Enteric Diseases of Food Animals: Enhanced Prevention, Control and Food Safety

Non-Technical Summary:<br/>
Estimates of foodborne illness can be used to direct food safety policy and interventions. The data from active and passive surveillance and other sources in the United States has revealed that each year 9.4 million episodes of foodborne illness occur in the United States leading to 55,961 hospitalizations, and 1,351 deaths. Food-borne illness is a concern in the United States due to large outbreaks as well as a high incidence of sporadic diseases however 1) Most food-borne illness events are of undefined etiology indicating the need for identification of emerging agents. 2) Since most food-borne disease agents are zoonotic in nature, investigation and control in the animal reservoir are required. 3) Several of these agents are also severe pathogens of animals and investigation of animal infections will be informative about human infections. In addition to human health considerations, animal diarrheal diseases are important causes of production loss. Continued research in support of food safety and of control of diarrheal diseases of livestock is necessary to optimize animal health and welfare.
<br/>In this project we will 1) develop improved detection methods for emerging causes of zoonotic enteric disease and enteric pathogens of cattle and poultry, 2) develop and improve interventions and preventative measures to reduce the incidence and prevalence of infections of cattle and poultry with enteric and food borne disease agents and 3) provide training and continuing education opportunities to students, producers, veterinarians and diagnostic laboratories.
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Approach:<br/>
Objective 1.Focus on emerging diseases: We will identify, characterize and develop improved detection and prevention methods related to newly recognized, novel or emerging causes of zoonotic enteric disease and enteric pathogens of food animals.The incidence of enteric diseases caused by specific pathogens and the genetic make-up of the pathogens themselves evolve over time. Similarly, constant changes occur in livestock management practices and animal genetics. Documentation of information regarding changes in the prevalence of zoonotic enteric disease and enteric pathogens of food animals is critical to understand the underlying processes and most importantly to make management adjustments and develop new preventative products or modify currently licensed preventive products. NC-1202 workers will 1) develop and distribute pathogen typing tools for enteric agent surveillance among cooperating stations; 2) monitor pathogen genotypes, antibiotic resistance and virulence determinants to identify trends in prevalence and to detect the emergence of new types; 3) archive strains for comparison with future isolates and for historic assessment of prevalence of virulence determinants yet to be discovered. Objective 2.Focus on preventions and interventions: We will develop and improve preventative measures and interventions to reduce the incidence and prevalence of infections of food animals with enteric and foodborne and waterborne pathogens.Strategies for prevention or management of zoonotic enteric disease and enteric pathogens of food animals must evolve to keep abreast of disease changes. NC1202 workers will develop, test and distribute new preventative tools and strategies or modify existing tools and strategies for prevention of zoonotic enteric disease and enteric pathogens of food animals among cooperating stations. Objective 3.Focus on disseminating knowledge: We will provide training or continuing education to disseminate new information to students, producers, veterinarians, diagnostic labs and others to implement interventions and preventative measures.Objective 4.Group interaction: The group will interact in a variety of ways to facilitate progress including direct collaborations with joint grant funding and publications, sharing of resources (pathogen strains, gene sequences, statistical analysis, bioinformatics information/expertise), and friendly feedback and facilitation for all research efforts at annual meetings.
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PROGRESS: 2013/01 TO 2013/09<br>
Target Audience: Scientists, producers, academic institutions, graduate and undergraduate students. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training of following graduate students was possible: Devendra H Shah (PI): Jake Elder, PhD (Salmonella), Kim Chiok, MS (Salmonella), Salma Al-Adwani, MS (Campylobacter) Thomas E. Besser (PI): Carrie Schmidt, PhD (E. coli O157:H7) Margaret Davis (PI): Niraj Suthar, MS (Campylobacter) Bill Sischo (PI), Afema Josephine, PhD (Salmonella) How have the results been disseminated to communities of interest? Via Publications and seminars What do you plan to do during the next reporting period to accomplish the goals? We will continue to work in the projects described above to meet our goals. Following are the examples of some of the specific projects: Currently we are investigating the vaccine potential of KsgA deficient mutant of Salmonella to induce a protective immune response in chickens. We are screening the remaining 245 CpG motifs of Salmonella and it’s expected that we will be able to identify several other immune-stimulatory CpG motifs. We are testing the validity of the real-time PCR for detection of SE on environmental drag swabs, eggs and chicken carcasses. We are continuing to test the hypothesis that high summertime seasonal shedding of O157 results from introduction of new season forage crops. We are sampling pens of dairy and feedlot cattle just prior to and just after introduction of the new forage crops. Trials to date have shown sometimes dramatic increases in O157 shedding following these introductions.
