Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics.

Aim of this study was to select environmental E. coli isolates that produced colicins against the swine pathogen E. coli K88+. In initial evaluation using a modified plate method with 18 colicinogenic E. coli constructs, colicins E3, E4, E5, E9, Ia, K and N were found to possess inhibitory activity against 12 ETEC K88+ strains. A total of 463 environmental isolates from cattle rumen, cattle feces, pig feces and hog manure-amended soil were screened for colicin production by a modified plate test. Further, colicinogenic isolates were screened for five toxin genes LT, STa, STb, VT1 and VT2 as well as K88 (F4) fimbriae using PCR reactions. Fourteen non-pathogenic isolates were subjected to characterization of colicin genes by PCR using 9 new primer sequences, antibiotic susceptibilities and substrate utilization. Two potential probiotic strains of E. coli, UM-2 and UM-7 which produced colicins that could utilize potato starch and inulin were selected for in-vitro competition with E. coli K88+ strain 2-12. In vitro competition between the synbiotics and E. coli K88+ revealed inhibition of E. coli K88+. Based on the present in vitro studies it could be concluded that carefully selected potential synbiotics should be further studied for their role in protecting piglets from post-weaning diarrhea without antibiotics.

Master of Science / Department of Animal Sciences and Industry / James M. Lattimer / Three experiments were conducted to determine the impact of novel probiotic (NP) supplementation in monogastric livestock and poultry. Experiment 1 evaluated effects of the probiotic on growth performance and cecal parameters in broiler chickens (n = 2520). Birds in treatment groups 1 and 2 were administered NP culture as either an oral gavage (OG) or an aerosolized mist (AM), respectively. Treatment group 3 served as a negative control (C). Growth performance and feed efficiency (FE) were unaffected by treatment (P > 0.10). Cecal pH was reduced in AM and OG birds compared to C (P < 0.05). This novel probiotic is most effective in altering cecal VFA profile before d 21 post-administration, after which no differences in VFA concentrations were detected between treatments. Reduction in cecal pH in response to NP indicates potential application of this probiotic as an acidifier, commonly used in poultry production as an antibiotic alternative. Experiment 2 was a swine performance study conducted to determine effects of treating sows or piglets or both with an oral gavage of NP. Factor 1 consisted of administration of NP oral gavage administered to sows or a negative control (n = 28). Factor 2 consisted of either a negative control or oral gavage of NP administered to litters of piglets shortly after birth and at weaning. Sow weight loss during lactation was unaffected by treatment (P > 0.10). Feed intake upon introduction to the nursery was greater in piglets that received NP directly than those that did not (P < 0.05). Large intestinal fermentation in both sows and piglets was affected by NP administration. Fecal VFA concentrations tended to be greater in animals that received NP either indirectly through the sow or directly via oral gavage (P < 0.05). Direct supplementation of piglets with NP is more effective in altering fermentation after d 21 than indirect administration, as VFA concentrations in piglets that received NP indirectly are similar to the control by d 28. Butyrate concentrations were greater in piglets that received NP; this VFA is important in prevention of intestinal atrophy and recovery from physiological stress of weaning. Experiment 3 examined effects of NP administered as either an oral drench (OD) or a lyophilized powder (LP) on equine cecal fermentation compared to a negative control (NC). Cecally cannulated horses (n = 8) were transitioned to a 1:1 roughage to concentrate diet over a period of 5 d. Cecal pH was greater than NC in OD and LP horses on d 5 and d 7, respectively (P < 0.10). Acetate:propionate ratio, valerate, and caproate concentrations also were greater in LP horses than NC on d 7 (P < 0.05). This may indicate improved gut health and fiber digestion in supplemented horses. The novel probiotic used in these studies is able to effectively alter large intestinal fermentation in monogastric species and may have potential to improve gut health and performance of these species.

