, chloramphenicol-cml, kanamycin-aphAI, and sulfisoxazole-sul2. All resistant Campylobacter isolates were tested for the presence of the tet(O) gene, using primers DMT1-F and DMT2-R following the protocol described previously. Genotyping diversity among all Salmonella and a representative 100 Campylobacter isolates was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), respectively. C. difficile isolates were further characterized using toxinotyping. PFGE was performed according to the PulseNet protocol. For MLST, seven housekeeping genes (aspA, glnA, gltA, glyA, pgm, tkt, uncA) were PCR amplified and sequenced. Sequence data was analyzed and queried against the pubmlst.org database and new allele sequences and sequence types were submitted. Statistical analyses were performed to compare prevalence, antimicrobial resistance and genetic relationships among Salmonella, Campylobacter, and C. difficile. The results of the study were disseminated in multiple international and national conferences and extension related activities. The details of the conference presentations are listed under the publication section. Under extension activities, the results of the study were disseminated to the swine producers through presentations and individual meetings. PARTICIPANTS: 1. Dr. Siddhartha Thakur (PI). Dr. Thakur was responsible for the overall coordination and successful completion of the project. 2. Dr. Morgan Morrow: His primary role was to talk to the swine companies and producers and get them to agree to participate in this USDA project. He was also involved in the extension related activities in the project. 3. Dr. Morgan Scott: He is an epidemiologist and his primary role was to help with the study design, conduct and oversight, as well as analysis and reporting of these studies in the project. 4. Dr. Maria Correa: She was responsible for statistical analysis of the data as well as performing the multivariate logistic regression for risk factor analysis. 5. Dr. Shivaramu Keelara: He is the graduate student who has contributed significantly to the project and has been involved in the sample collection, processing and characterizing of the isolates. TARGET AUDIENCES: These include the scientific community, swine and other food animal industry professionals swine producers, veterinarians, national food animal institutions including the National Pork Board. PROJECT MODIFICATIONS: Not relevant to this project.

    Impacts
    A total of 59 Salmonella isolates were recovered from ABF pigs, environment at farm, and slaughter. Salmonella prevalence was low in ABF pigs (0.16%; n=2) and environment (0.62%; n=5) (P < 0.01). At slaughter, Salmonella was isolated from all the stages including post chill. The isolates exhibited highest frequency of resistance to tetracycline including ABF pigs (60.0%) and its environment (21.4%). The PFGE analysis identified 100% similarity among the pig and environmental Salmonella isolates in both at farm and slaughter. Therefore, there is definite role of the environment in transmission of AR Salmonella in the two production systems. Clostridium difficile was isolated predominantly in the farrowing stage of production (23%) with a significant reduction in prevalence at the nursery (1.4%) and finishing stages (0.2%; P=0.001). At slaughter, C. difficile was isolates rarely in the PE (2.2%) and PC (2.5%) swabs, and not in the MLN. Repeated sampling of the farm environment revealed higher C. difficile prevalence in soil (17.9%) and swab (40%) samples at the farrowing stage, which decreased significantly over time. C. difficile was isolated from a single feed sample at farrowing, but not from any water provided to the pigs at any production state. All environmental samples collected at the finishing stages were negative. The majority of isolates collected were tcdA+tcdB+cdtb+ (96.3%) and Toxinotype V (81.3%). The remaining isolates were Toxinotype XIII. Campylobacter coli was the predominant species identified in this study (n=1235, 99.8%), with the remaining isolates C. jejuni (n=4, 0.2%). Overall, Campylobacter prevalence in environmental samples collected on farm mirrored the trends seen in fecal prevalence, with a higher number of isolates recovered from the nursery and finishing stages of production. At slaughter, Campylobacter was detected in 73% of PE, 11% of MLN and 1% of PC samples. In the slaughter environment, Campylobacter was recovered from 21% of lairage swabs but not from the truck transporting pigs from farm to slaughter. A total of 100 representative C. coli isolates selected from fecal, environmental, and carcass samples were typed by multilocus sequence typing (MLST). The majority of the C. coli isolates belonged to ST-854 (n=31), and were grouped in clonal complex ST-828 (68%). The index of association (IsA) for the ABF was IsA= 0.1513. C. coli populations were close to linkage equilibrium, indicative of a freely recombining population. A minimum spanning tree revealed the close clustering of C. coli STs that originated from swine and carcass with those from the environment. Our results highlight the role of the environment in the persistence and dissemination of AR Campylobacter, Salmonella, and C. difficile in alternative swine production systems that do not use antimicrobials. The environmental reservoirs present in ABF farms could potentially explain resistance in pigs that were not exposed to antimicrobials. The prevalence of AR bacterial strains in food animals raised in the ABF production systems where no antimicrobials are used for either treatment or prophylaxis is a cause for concern.

    Publications

    • Quintana-Hayashi, MP and Thakur, S, 2012. Longitudinal study of the persistence of antimicrobial-resistant Campylobacter strains in distinct swine production systems on farms, at slaughter, and in the environment. Appl. Environ. Microbiol. 78(8): 2698-2705.
    • Quintana-Hayashi, MP, Thakur, S, 2012. Phylogenetic analysis reveals common antimicrobial resistant Campylobacter coli population in antimicrobial free (ABF) and commercial swine systems. Plos One 7(9): e44662.
    • Susick, E, Putnam, M, Bermudez, DM, Thakur, S, 2012. Longitudinal study comparing the dynamics of Clostridium difficile in conventional and antimicrobial free pigs at farm and slaughter. Vet. Microbiol. 157: 172-178.
    • Keelara, S, Gebreyes , WA, Morgan Morrow, WE, Correa, M, Scott, M and Thakur, S. 2013. Role of Environment in Occurrence of Antimicrobial Resistant Salmonella in Antimicrobial-free and Conventional Pigs at Farm and Slaughter (Manuscript in preparation for Applied and Environmental Microbiology)
    • Keelara, S, and Thakur, S. 2013. Genotypic Diversity of Plasmids Among Salmonella Isolates from Pigs and their Environment at farm and Slaughter (Manuscript in preparation)


