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ARS Home » Midwest Area » Ames, Iowa » National Laboratory for Agriculture and The Environment » Agroecosystems Management Research » Research » Research Project #433200

Research Project: Reducing Production Losses due to Oxidative Stress and Bacterial Pathogens in Swine

Location: Agroecosystems Management Research

2022 Annual Report

The objectives of the project are to develop an understanding of the interrelationships between non-antibiotic feed additives and pathogen translocation and shedding in growing pigs, obtain critical measures of oxidative stress in growing pigs and reproducing swine, and develop interventions to reduce or prevent pathogen colonization and disease in swine in an effort to maximize production efficiency but minimize environmental impact. Objective 1: Evaluate alternatives to antibiotics (butyric acid, resistant starch, inulin, etc.) for maintaining growth performance and reducing intestinal bacterial translocation and shedding in growing pigs. Objective 2: Determine the inherent variation in oxidative stress measures in breeding and lactating gilts, and correlate to measures of sow lifetime productivity. Objective 3: Identify markers associated with oxidative stress and correlate to changes in pig growth and feed intake, using peroxidized soybean oil as the inducer of oxidative stress. Objective 4: Determine whether vaccination of swine with a Salmonella DIVA vaccine can prevent/reduce colonization and improve growth following transmission of Salmonella from actively shedding pigs.

Alternatives to antibiotics for growing pigs will be assessed through the evaluation of compounds which have the potential to affect gastrointestinal function and microbial ecology which would, therefore, affect pig performance. Compounds to be evaluated include resistant starch, soluble dietary fiber, short- and medium-chain fatty acids, phytogenics, inorganic minerals, and beta-glucans; all of which have been suggested to affect gastrointestinal function and microbial ecology. Determination of the inherent variation in oxidative stress measures in breeding and lactating gilts will be assessed by collecting biological samples from gestating and lactating sows at 4 critical time points during these reproduction phases, and measuring key oxidative stress parameters (DNA, protein, and lipid damage) as well as antioxidant status (plasma vitamin E). In growing pigs, identification of markers associated with oxidative stress and impact on pig performance will be assessed by feeding peroxidized soybean oil to nursery, grower, or finishing pigs. Peroxidized soybean oil will be generated by heating soybean oil at 45°C for 288 h, 90°C for 72 h, or 180°C for 6 h, in comparison to unheated (22.5°C) soybean oil. Oxidative stress will be assessed measuring key oxidative stress parameters (e.g., DNA, protein, and lipid damage) as well as antioxidant status (e.g., plasma vitamin E) while performance effects will be measured by growth over a predetermined period. An attenuated Salmonella vaccine was previously designed and constructed to provide broad protection against numerous Salmonella serovars in food-producing animals. A Salmonella transmission trial will be performed in swine to determine whether vaccination against Salmonella can prevent or reduce Salmonella colonization and improve growth performance following exposure to pigs actively shedding Salmonella.