Probiotics are an alternative to increase broiler productivity and flock health. For this reason, the objective of the first study was to evaluate commercially available Bacillus spp. based probiotic products on their ability to reduce Salmonella Heidelberg, in vitro. Results showed that most products reduced the lower concentrations of S. Heidelberg and maintained a reduction for up to 8 h. The second study was conducted to determine if bacterial colonization occurred in a broiler chick after in ovo injection. Using bioluminescence, it was demonstrated that bacteria could migrate and colonize the embryo within 2 h after in ovo injection into the amniotic fluid. The results of these studies demonstrated that probiotics could reduce pathogenic microorganisms and can potentially have a positive impact on the chick before it even hatches and is placed in a broiler house.

The primary objectives of the following experiments were to: 1) determine the impact of a directed microbial (DFM) blend on digestibility and microbial populations on horses fed low (LQ) and high (HQ) quality hay, 2) evaluate the differences in diet digestibility, microbial populations, and blood metabolites due to DFM supplementation to the exercising horse, 3) assess the impact of short-chain fructooligosaccharide (scFOS) supplementation on digestibility and the fecal microbial ecosystem in senior (SR) vs. mature (MA) horses, and 4) determine the relationship between insulin dysregulation (ID) and morphometric neck measurements (MNM) in the non-obese stock-type horse. Results indicated that: 1) Supplementation with DFM tended to be beneficial in enhancing CP digestibility. Feeding CP beyond requirements may contribute to excess excretion of Escherichia coli (EC) in HQ which was supported by 16S rRNA analysis. Differences in the fecal microbial ecosystem were detected between LQ and HQ. The phylum Saccharibacteria was identified in both hay qualities even though it has been lightly reported in equine literature. 2) Supplementing DFM to moderately exercised horses tended to enhance DM and ADF digestibility but there was no evidence of a dietary modulation to the fecal bacteria isolated. 3) No differences were found between MA and SR when fed scFOSded ration balancer which indicates that scFOS may help mitigate decreases in digestibility experienced by SR. Senior horses had less diversity in their fecal bacterial population, which may help explain why SR are more prone to ailments such as colic and impaired immune function. 4) Obesity is an important genetic factor for ID however it should not be the only determining factor, as ID can occur in non-obese individuals. One individual out of 62 tested horses was identified as ID. Morphometric neck measurements (MNM) may help pinpoint horses that are at a greater risk for ID, but more research is needed to validate MNM in both obese and non-obese stock-type horses. There were correlations between glucose sampled before an oral sugar test (OST) and insulin post-OST but a fasting OST would still be recommended in assessing ID.

The effects of dietary antimicrobial removal and Bacillus subtilis supplementation on the growth and intestinal health of male broilers were investigated. Birds were fed either a control, antimicrobial, or a B. subtilis probiotic diet at different feeding phases. Birds were challenged with a 10 × dose of a coccidiosis vaccine. Supplementation of B. subtilis in for antimicrobials in the late grower and early finisher phases improved growth similar to birds fed antimicrobials until withdrawal, while antimicrobial removal without B. subtilis supplementation in those periods hindered growth. The improved growth suggests that the probiotic was able to alleviate the stress of the challenge compared to antimicrobial removal. Processing yields were improved with antimicrobial removal and B. subtilis supplementation in late grower and early finisher phase. Intestinal health was improved with lower intestinal lesions when antimicrobial were removed and B. subtilis supplemented suggesting the reduction of Eimeria species from colonizing the intestine.

