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Previous reports have shown that some bacteria utilize a dissimilatory nitrate reductase enzyme (NR) in anaerobic environments. This enzyme reduces nitrate to nitrite and also has been shown to co-metabolize chlorate to cytotoxic chlorite. A commercially available competitive exclusion (CE) product was evaluated for its nitrate reductase activity and therefore its experimental chlorate product (ECP) sensitivity. Of the 29 constituent bacteria of the CE culture, 11 had slight utilization of NR, 3 had moderate utilization of NR; the remaining were NR negative (with slight and moderate utilization: >0.1 to < 1.0 mM and > 1.0 mM nitrate utilized within 6 h, respectively). In vivo studies utilizing CE and ECP showed significant reductions in Salmonella. Although some of the bacteria were affected by ECP, the combined effect of the CE culture and ECP were effective in reducing Salmonella. Clostridium perfringens (CP) is a pathogen in the commercial poultry industry, which is the etiologic agent of necrotic enteritis (NE). Day-of-hatch broilers were fed a wheat diet and assigned to the following groups: control, commercial coccidia vaccine, commercial bursal disease vaccine, or the combination of the two, and challenged with CP in order to develop a disease model. Broilers in each treatment group had significant increases (P≤ 0.05) in lesion scores, mortality, and CP incidence. As pressure mounts for discontinuing the use of antibiotics in the agriculture industry, it is important to develop new strategies to combat these costly enteric pathogens. In vitro investigations evaluated a mixed gut culture with CP and the ECP at 5 mM or a 10 mM concentrations, over time. By 3 h there was a reduction (P≤ 0.05) in the 5 mM ECP and 10 mM ECP treatment groups. In vivo studies showed significant reductions in the incidence of CP and populations of intrinsic E. coli in all of the chickens provided ECP in the drinking water. Birds administered ECP in the feed showed significant reductions in lesion scores, incidence of CP and also had reduced E. coli log10 values. These results show that an ECP could provide the industry with a new management tool for controlling NE.

A series of experiments were conducted to examine the effect of chemical composition of the diet on intestinal <i>Clostridium perfringens</i> populations and necrotic enteritis (NE) in broiler chickens. In the first experiment, birds were fed high concentrations of dietary protein (fish meal or soy protein concentrate) and soluble fiber (guar gum). Clinical NE was not observed, however, there was a high level of <i>C. perfringen</i> colonization especially in guar gum fed birds. The next set of experiments examined the effect of various levels of DL-Met or MHA-FA on <i>C. perfringen</i> and other intestinal microbes. These experiments demonstrated a significant reduction (P < 0.05) in <i>C. perfringen</i> growth with methionine supplementation in ileum and cecum. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of <i>C. perfringen</i> in broiler chickens when used in high concentrations. The next three experiments were conducted to examine the effect of dietary glycine levels on gut <i>C. perfringen</i> populations, α-toxin production and NE lesion scores. Majority of birds showed clinical signs of disease with 4.16-8.33% mortality. There was a direct correlation between intestinal <i>C. perfringen</i> populations, NE lesions scores and mortality with dietary glycine level. However, due to the use of gelatin as the dietary source of glycine in these experiments, the diets also contained high proline levels which confounded our results. The last study was conducted to establish a direct causative relationship between dietary glycine concentration and <i>C. perfringen</i> growth and/or NE in broiler chickens using encapsulated amino acids. Birds fed diets containing high levels of encapsulated glycine had higher NE lesion scores than those fed encapsulated proline or no encapsulated amino acids, thus demonstrating a direct effect of glycine on intestinal <i>C. perfringen</i> growth. It is concluded that amino acid composition of dietary protein is an important determinant of intestinal microbial growth, particularly <i>C. perfringen</i>, and could affect incidence of NE in broiler chickens.