Progress Report
This is the final report for project 5030-31000-006-00D. Over the life of the five-year project, substantial results were realized in each of the following four objectives. Objective 1: Non-antibiotic feed additives such as soluble dietary fiber, resistant starch, and short- and medium-chain fatty acids have been reported to modulate intestinal microbiota, improve nutrient digestibility, improve gastrointestinal health, and ultimately improve productivity. Research conducted with several of these feed additives verified that intestinal microbial ecology was altered but did not impact nutrient digestibility or animal productivity when tested in a university research-based setting in the absence of a health challenge. In addition, the metals copper and zinc are frequently used in animal feed during the early stages of swine production as alternatives to antibiotics to suppress pathogens, protect animal health, and minimize losses in productivity. However, the emergence of metal-tolerant, opportunistic pathogens in animal production may be an unintended consequence of metal usage. In 2015, a foodborne outbreak due to multidrug-resistant (MDR) Salmonella enterica serovar I 4,[5],12,i:- was linked to pork with 188 human infections and 30 hospitalizations. Non-typhoidal Salmonella serovars like Salmonella serovar I 4,[5],12,i:- are a leading cause of foodborne disease in the U.S. and serovar I 4,[5],12,i:- has emerged as one of the top five serovars to cause disease. To gain a better understanding of the emergence of MDR Salmonella serovar I 4,[5],12,i:- in swine production and the potential impact of in-feed metal usage, we performed genetic and phenotypic characterization of a pork outbreak-associated isolate USDA15WA-1 (FSIS1503788). The genome of MDR serovar I 4,[5],12,i:- isolate USDA15WA-1 was sequenced and shown to have two genetic insertions that encode genes for resistance to multiple antimicrobials (ampicillin, streptomycin, sulfisoxazole, and tetracycline) and tolerance to heavy metals (mercury, copper, zinc, and arsenic). Construction and evaluation of an isogenic mutant of strain USDA15WA-1 with a deletion of the metal tolerance island indicated that the metal deletion mutant had reduced growth in the presence of copper, arsenic, and antimony compared to the wildtype strain, indicating that the presence of the metal tolerance genes in wildtype USDA15WA-1 increases pathogen survival in environments containing these metals. In addition to metal tolerance genes, the metal tolerance island contains DNA mobilization and transfer genes that facilitated transfer of the island to another Salmonella strain that did not initially contain the metal tolerance island. These experiments demonstrate horizontal gene transfer of the metal tolerance island from the pork outbreak-associated Salmonella serovar I 4,[5],12:i:- isolate to another Salmonella strain, resulting in the recipient acquiring an increased tolerance to multiple metals. The inclusion of zinc and copper in swine diets at levels much greater than concentrations required to support optimal pig growth, have been utilized in animal production as alternatives to antibiotics. The ability of zinc and copper administration to reduce gastrointestinal colonization and fecal shedding of the multidrug-resistant, metal tolerant strain of Salmonella serovar I 4,[5],12:i:- was assessed. The swine fecal shedding and gastrointestinal colonization of serovar I 4,[5],12:i:- was not significantly reduced in pigs administered zinc and copper at elevated levels compared to pigs receiving the control diet. Furthermore, the rate of decline in the level of Salmonella fecal shedding over the duration of the study was significantly slower in swine receiving elevated zinc and copper in their diet compared to control pigs suggesting that inclusion of metals in the diet may provide selective pressure for colonization of metal tolerant Salmonella in swine. This swine study indicates that elevated levels of zinc and copper as alternatives to antibiotics do not reduce gastrointestinal colonization and fecal shedding of a metal tolerant strain of Salmonella serovar I 4,[5],12:i:- and in contrast to their intended use, these metals may prolong gastrointestinal colonization and fecal shedding of this pathogen. Future research to evaluate alternatives to antibiotics to enhance swine production should focus on feed additives that are not dependent upon metals for suppression of pathogens. Objective 2: Female swine need to remain in the breeding herd for multiple parities for pork production to be financially and socially sustainable. It has been suggested that sow lifetime productivity needs to improve by 30% over the next 10 years, with one factor assumed to be important in this effort being oxidative stress within the animal during its breeding, gestation, and lactation periods. Research conducted showed that there were oxidative stress differences between gilts and sows and that oxidative stress differed between gestation, lactation, and rebreeding, but there were no consistent relationships between measures of oxidative stress and sow reproductive performance. Objective 3: Research conducted indicated that feeding growing pigs unsaturated oils which have been thermally stressed in the presence of air generates peroxides and aldehydes in the oil, which when added to diets and consumed by growing pigs reduced feed intake and subsequent growth. In contrast, feeding pigs saturated fats which had been thermally stressed in the presence of air did not generate peroxides and aldehydes in the fat, and therefore, had no impact on feed intake or growth performance. Objective 4: Interventions are needed to reduce opportunistic pathogens in swine production to protect animal health and limit the use of antimicrobials for disease treatment. A vaccine trial was performed to determine if vaccination with an attenuated Salmonella vaccine strain would reduce pig colonization following transmission of Salmonella from swine donors actively shedding Salmonella. Salmonella shedding in the feces of pigs intranasally inoculated with the pathogen was highly variable as a model of Salmonella transmission to naïve pen mates. The quantity of Salmonella present in swine cecal contents were significantly reduced in vaccinated pigs compared to unvaccinated control swine 14 days following the initiation of Salmonella transmission from actively shedding donors.

1. Feeding oxidized lipids have performance-depressing effects in growing pigs. Past research conducted by ARS researchers in Ames, Iowa, has shown that heating unsaturated lipid sources (e.g., soybean oil) in the presence of infused air results in the generation of peroxides and aldehydes, each being time and temperature dependent. In contrast, heating a saturated lipid source (e.g., beef tallow) is less susceptible to the formation of lipid oxidation products. Experiments which varied the level of peroxidation products included in complete diets clearly showed that when peroxidized lipids were fed to growing livestock there was a reduction in feed intake and growth up to 25% in pigs and up to 10% in growing broilers. Combining the data from several experiments suggests that knowing both the peroxide value and anisidine value of a lipid, and the inclusion level of a lipid in a diet, can allow one to predict the reduction in animal performance that would be observed by feeding a peroxidized lipid compared to feeding an unadulterated lipid. This data is valuable to the livestock industry on determining the detrimental effects if growing animals are fed peroxidized lipids.

