Location: Food Safety and Enteric Pathogens Research
2021 Annual Report
Objectives
Our objectives target three factors that influence Salmonella colonization, pathogenesis, and persistence. These factors include virulence mechanisms of Salmonella, the tactical response from the host, and interactions with the microbiota residing within the host. Our systematic approach integrates these research areas into three complementary objectives:
Objective 1: Investigate the impact of antibiotic usage on influencing Salmonella virulence mechanisms and enhancing antibiotic resistance.
Objective 2: Develop novel non-antibiotic intervention strategies such as beneficial microbes and vaccines to limit Salmonella colonization, persistence and shedding.
Objective 3: Evaluate immune networks and identify porcine genes for their relationship with the host microbiota to reduce Salmonella colonization, persistence, and shedding.
Approach
The common goal of each research objective is to identify targets for the development of novel antibiotic alternatives to reduce both Salmonella transmission through the food chain and antibiotic usage on the farm. To accomplish these objectives, experiments are planned to examine molecular mechanisms in Salmonella that are influenced by antimicrobial resistance and host colonization, elucidate porcine genetic pathways associated with decreased Salmonella colonization, and investigate interactions between Salmonella and host microbiota that affect Salmonella colonization and persistence. We plan to: 1) identify antibiotics that enhance virulence properties in multidrug-resistant (MDR) Salmonella, as well as those antibiotics that have no effect on virulence; this useful information will aide producers and veterinarians when determining antibiotic therapy for the treatment of infectious diseases; 2) genome sequence and transcriptionally analyze MDR Salmonella isolates that phenotypically respond to antibiotic exposure; 3) measure the effect chlortetracycline treatment has on limiting or exacerbating Salmonella shedding and altering the microbiota in swine; 4) evaluate a cross-protective Salmonella vaccine in turkeys for reduction of Salmonella colonization and transmission; 5) systematically characterize changes in the porcine immune response and gastrointestinal microbiota during Salmonella colonization; 6) assess biotherapeutic treatments as alternatives to antibiotics for treatment of swine colonized with Salmonella.
Progress Report
Considerable results were achieved over the five years of the project and some projects are continuing in the new replacement project 5030-32000-227-00D “Analysis of Genetic Factors that Increase Foodborne Pathogen Fitness, Virulence, and Antimicrobial Resistance Gene Transfer, to Identify Interventions against Salmonella and Campylobacter in Food Animals”. Additional information can be found in the bridging project report 5030-32000-226-00D, “Analysis of Virulence and Antibiotic Resistance Mechanisms of Salmonella and Development of Intervention Strategies” for additional information.
Under Objective 1, antibiotic exposure of multi-drug resistant (MDR) Salmonella induced expression of disease-associated genes and enhanced tonsil colonization in pigs. Chlortetracycline is an antibiotic commonly used in veterinary medicine for respiratory and gastrointestinal infections, but many MDR Salmonella isolates are resistant to chlortetracycline. Exposure of MDR Salmonella isolates to chlortetracycline changed the expression levels of over 50 percent of Salmonella genes, including genes involved in the ability of Salmonella to move (motility), invade host cells (invasion), and survive and replicate inside host cells (virulence). Furthermore, pigs exposed to MDR Salmonella and given a therapeutic dose of chlortetracycline had higher levels of Salmonella in the oral cavity (tonsils) and feces compared to pigs that did not receive chlortetracycline. Thus, consideration for Salmonella status may be important when administering therapeutic antibiotics because animals that are unknowingly colonized with MDR Salmonella and receive therapeutic chlortetracycline for an unrelated infection may have prolonged Salmonella host colonization and fecal shedding, thereby increasing environmental or pork product contamination and posing a risk to public health.
Also in Objective 1, investigation of multidrug resistant (MDR), pork-associated Salmonella enterica serovar I 4,[5],12:i:- outbreak isolates revealed the acquisition of genes for metal tolerance. To identify a cause for the increased prevalence of this particular MDR Salmonella serovar in swine, genome sequencing of a pork-associated outbreak isolate of MDR Salmonella serovar I 4,[5],12:i:- revealed the acquisition of several genes involved in metal tolerance (copper, silver, arsenic, and mercury). Elevated tolerance to copper and silver was measured in the outbreak isolate, and exposure of the isolate to copper increased the expression of multiple metal tolerance genes. Because metals are frequently used in swine production as an antimicrobial feed additive, a study was conducted in pigs to evaluate the impact of elevated levels of in-feed zinc and copper concentrations on MDR Salmonella I 4,[5],12:i:- colonization and shedding levels. Administration of high levels of zinc and copper in the diet did not reduce fecal shedding or tissue colonization of the Salmonella serovar I 4,[5],12:i:- isolate in pigs. In fact, at three weeks post-inoculation with the outbreak isolate, higher fecal shedding levels of Salmonella were observed in the zinc/copper fed pigs compared to the pigs that did not receive high levels of zinc and copper in their feed. Thus, metals as an antimicrobial feed additive in the swine diet may have contributed to the increased prevalence and persistence of Salmonella serovar I 4,[5],12:i:- in the pigs (i.e. selective pressure).