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IMPACT: 2013/01 TO 2013/09<br>
What was accomplished under these goals? Virulence characterization of Salmonella Enteritidis (SE) mutants in orally inoculated day-old chickens: Fourteen mutant strains of SE had significantly reduced invasiveness in human intestinal epithelial cells (Caco-2), chicken macrophages (HD-11) and chicken hepatocellular epithelial cells (LMH). SE mutants SEN0034::Tn5, fliH::Tn5, SEN1393::Tn5 and spvR::Tn5 were indistinguishable from the isogenic wild-type strain when orally inoculated in chickens whereas the mutants CsgB::Tn5 and PegD::Tn5 were defective for intestinal colonization (P < 0.05). eight mutants (hilA::Tn5, SEN3503::Tn5, SEN0803::Tn5, SEN2278::Tn5, fljB::Tn5, rfbM::Tn5, rfbN::Tn5 and pipA::Tn5) showed significant in vivo attenuation in more than one organ (P < 0.05). Virulence and phenotypic characterization of dimethyl adenosine transferase (KsgA) deficient mutant of SE: We characterized KsgA mutant of SE. KsgA mutant showed impaired invasiveness in human and avian epithelial cells, and significantly reduced intestinal colonization and organ invasiveness in chickens, reduced respiratory activity with respect to a number of carbon, nitrogen, phosphate, sulfur and peptide nitrogen sources, and significantly reduced tolerance to high osmolarity, oxidative stress and chloramphenicol, but increased resistance to kasugamycin. Identification and characterization of the immunomodulatory activity of CpG motifs of Salmonella: We have identified a total of 256 CpG motifs (6-mers) in the Salmonella pan-genome that can potentially modulate host immune response. In a preliminary screen, 11 out of 256 CpG motifs were tested for their immune-stimulatory properties using in vitro grown avian macrophages, and 4 induced production of IL-1β Development of a multiplex real-time qPCR for the serotype-specific detection of SE (SE): We optimized TaqMan PCR reactions targeting invA, sdfI and prot6E genes of SE. Sensitivity was determined in serially diluted DNA from SE; specificity was assessed in 37 non-SE Salmonella strains and in 9 non-Salmonella strains. The PCR assay correctly identified all SE isolates, distinguished them from all non-SE strains and discriminated plasmid from non-plasmid bearing strains. Detection limit of the assay was 1 CFU in clean drag-swab samples. In environmental drag swabs (n=14), the assay displayed 100% sensitivity, specificity, accuracy, PPV and NPV. Campylobacteriosis epidemiology in Washington State dairy counties: Conditional logistic regression analysis of case-control data from two counties in WA showed that living or working on a dairy farm (6.7, 1.7 – 26.4) and Hispanic ethnicity (5.9, 2.9 – 12.0) were associated with campylobacteriosis. Hispanic ethnicity, (OR 9.3, 95% CI 3.9 – 22.2), contact with cattle (OR 5.0, 95% CI 1.3 – 19.5) and pet ownership (OR 2.6, 95% CI 1.1 – 6.3) were found to be independent risk factors for disease. Genetic comparison of Campylobacter jejuni isolates from human (n=65), bovine (n=28), and retail poultry (n=27) showed common genotypes between human and bovine isolates. Veterinary nosocomial infections caused by antibiotic resistant: We developed an individual-based model to incorporate movement of canine patients within the veterinary teaching hospital at WSU. A multiple regression demonstrated that the risk of carriage for resistant pathogens was decreased with reduction in: (i) length of patient stay in the hospital (P < 0.0001); (ii) intervals between decontamination events for healthcare workers and transmission points (P < 0.0001); and (iii) probability of colonization of patients given better healthcare worker hygiene compliance (P < 0.0001). Cefepime resistance in bovine E. coli: We visited 30 Washington State Dairy farms and found high prevalence of cefotaxime, cefepime resistant E. coli on 28 farms. Cefepime resistance was highly correlated with the presence of blaCTX-M. Risk factors for high