2. Reduced performance caused by enteric challenges is not mediated by oxidative stress in growing swine. In swine, enteric challenges may have effects on in vivo markers of oxidative stress, which relates to the balance between free radicals or reactive oxygen-, nitrogen- or sulfur-species and the antioxidant defense system within an animal. Because free radicals or reactive species need electrons to become less reactive, they scavenge electrons from proteins, lipids, or DNA, causing damage. Antioxidant defense systems intercept these reactive compounds, but, if the presence of free radicles or reactive oxygen species overwhelms the antioxidant defense system in the animal, protein, lipid, DNA damage can occur leading to oxidative stress. Research conducted by ARS researchers in Ames, Iowa, indicated that enteric challenges such as the consumption of mycotoxins, oxidized proteins, and peroxidized lipids can reduce animal performance; however, the effects of these compounds altering specific markers of oxidative stress (i.e., protein, lipid, or DNA damage indicis) were either not affected or inconsistently affected, suggesting that oxidative stress was not a major mechanism for the observed reduction in animal performance. This data is valuable to the livestock industry in determining that measuring oxidative stress in the animal is not always a valuable measure on how an enteric challenge may affect animal productivity.

3. Oxidative stress in breeding swine has little relationship to reproductive productivity. It is both economically and socially important to retain reproducing females in a swine herd. However, during breeding, gestation, and lactation, reproducing female pigs are assumed to be under a great deal of oxidative stress due to the extensive amount of energy and protein metabolism occurring for mammary, uterine, placental, fetal metabolism and milk production. It has been suggested that one of the reasons that a breeding female is removed from the swine herd is in part due to high levels of oxidative stress due to this increase in energy and protein metabolism. Research conducted by ARS researchers in Ames, Iowa, at a commercial swine facility showed that gilts exhibited greater oxidative stress compared to sows and that oxidative stress differed between gestation, lactation, and rebreeding periods, but there were no correlations between various measures of oxidative stress and sow reproductive performance. This data is valuable to the livestock industry in determining that measuring oxidative stress in reproductive animals is not a valuable measure in understanding reproductive productivity in swine.

4. Resistant potato starch as an alternative to antibiotics increases swine intestinal short chain fatty acids and reduces Salmonella fecal shedding. Due to the U.S. ban on the use of clinically important antibiotics for animal growth promotion in 2017, there is a need for the development and evaluation of alternatives to antibiotics for suppression of opportunistic pathogens in swine production. Resistant potato starch is a prebiotic that supports the growth of beneficial bacteria in the gastrointestinal tract including microorganisms that produce short chain fatty acids (FAs) which promote favorable intestinal health. ARS researchers in Ames, Iowa, determined that administration of resistant potato starch to pigs increased accumulation of intestinal short chain FAs and decreased fecal shedding of the opportunistic pathogen Salmonella compared to a control group receiving a non-amended swine diet. The pigs on the resistant potato starch diet with the highest amounts of short chain FAs had the lowest levels of Salmonella shedding, but bacteria that utilize resistant potato starch for production of short chain FAs appear to be necessary to modulate these effects. The data suggest that feeding pigs resistant potato starch as an alternative to antibiotics may provide beneficial outcomes for swine producers by modulating intestinal metabolism to minimize the presence of opportunistic pathogens.

Review Publications
Trachsel, J.M., Bearson, B.L., Kerr, B.J., Shippy, D.C., Byrne, K.A., Loving, C.L., Bearson, S.M. 2022. Short chain fatty acids and bacterial taxa associated with reduced Salmonella enterica serovar I 4,[5],12:i:- Shedding in swine fed a diet supplemented with resistant potato starch. Microbiology Spectrum. 10(3). Article e0220221.
Trachsel, J.M., Bearson, B.L., Brunelle, B.W., Bearson, S.M. 2022. Relationship and distribution of Salmonella enterica serovar I 4,[5],12:i:- strain sequences in the NCBI Pathogen Detection database. Biomed Central (BMC) Genomics. 23(1). Article 268.