A series of experiments were conducted to examine the effect of chemical composition of the diet on intestinal <i>Clostridium perfringens</i> populations and necrotic enteritis (NE) in broiler chickens. In the first experiment, birds were fed high concentrations of dietary protein (fish meal or soy protein concentrate) and soluble fiber (guar gum). Clinical NE was not observed, however, there was a high level of <i>C. perfringen</i> colonization especially in guar gum fed birds. The next set of experiments examined the effect of various levels of DL-Met or MHA-FA on <i>C. perfringen</i> and other intestinal microbes. These experiments demonstrated a significant reduction (P < 0.05) in <i>C. perfringen</i> growth with methionine supplementation in ileum and cecum. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of <i>C. perfringen</i> in broiler chickens when used in high concentrations. The next three experiments were conducted to examine the effect of dietary glycine levels on gut <i>C. perfringen</i> populations, α-toxin production and NE lesion scores. Majority of birds showed clinical signs of disease with 4.16-8.33% mortality. There was a direct correlation between intestinal <i>C. perfringen</i> populations, NE lesions scores and mortality with dietary glycine level. However, due to the use of gelatin as the dietary source of glycine in these experiments, the diets also contained high proline levels which confounded our results. The last study was conducted to establish a direct causative relationship between dietary glycine concentration and <i>C. perfringen</i> growth and/or NE in broiler chickens using encapsulated amino acids. Birds fed diets containing high levels of encapsulated glycine had higher NE lesion scores than those fed encapsulated proline or no encapsulated amino acids, thus demonstrating a direct effect of glycine on intestinal <i>C. perfringen</i> growth. It is concluded that amino acid composition of dietary protein is an important determinant of intestinal microbial growth, particularly <i>C. perfringen</i>, and could affect incidence of NE in broiler chickens.

Previous reports have shown that some bacteria utilize a dissimilatory nitrate reductase enzyme (NR) in anaerobic environments. This enzyme reduces nitrate to nitrite and also has been shown to co-metabolize chlorate to cytotoxic chlorite. A commercially available competitive exclusion (CE) product was evaluated for its nitrate reductase activity and therefore its experimental chlorate product (ECP) sensitivity. Of the 29 constituent bacteria of the CE culture, 11 had slight utilization of NR, 3 had moderate utilization of NR; the remaining were NR negative (with slight and moderate utilization: >0.1 to < 1.0 mM and > 1.0 mM nitrate utilized within 6 h, respectively). In vivo studies utilizing CE and ECP showed significant reductions in Salmonella. Although some of the bacteria were affected by ECP, the combined effect of the CE culture and ECP were effective in reducing Salmonella. Clostridium perfringens (CP) is a pathogen in the commercial poultry industry, which is the etiologic agent of necrotic enteritis (NE). Day-of-hatch broilers were fed a wheat diet and assigned to the following groups: control, commercial coccidia vaccine, commercial bursal disease vaccine, or the combination of the two, and challenged with CP in order to develop a disease model. Broilers in each treatment group had significant increases (P≤ 0.05) in lesion scores, mortality, and CP incidence. As pressure mounts for discontinuing the use of antibiotics in the agriculture industry, it is important to develop new strategies to combat these costly enteric pathogens. In vitro investigations evaluated a mixed gut culture with CP and the ECP at 5 mM or a 10 mM concentrations, over time. By 3 h there was a reduction (P≤ 0.05) in the 5 mM ECP and 10 mM ECP treatment groups. In vivo studies showed significant reductions in the incidence of CP and populations of intrinsic E. coli in all of the chickens provided ECP in the drinking water. Birds administered ECP in the feed showed significant reductions in lesion scores, incidence of CP and also had reduced E. coli log10 values. These results show that an ECP could provide the industry with a new management tool for controlling NE.

Necrotic enteritis (NE) is an economically important, but poorly understood, disease of poultry, typically caused by Clostridium perfringens Type A strains that carry the NetB toxin gene. The objective of the current research was to identify additional genes associated with NE-causing C. perfringens strains, and thus putatively involved in virulence. To identify novel NE-associated genes, the draft genome sequences of seven C. perfringens NE isolates and one isolate from a healthy chicken were compared against nine non-poultry genomes, and three highly-conserved NE-associated loci (NELoc-1 – 3) were identified. The largest locus (NELoc-1) encoded 37 putative proteins, including NetB, an internalin-like protein, a ricin-domain protein, two leukocidins, several cell-surface proteins and a cyclic-di-guanidine monophosphate (c-di-GMP) signaling system. NELoc-1 and -3 were both localized to separate plasmids that are both predicted to undergo conjugative transfer. These findings suggest that NE pathogenesis involves multiple virulence factors that are encoded on discrete pathogenicity loci, some of which are plasmid-borne. To further elucidate the genetic basis of NE pathogenicity, a microarray was developed based on two of the sequenced NE bird isolates, and used to assess the gene content of 54 isolates from chickens with and without NE. Variable genomic regions associated with netB-positive isolates were identified, including several chromosomal fitness-related loci, such as a carbohydrate ABC transporter, ferric-iron siderophore uptake system, and adhesion locus. Additional loci were related to plasmid maintenance. This study suggests that chromosomal background confers a selective advantage to NE-causing strains, possibly through mechanisms involving iron acquisition, carbohydrate metabolism and plasmid maintenance Finally, the relationship between netB presence, NetB production and host NE status was examined to assess the hypothesis that netB-positive isolates from healthy birds frequently do not express NetB toxin. The expression of NetB toxin was determined in 57 poultry isolates, demonstrating that NetB expression is closely correlated with the presence of netB, and independent of host disease status. In conclusion, these studies have identified a number of C. perfringens genes predicted to play a role in NE pathogenesis, and suggest that NE is a complex, multifactorial disease involving both host and plasmid-encoded virulence factors.