Celiac disease (CD) is an autoimmune disorder that affects approximately 1% of the population [55]. CD is characterized by intestinal villus atrophy after consumption of gluten from wheat, barley, or rye. Patients with CD often experience abdominal pain, diarrhea, malnutrition, fatigue, and a failure to thrive. There is currently no treatment for CD. Patients must live on a strict lifelong exclusion of dietary gluten. Due to the high content of gluten in western diets and poor labeling of gluten content, adherence to a gluten free diet (GFD) is difficult [15].Nearly all the enzymes that can digest the gluten peptide are sensitive to the stomach's low pH . As a result, dietary supplementation with enzymes to digest gluten has yet to produce a viable alternative treatment to a GFD. We propose to use a resident microbe of the human intestinal tract to express a peptidase to digest the immunoreactive gluten fragments. The bacteria, L. reuteri, will colonize the host's intestines and digest the gluten peptides before causing an autoimmune response. To accomplish this task, this thesis describes a food grade, plasmid based system to integrate genes into the genome of L. reuteri. The plasmid system utilizes an origin of replication that requires a protein, RepA, to propagate itself. A helper plasmid provides the RepA protein in trans to an integration plasmid that cannot provide RepA to itself. The integration plasmid carries a homologous region to the genome of L. reuteri allowing for targeted genomic integration. The integration plasmid will not replicate on its own, and will be integrated into the genome if the helper plasmid is absent. To select for these genomic integrants the integration plasmid expresses an erythromycin resistance marker. Using the Cre/Lox system the antibiotic resistance will be removed from the bacterial genome to re-establish the L. reuteri's food grade status. This thesis describes the construction and verification of the above mentioned plasmid tool kit containing the helper, integration, and Cre expression plasmids to integrate genes into the L. reuteri genome.

The use of antibiotics as growth promotors in the food animal industry has led to the generation of antibiotic resistant microorganisms, which are a major health risk to humans. Therefore, bans and limitations, including the FDA’s veterinary feed directive, have been placed on the use of antibiotics as growth promotors, and there has been a push to find an adequate alternative. In the laying hen, probiotics and/or prebiotics have shown promise as they help promote the colonization of beneficial bacteria in the GI tract of the host, increase feed efficiency, increase egg production, and provide protection against pathogenic bacteria. In this feeding trial, Leghorn HyLine W36 hens were fed diets supplemented with either 0.05% IMW50(prebiotic), 0.05% CALSPORIN (probiotic), 0.05% IMW50 and 0.05% CALSPORIN, or a control diet with no prebiotic or probiotic additive. The birds were maintained on this diet from 1 day to 66 weeks of age, over which time feed intake and egg production was measured. From 37 to 65 weeks of age, every 3-5 weeks, egg quality was evaluated through specific gravity, egg weight, albumen height, and eggshell breaking force measurements. At 6, 16, 32, and 64 weeks of age, the microbiota population in the ileal digesta was evaluated using T-RFLP analysis methods. Then at 64 weeks of age, splenic IFN-γ, IL-6, IL-10, and IL-12 expression was measured by RT-qPCR. Unexpectedly, fluctuations in feed disappearance were observed, potentially linked to factors such as changes in diet type, temperature variations, feed wastage, or a technical error. Birds on CALSPORIN-containing diets showed resilience and maintained egg production during a decline in feed disappearance, suggesting enhanced capabilities in digesting larger particle sizes. However, there were trade-offs, as egg quality parameters slightly decreased in supplemented diets. CALSPORIN alone showed a promising effect on survival probability. Microbial diversity in the ileum increased with CALSPORIN, but the IMW50 alone led dysbiosis, possibly impacting infection resistance. Additionally, the combined supplementation of IMW50 and CALSPORIN may have introduced a disruption in immunological homeostasis.

No description available.

Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are opportunistic pathogens that can be transmitted via drinking water, when tiny droplets containing the bacteria are aerosolized and inhaled during activities such as showering. The resulting respiratory illnesses, Legionnaires' Disease and NTM lung disease, are among the leading sources of drinking water associated disease in the United States and other parts of the world. Lp and NTM are both difficult to control, because they establish as part of natural biofilms that form within the interiors of pipes and fixtures that deliver drinking water to the point of use. These pathogens are especially problematic within premise (i.e., building) plumbing, where intermittent use throughout the day leads to long periods of stagnation, increased water age, warmer temperatures, and depleted disinfectant residuals that exacerbate bacterial growth. The recent advent of high throughput DNA sequencing has led to the discovery that drinking water microbiomes are diverse, complex, and largely comprised of non-pathogenic microbes. This has further led researchers to hypothesize that the microbial ecology of this diverse microbiome could be harnessed as a natural means to control Lp and NTM, i.e., a "probiotic" approach, but such an approach has not yet been demonstrated. The objective of this study was to assess this hypothesis by utilizing biologically active granular activated carbon (GAC) filters, which are already a widely used drinking water treatment both at the municipal and household scale, as a means to naturally shape the microbial ecology of downstream premise plumbing and inhibit Lp and NTM proliferation. GAC has an extremely high surface area that aids removal of organic carbon via adsorption but also provides an ideal habitat for establishment of biofilms, which removes organic carbon from the water via biodegradation. Convectively-mixed pipe reactors (CMPRs) were used for replicable simulation of premise plumbing distal taps. The CMPRs consisted of four-foot-long closed polyvinyl chloride (PVC) pipe segments with the sealed bottom portion resting in a ~48 °C water bath and with the top portion plugged and exposed to the cooler, ambient atmosphere (25 °C in this study), inducing convective mixing and resulting in an internal water temperature of 37 °C. PVC was chosen because it is common in premise plumbing and generally leaches the least organic carbon among the different types of plastic pipe. Four different influent water conditions were implemented in the experimental design: 1) Untreated, dechlorinated municipal tap water with high organic carbon and low biomass; 2) GAC-treated tap water with low organic carbon and elevated, viable biomass; 3) GAC-treated + 0.22-m pore size membrane-filtered tap water to remove both nutrients and biomass; 4) GAC-treated tap water pasteurized at 70 °C with low nutrients and elevated, killed biomass. The 0.22-m pore size membrane filter simulated the use of a building scale particle filter, while pasteurization simulated water passing through a hot water heater at an elevated temperature recommended for pathogen thermal disinfection. To understand the influence of these experimental conditions on older pipes containing mature biofilms versus new pipes that leach more organics and are being freshly colonized, a set of older pipes colonized with mature ~4-year-old biofilms were compared to newly purchased pipes. Each set of pipes was tested in triplicate for the four different experimental conditions with the full volume replaced three times a week for eight months, simulating infrequently used taps containing warm, continuously mixing water thought to create conditions at a very high risk for opportunistic pathogen growth. In the aged CMPR bulk water effluents, droplet-digital-polymerase-chain-reaction measurements showed a one-log reduction of Lp and NTM when receiving GAC-treated or GAC-treated + particle-filtered influent water versus receiving dechlorinated municipal tap water or GAC-treated + pasteurized water. These findings suggest that decreased biodegradable dissolved organic carbon achieved by GAC filtration acted to suppress Lp and NTM growth, while the additional step of biomass removal by particle filtration provided a more modest benefit. In the CMPRs consisting of new pipes, concentrations of Lp and NTMs in the effluent bulk water were similar among the experimental conditions, except that the CMPRs receiving the GAC-treated + particle-filtered influent water experienced a two-log reduction in NTMs. These results demonstrate that the colonization and proliferation of NTM within premise plumbing can be significantly controlled by limiting nutrients and biomass in the influent water. This work demonstrates the potential of harnessing GAC-treatment as a means to Control Lp and NTM in premise plumbing via nutrient removal. In scenarios where chemical disinfectants have been depleted, off-the-shelf GAC-treatment used as point-of-entry treatment to large buildings with recirculating plumbing could provide benefits that have previously been unrecognized. Alternatively, pasteurization in very hot water heaters could provide a short-term disinfection benefit, but eventually the nutrients embodied in the dead biomass undermine the positive influence of the nutrient removal provided by the GAC-treatment. Improved