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

    Outputs
    OUTPUTS: The objective of this study is to determine the dynamics and the risk factors that aid in Salmonella transmission in the outdoor ABF pigs. In this study, every pig selected is being sampled five times as it moves from farrowing to the finishing farms. In addition, the pigs are also sampled in the small scale slaughter facilities that specifically cater to these outdoor ABF reared pigs. We have completed sampling of eight ABF farms. A total of 1239 pig fecal and 797 environmental samples have been collected on the farms. At slaughter, we have sampled 565 carcasses and 115 environmental samples. The pig prevalence on farm has been very low (0.4%, n=5 pigs) while 6% (n=33) carcasses tested positive for Salmonella. A high frequency of environmental samples (18%) from slaughter tested positive. The isolates were tested as per the CLSI recommended protocol against a panel of 15 antimicrobials and the highest frequency of resistance was detected in pigs to tetracycline (29%), followed by kanamycin (10%) and in environment highest frequency of resistance was detected to tetracycline (26%) followed by cefoxitin, sulfisoxazole and streptomycin (3%) each. Tetracycline-resistant strains tested PCR positive for the tetB (45%) and tetC (53%) resistance genes. One out of three cefoxitin resistant isolate harbored the blaCMY-2 resistance gene. DNA fingerprinting revealed the role played by the slaughter environment in Salmonella transmission at slaughter. Questionnaires information at farm and slaughter is being collected for risk factor analysis. PARTICIPANTS: 1. Dr. Morgan Morrow: 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 in this project. 3. Dr. Maria Correa: She is responsible for statistical analysis of the data as well as performing the multivariate logistic regression for risk factor analysis. 4. Dr. Shivaramu Keelara: He is the graduate student who has contributed significantly to the project and has been involved in the sample collection, processing and characterizing of the isolates. 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 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


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

    Outputs
    OUTPUTS: The objective of this study is to determine the dynamics and the risk factors that aid in Salmonella transmission in the outdoor ABF pigs. In this study, every pig selected is being sampled five times as it moves from farrowing to the finishing farms. In addition, the pigs are also sampled in the small scale slaughter facilities that specifically cater to these outdoor ABF reared pigs. At this stage we have completed sampling of eight out of the 10 ABF farms to be sampled. A total of 1063 pig fecal and 697 environmental samples have been collected on the farms. At slaughter, we have sampled 406 carcasses and 95 environmental samples. The pig prevalence on farm has been very low (0.5%, n=5 pigs) while 7% (n=29) carcasses tested positive for Salmonella. A high frequency of environmental samples (21%) from slaughter tested positive. 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. DNA fingerprinting revealed the role played by the slaughter environment in Salmonella transmission at slaughter. Questionnaires information at farm and slaughter is being collected for risk factor analysis. PARTICIPANTS: Dr. Morgan Morrow: 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 in this project. Dr. Maria Correa: She is responsible for statistical analysis of the data as well as performing the multivariate logistic regression for risk factor analysis. Dr. Shivaramu Keelara: He is the graduate student who has contributed significantly to the project and has been involved in the sample collection, processing and characterizing of the isolates 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 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

    • Veerappa, S. K., and Thakur, S. 2010. Molecular characterization of multidrug resistant (MDR) Salmonella Typhimurium from swine and human origin by phage types, resistance genes, integrons and PFGE. Conference of Research Workers in Animal Diseases (CRWAD). December 5-7, Chicago, Illinois.
    • 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., Veerappa, S. K., and Morrow, W. E. 2010. Influence of Pre Harvest environment on the status of Salmonella in Conventional and Antimicrobial free (ABF) swine at farm and slaughter. American Association of Swine Veterinarians, Phoenix, Arizona.


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

    Outputs
    OUTPUTS: Sample Collection at the antimicrobial free (ABF) swine production systems at farm and slaughter 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. 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: Salmonella was isolated at farm from a total of two out of 706 pigs sampled in the outdoor ABF farms (prevalence: 0.28%). Salmonella prevalence at slaughter level was higher than at the farm with a prevalence of 15.6% (35 positive out of 223 samples). At the post chill stage, we isolated the pathogen from five carcasses. At this stage, we are characterizing three Salmonella isolates from every positive sample. Therefore, we have a total of 122 isolates including four from the pigs at farms and another 18 isolates from the farm environment. At slaughter, we have isolated a total of 52 Salmonella isolates from the carcass at post-evisceration and post-chill stages and another 48 isolates from the slaughter plant environment (lairage). Antimicrobial susceptibility testing of Salmonella isolates: We are in the process of susceptibility testing for Salmonella isolates. 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 122 isolates. Salmonella isolates from slaughter exhibited maximum frequency of resistance against tetracycline (69.09%, n=38) where as only two out of four from farm were resistant to tetracycline, streptomycin and kanamycin. PARTICIPANTS: Individuals 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. Dr. Maria Correa: She is responsible for statistical analysis of the data as well as performing the multivariate logistic regression for risk factor analysis. She will focus on identifying risk factors that aid in Salmonella transmission in pigs at farm and slaughter. We are currently in the process of collecting this data and have used her help in designing the questionnaires. 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. Dr. Shivaramu Keelara: He is the graduate student whom we have hired through this current USDA support. Dr. Keelara has contributed significantly to the project and has been involved in all the stages including sample collection, processing and characterizing the Salmonella isolates. 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 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