Under Objective 2, vaccination with a live-attenuated vaccine significantly reduced Salmonella levels in turkeys. Causing an estimated 1.2 million illnesses/year in the U.S., Salmonella is a major foodborne pathogen that often resides in the gastrointestinal tract of food animals including pigs, poultry and cattle without causing disease, but can cause significant disease in humans when contaminated products from food animals are consumed. A Salmonella vaccine was created with genetic mutations in the bacterial genome to limit Salmonella serotype-specific immunity (greater than 2,500 Salmonella serotypes exist) and instead, induce an immune response that would be cross-protective against diverse Salmonella serotypes. In addition to designing a vaccine that would protect against multiple Salmonella serotypes, the intent was also for application in multiple food-producing animal species. Effective reduction of Salmonella disease, colonization and fecal shedding had previously been determined in vaccinated swine. To highlight utility of the vaccine, the vaccine was tested in turkeys and showed a reduction in systemic and intestinal colonization of vaccinated turkeys following challenge with multi-drug resistant Salmonella Heidelberg. Enhancing pre-harvest control of Salmonella in food-producing animals can protect animal health, limit antibiotic usage, decrease environmental contamination, reduce Salmonella carriage into the human food chain, and diminish the cost of meat product recalls to producers.
Under Objective 3, enhancing porcine innate immune defenses reduced Salmonella in swine. Development of intervention strategies that are effective against diverse Salmonella serovars are desired to limit foodborne illness. Neutrophils are cells of the immune system that play a vital role in combatting bacterial infection, and the immune protein that induces neutrophil production in the animal is granulocyte-colony stimulating factor (G-CSF). The research team demonstrated that pigs injected with an engineered vector expressing the porcine G-CSF (Ad5-G-CSF) had significantly reduced Salmonella fecal shedding and tissue colonization, and less Salmonella-induced disturbance of their intestinal microbiota compared to the Salmonella-exposed pigs that did not receive Ad5-G-CSF. The data suggest that delivery of a targeted immunostimulant to enhance innate immunity may be a strategy to reduce Salmonella colonization during periods of immunological and production stress.
Also under Objective 3, swine saliva was evaluated to signal prior Salmonella exposure. Greater than 50% of U.S. swine herds are positive for the human food-borne pathogen Salmonella. Because pigs are often colonized with Salmonella without showing clinical signs (i.e. carriers), an important first step in reducing the incidence of Salmonella on the farm is to promptly and easily detect when a herd has been exposed. The swine industry would benefit from a surveillance tool that would identify Salmonella-exposed pigs and determine herd level immunity. Oral fluids (i.e. saliva) from pigs were used to detect antibodies against Salmonella. Oral fluid samples are collected by allowing pigs to chew on cotton ropes, which is non-invasive, inexpensive, and can be repeatedly collected from live animals. The samples can be easily stored and used in an enzyme-linked immunosorbent assay (ELISA) to detect the presence of Salmonella-specific antibodies. The data support oral fluids serving as a repeatable sample during swine production to provide not only timely surveillance information on Salmonella exposure and herd immunity, but also to evaluate the effectiveness of disease intervention strategies against Salmonella.
Accomplishments
Review Publications
Bearson, B.L., Trachsel, J.M., Shippy, D.C., Sivasankaran, S.K., Kerr, B.J., Loving, C.L., Brunelle, B.W., Curry, S.M., Gabler, N.K., Bearson, S.M. 2020. The role of Salmonella Genomic Island 4 in metal tolerance of Salmonella enterica serovar I 4,[5],12:i:- pork outbreak isolate USDA15WA-1. Genes. 11(11). Article 1291. https://doi.org/10.3390/genes11111291.
Sylte, M.J., Shippy, D.C., Bearson, B.L., Bearson, S.M. 2020. Detection of Campylobacter jejuni liver dissemination in experimentally colonized turkey poults. Poultry Science. 99(8):4028-4033. https://doi.org/10.1016/j.psj.2020.03.042.