mechanistic understanding of probiotic strategies to opportunistic pathogen control would be needed to overcome inherent limitations to the approaches examined herein, if more effective control is desired in the absence of thermal or chemical disinfection. / Master of Science / Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are bacterial pathogens that are the leading source of drinking water-associated disease in the US. Unfortunately, they are not effectively controlled by protections put in place by the US Safe Drinking Water Act (SDWA). Firstly, they cause respiratory infections, which are spread when tiny droplets of water are inhaled during activities such as showering, whereas the SDWA is specifically designed to protect against ingested pathogens. Secondly, unlike fecal-derived organisms (e.g. E. coli) that are the focus of the SDWA, Lp and NTM grow naturally in drinking water distribution systems, especially in premise (i.e., building) plumbing, where water is warmer and more stagnant. Therefore, even if water leaving the treatment plant is devoid of Lp or NTM, this does not guarantee that the consumer's tap water will be Lp- or NTM-free. Also, even though chlorine or other chemical disinfectant is required by the SDWA to be added to the water leaving the treatment plant to control downstream microbial growth, the disinfectant can be depleted or absent within the premise plumbing itself. Additionally, both Lp and NTM tend to more naturally resist chemical disinfectants than fecal-derived organisms. This research is aimed at overcoming these challenges, opening the door to new approaches to controlling Lp and NTM in premise plumbing. Historically, any microbial growth occurring in drinking water has been viewed as problematic, as it usually indicates the chemical disinfectant is inadequately protecting consumers. However, this work explores whether having an abundant community of beneficial bacteria could improve microbial water quality by competing against pathogens for limited space for attachment and nutrients. Such an approach would be analogous to the use of probiotics in humans, to establish a beneficial gut flora that is less susceptible to pathogen invasion. Granular activated carbon (GAC) filters are often used at drinking water treatment plants and by consumers as a point-of-use (e.g., installed on the kitchen tap or in a refrigerator) or whole-house treatment to remove any contaminants of concern and improve the taste and odor of tap water. The granules within GAC filters have a high surface area that helps remove contaminants, but also provides an environment where microbes can live and thrive. As water enters the filter, beneficial microbes can break down any remaining nutrients in the water (e.g., organic carbon and nitrogen). Additionally, the water leaving the filter carries high levels of microbes that grow on the GAC filter that are shed as water passes through. The resulting water with reduced nutrients and higher concentrations of potentially beneficial microbes could create a competitive environment that alters growth of harmful bacteria, like Lp and NTM, in downstream portions of plumbing. The incoming cold water is also warmed by the building envelope, which increases bacterial growth rates. Thus, the underlying hypothesis of this research is that GAC treatment could provide a combination of reduced nutrients and competitive microbes as water enters downstream premise plumbing and reduce the growth of Lp and NTM. However, GAC-treated water within a building can be further altered by other treatments, like a very hot water heater, which would heat and kill the microbes flowing through it, or a particle filter, which could remove the microbes in the water. This work also seeks to understand how these additional treatments might improve or interfere the nutrient reduction and addition of competitive microbes provided by GAC treatment. This research explores how all these different scenarios affect the growth of Lp and NTM using a lab-scale simulated premise plumbing system constructed out of polyvinyl chloride (PVC) pipe that is a common plumbing material used in homes. Water that was added to the pipes was prepared in four different ways to test the probiotic control hypothesis across distinct experimental conditions that replicate the different influent water scenarios. The four conditions were implemented over the course of eight months with regular chemical and biological analyses conducted to understand the effects of the different influent waters on Lp and NTM. It was discovered that premise plumbing with mature biofilms receiving GAC-treated water or GAC-treated + particle-filtered water contained ~90% less Lp and NTM than premise plumbing receiving non-filtered municipal tap water. However, if the GAC-treated water passes through a water heater, the capacity to limit Lp or NTM growth was lost. While GAC filters are currently thought of as an instantaneous treatment that removes contaminants from water, this work demonstrates how GAC treatment might provide prolonged benefits to water, after it has passed through the filter on its journey to a shower head or faucet. Increased understanding of the exact mechanisms of limited pathogen growth gained by this research can lead to new and effective approaches to protect people from contracting diseases caused by Lp and NTM.