Diet composition and nutrient balance can have a critical impact on intestinal integrity during exposure to enteric pathogens. Researchers have extensively reported benefits on nutrient availability and broiler performance as a consequence of the impact of phytase supplementation. However, the poultry industry has little information on the effects of phytase supplementation in disease settings. The objective of these studies was to evaluate phytase supplementation impact on bird performance, intestinal morphology and pH, nutrient digestibility and bone mineralization during necrotic enteritis (NE). In each experiment, Cobb 500 broilers were obtained from a commercial hatchery and housed in floor pens at the Virginia Tech Turkey Research Center. Birds were placed on used litter from a previous flock that had presented clinical signs of NE. Broilers were fed non-medicated diets formulated to meet NRC (1994) nutrient requirements, except for calcium and phosphorus. In the first experiment, birds began exhibiting clinical signs of NE on d 9, and elevated NE-associated mortality persisted until d 26. Mortality was influenced by the main effects of dietary Ca or phytase. Dietary Ca supplemented at 0.9% or 1000 FTU/kg of phytase increased mortality compared to 0.6% Ca or 0 FTU/kg phytase, respectively, from d 0 to 19. Feed intake (FI) and feed conversion (FC) were affected by Ca x P interaction. From d 0 to 19, birds fed 0.9% Ca and 0.3% available P (avP) had decreased FI and improved FC compared to birds fed 0.9% Ca and 0.45% avP, while FI and FC were similar in birds fed diets with 0.6% Ca, regardless of avP level. Calcium x P x phytase interaction influenced BW or BWG from d 0-12. In general, birds fed 0.9% Ca and 0.45% avP with phytase were heavier compared to birds fed 0.6% Ca, 0.45% avP, and phytase. Calcium at 0.9% increased gizzard (d 19) and jejunum (d 12) pH. Dietary Ca supplemented at 0.9%, avP supplemented at 0.45%, and 1,000 FTU/kg phytase significantly increased tibia ash weight compared to 0.6% Ca, 0.3% avP, and 0 FTU/kg phytase, respectively, on d 12. A 3-way interaction was observed on d 35 for tibia ash percentage; birds fed 0.9% Ca and 0.45% avP had a significant increase in tibia ash percentage, regardless of phytase supplementation. A 3-way interaction was also observed for Ca and P digestibility on d 35. Phytase supplementation significantly increased Ca digestibility regardless of Ca and P levels of the diets. In addition, diets containing 0.6% Ca and 1,000 FTU/Kg of phytase resulted in a significant increase in P digestibility, regardless of P levels. In the second experiment, birds also began exhibiting clinical signs of NE on d 9, and elevated NE-associated mortality persisted until the end of the trial (d 21). Mortality was significantly affected by an interaction between Ca source and Ca levels. Significantly higher mortality was observed when animals were fed 0.9% Ca diets formulated with calcified seaweed from d 0-21 compared to 0.6% Ca diets (regardless of Ca source). From d 0-7, birds fed 0.6% Ca in diets supplemented with phytase had heavier BW than the other treatments regardless of Ca source. From d 0-14 and 0-21, animals fed diets with calcified seaweed had significantly higher FC than animals fed diets with limestone. On d 21, the gizzard of birds fed 0.9% Ca was significantly less acidic than the gizzard of birds fed 0.6% Ca. In conclusion, reducing dietary levels of Ca associated with phytase supplementation improved bird performance and nutrient digestibility. In addition, these experiments indicate that Ca is an important dietary factor in the pathogenesis of NE. / Ph. D.

Necrotic enteritis in poultry is caused by Clostridium perfringens type A, and is estimated to cost the worldwide poultry industry approximately $2 billion dollars a year, due to increased mortality and decreased feed conversion and weight gain. Very little is known about the pathogenesis of this disease due to the lack of a consistently reproducible experimental model. This dissertation outlines the development of an effective and consistent experimental model for necrotic enteritis in broiler chickens. It was also found that in vivo passage through the chicken's intestinal tract let to increased virulence; we increased the proportion of birds developing disease from 34.6% to 81.4%. Researchers have proposed that alpha toxin (CPA) is believed to be the critical virulence factor of the disease. All type A isolates have the potential to produce CPA, thus we challenged birds with numerous type A isolates that are virulent in other animal hosts. However, we found that they did not produce necrotic enteritis in broiler chickens. In addition, challenge with culture supernatant alone failed to produce gross lesions in the birds, although challenging with washed whole cell cultures did do so. Vaccinating birds with HIS-tagged recombinant CPA provided partial protection against disease; there was a 42.0% decrease in lesion development. The conclusion of this doctoral research is that CPA does have a role in the pathogenesis of necrotic enteritis in broiler chickens, but there are apparently other critical virulence factors involved in the development of disease.