prevalence of blaCTX-M-bearing E. coli included recent animal movements on the farm, region of Washington (eastern Washington farms were at higher risk than northwestern Washington farms), use of residual fly sprays (lack of use created a higher risk), direction of feeding calves (youngest to oldest having a lower risk), frequency of adding new bedding to calf hutches (higher frequency, lower risk) and use of florfenicol on calves (any use, lower risk). Any ceftiofur use in calves was not associated with a higher risk. Development of a genotyping method for E. coli: 75 blaCTX-M-bearing E. coli strains were typed using the established 7-locus method and a new 2-locus method. The congruence between the two methods using the adjusted Rand coefficient was 0.417 (0.27 – 0.56). The Adjusted Wallace Index indicated that the 7-locus method predicted partitioning by the 2-locus method (AW =0.86 (0.78 – 0.93) much better than the 2-locus method predicted 7-locus MLST partitioning (AW = 0.28 ( 0.17 – 0.38). We participated in a large collaborative comparative study of E. coli O157:H7 isolated from cattle and from humans in the USA and Australia. Over 600 isolates were genotyped using Shiga toxin encoding bacteriophage insertion sites (SBI), lineage specific polymorphisms (LSPA-6), multi-locus VNTR analysis (MLVA) and the tir 255T>A polymorphism. The results clearly showed phylo-geographic structuring of this bacterium, and divergent evolution of this serotype on these two continents. As a follow-up to the US-Australia study, we are conducting a comparative study involving those two countries plus Argentina, using SNP and SBI genotyping. Preliminary results indicate strong further support for phylo-geographic structuring, and in addition, strong correlation between the existence of strains carrying the Stx2a-encoding bacteriophage and the incidence of human O157-related disease. We participated in a second collaborative case-control study of E. coli O157:H7 to identify sources of human infection in New Zealand using SBI and PFGE genotyping approaches. The results showed 1) environmental and animal contact as the major source of human infection (rather than foods), and 2) phylo-geographical structuring of the pathogen between the North and South Islands. We have developed a 48-plex chromosomal backbone SNP allele assay for E. coli O157:H7 on the Illumina GoldenGate platform to efficiently type E. coli O157 isolates into the eight lineages defined by Bono et al., as well as 4 sub-lineages. Using the SNP assay, we have genotyped >2000 isolates of known sources, with the goal of evaluating seasons, production systems, hosts of origin, interventions, etc., for effects on clinical and bovine-biased genotypes. A previously observed effect, >90% of feedlot cattle isolates, was clarified to be the result of a single over-represented feedlot. By expanding the numbers of isolates from additional feedlots, and by including the premises as a random variable, we determined that the feedlot operation type was not strongly associated with clinical genotypes of O157. We conducted a switch-back cattle challenge trial to test whether grain diets selected for clinical vs bovine-biased genotypes of O157. The results did not show any effect of grain vs forage rations on the shedding of clinical genotypes of O157, but did show that grain rations were associated with lower shedding and more rapid clearance of O157 infection than were forage-based rations. We completed an experimental challenge study on seasonal variation of O157 shedding by cattle that involved 4 serial challenges of 20 cattle with standardized doses of E. coli O157 at 6 month intervals, with the doses timed to coincide with the peak and trough seasonal periods. Results indicate no significant seasonal variation in the amount or duration of shedding, given equal oral challenge. These results indicate that seasonal variation is principally due to seasonal variation in oral exposure of cattle to this pathogen.