The objective of this study was to determine the effect of time, temperature and organic matter on disinfectant efficacy. Staphylococcus aureus (SA) and Salmonella Typhimurium (ST) were used as organisms to represent Gram positive and Gram negative bacteria, respectively, commonly found in poultry housing. Three independent experiments evaluated the effect of temperature, time, and organic matter on the efficacy of working concentrations of disinfectants against representative organisms found in commercial poultry housing. Quaternary ammonium, chlorhexidine, phenolic and binary ammonium based solutions represented disinfectants commonly used within the poultry industry. Results from these experiments indicated that long term storage of disinfectants will reduce their efficacy against SA. However, a reduction (p ≤ 0.05) in efficacy was observed with the phenolic compound against ST at elevated temperatures. Following the inclusion of organic matter (OM), reduced (p ≤ 0.05) efficacy of all disinfectants was observed in a dose dependent manner against both organisms, with the exception of the phenolic compound against SA. Fresh disinfectant performed better (p ≤ 0.05) in the presence of OM than 30 wk old disinfectant. These results emphasize the need to use fresh disinfectants and that OM should be removed prior to disinfection. We also evaluated the effect of bismuth citrate, lactose and citric acids on the development of necrotic enteritis in broilers. Clostridium perfringens’ associated necrotic enteritis in poultry causes significant loss and increased morbidity in the industry. Due to the reduced usage of antibiotic growth promoters, the incidence of necrotic enteritis has increased. These experiments evaluated different levels of bismuth citrate and bismuth citrate with lactose or citric acid added, on lesion development, bacterial intestinal colonization of C. perfringens and pH levels in the gut of broilers orally challenged with C.perfringens. Results from this investigation indicate that bismuth citrate at 100 ppm and 200 ppm caused a reduction (p ≤ 0.05) in C. perfringens colonization and intestinal lesion development. The addition of dietary lactose to bismuth citrate enhanced the effect of bismuth citrate on intestinal lesion development. These data suggest that bismuth citrate alone or in combination with dietary lactose will reduce intestinal lesion development in broilers with necrotic enteritis.

Necrotic enteritis (NE) is an economically important enteric disease of broiler chicken primarily caused by a-toxin (Cpa) secreted by C. perfringens type A. Mice immunized with recombinant C-terminal domain of Cpa (CpaCD) had transient and fewer localized lesions upon challenge with C. perfringens type A. These results demonstrate the usefulness of CpaCD as an immunogen for vaccine development against NE for chickens. Chicken CD40 (chCD40) is mainly expressed on the surface of chicken antigen-presenting cells (APCs), and the interaction of chCD40 and chCD40L (natural ligand for chCD40) provides crucial activation signals for chicken B-cells. A hypothesis was proposed that in ovo vaccination with an adenovirus-vectored CpaCD vaccine capable of targeting immunogen to APCs through the CD40 pathway will improve protection against NE in chickens. One agonistic monoclonal anti-chCD40 antibody (designated 2C5) was produced and characterized. 2C5 not only detected expression of chCD40 on chicken APCs, but also induced NO synthesis in chicken HD11 macrophages and enhanced proliferation of serum-starved chicken DT40 B-cells. This demonstrated substantial functional equivalence of 2C5 with chCD40L. The potential of 2C5 as an immunological adjuvant was further assessed by targeting a hapten to chicken APCs in hopes of enhancing an effective IgG response. Seven-week old chickens were immunized subcutaneously once with a complex consisting of 2C5 and peptide, and relative quantification of the peptide-specific IgG response showed that this complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. An agonistic anti-chCD40 single-chain variable fragment (designated DAG1) was combined with an adenoviral delivery system to create a vaccine, Ad-(DAG1-Cp aCD-FLAG), for in ovo administration. The efficacy of in ovo vaccination of broilers with Ad-(DAG1-Cp aCD-FLAG) in controlling NE was evaluated by C. perfringens type A challenge at 18 days post-hatch. Neither statistically significant IgA / IgG response nor protection against C. perfringens type A challenge was found in the vaccinated birds. These preliminary data suggest that a super-optimal dose of Ad-(DAG1-Cp aCD-FLAG) may be the main issue, because Cpa-specific B-cells may undergo apoptosis through the CD40 pathway.