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.

Typescript (photocopy) / iv, 206 leaves, [4] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Entomology, 1980

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.

As the principal antigen-presenting cells to T cells, dendritic cells (DCs) have a key role in the balance of immunity and autoimmunity. They are essential in two major, converse roles - eliciting T cell immune responses to pathogenic material, and maintaining peripheral tolerance to self-tissue by inhibiting self-reactive T cells. These functions involve the processing of pathogenic or self antigens and subsequent presentation of antigenic peptides on MHC to antigen-specific T cells. DC recognition of conserved pathogenic markers induces a mature phenotype that governs immunogenic presentation to T cells and, consequently, the adaptive immune response. In contrast, DC recognition of self tissue suppresses maturation, instead inducing a tolerogenic phenotype that induces self antigen-specific T cell to die, become anergised, or converted to T regulatory cells. Apoptotic cells are the major source of self-antigen for the maintenance of peripheral tolerance, and their defective clearance by DCs is implicated in autoimmunity. Apoptotic cells are thought to actively suppress maturation of DCs and inhibit the possible immune responses promoted by proinflammatory mediators released from necrotic cells. However, the immune function of apoptotic cells and their relative influence over necrotic cells are highly contested, partially due to the complex nature of immunogenicity arising from the sourcing and generation of apoptotic cells. In this investigation, various methods of inducing apoptosis and necrosis are evaluated. Definitive methods of inducing well-characterised cell death are then employed to compare the effects of apoptotic and necrotic cells on dendritic cells and in vitro and in vivo immune responses. Reported here are in vitro findings that support previous reports of the anti-inflammatory response of DCs to apoptotic cells, and the inflammatory response of DCs to necrotic cells. The previously-reported inhibitory effect of apoptotic cells on LPS-induced secretion of Th1 cytokines is supported here, but the inhibitory effect of apoptotic cells on LPS-induced upregulation of co-stimulatory molecules is contested. Novel findings describe the upregulation of DC expression of co-inhibitory molecules induced by both apoptotic cells and necrotic cells. Apoptotic cells, but not necrotic cells, had a suppressive effect on CpG-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokines. Apoptotic cells suppressed the capacity of untreated and CpG-treated, but not LPS-treated, DCs to elicit IFNγ production by T cells. Apoptotic cells, but not necrotic cells, induced regulatory T cells and partially restored their CpG-suppressed induction. Finally, apoptotic cell-modulation of DCs inhibited the induction of autoimmunity in a novel modification of an in vivo model of diabetes. Interestingly, novel evidence for the possibility of necrotic cell-induced tolerance by means of direct T cell killing is addressed.

Intensified poultry production to meet global food demands has faced challenges associated with the removal of in-feed antibiotics due to concerns over antibiotics resistance. The reduction of low-dose antibiotics in feed has allowed for reemergence of intestinal diseases that diminish animal welfare and producer economics. Alternative mechanisms to preventing disease are therefore required. The objective of this dissertation was to examine factors that contribute to chicken development and health including intestinal structure and function, environment, and genetic selection. Chapter 2 investigated the host response to infection of the parasite Eimeria maxima that predisposes chickens to a bacterial infection Clostridium perfringens. Intestinal structure, function, inflammatory response, and epithelial composition was examined during a mild subclinical infection. Analysis of E. maxima and C. perfringens as individual infections revealed how each pathogen contributes to a co-infection. E. maxima caused a more severe inflammatory response, increasing pathology scores, shortening intestinal villi, and elongating crypts in the jejunum at peak infection. C. perfringens was shown to manipulate intestinal epithelial composition by influencing stem cells to differentiate into secretory goblet cells. The most deleterious effects were observed when the pathogens were introduced together, increasing pathology scores further, damaging intestinal villi, and increasing crypt depth. The introduction of C. perfringens and E. maxima also increased signaling for the production of reactive oxygen species, stimulation of tumor necrosis factor- that is involved in innate immunity, and decreased transcription of Hes1, which is involved in Notch signaling towards absorptive cell differentiation. Hes1 has previously been shown to be involved in the inflammatory response and could be an area of interest in determining new treatments to prevent or relieve the effects of E. maxima and C. perfringens. Chapter 3 applied an environmental perspective to disease prevention and examined the properties of C. perfringens that allow it to persist in the poultry house environment. Spores resist treatments used to sanitize poultry houses and litter has been shown to be a reservoir for disease, potentially increasing occurrence in certain houses. The metabolic and physiological properties of C. perfringens were utilized to separate the microbe from other poultry litter bacteria to enumerate spores within houses. A selective and differential medium combined with a heat treatment was developed to isolate C. perfringens spores from poultry litter samples. On average, houses that had histories of necrotic enteritis harbored a greater abundance of C. perfringens spores. Colonies that were isolated on the specialized medium were confirmed using PCR as C. perfringens. Lastly, Chapter 4 examined how genetic selection for multiple traits has influenced early intestinal development compared to divergently selected lines based on eight-week body weight. This study showed the morphological and gene expression differences between lines and revealed that most pathways involved in intestinal development are conserved through genetic selection. The major differences between lines were an increase in peptide transporter PepT1 on d5 and d7 in chicks selected for low eight-week body weight (LWS) compared to high weight selected (HWS) chicks and modern broiler Cobb500 chicks. In HWS chicks, the opposite mechanism was observed with an increase in expression of secretory goblet cell marker Muc2. The findings of these studies give multiple perspectives into poultry production and how major factors in management including nutrition, environment, and genetics can be used to increase efficiency while preventing disease. / Doctor of Philosophy / In poultry production, it is important to use management methods that help chickens grow efficiently while preventing illnesses. A few factors that contribute to the success of a producer include the use of nutrition to enhance intestine health and efficiency, a healthy environment in the poultry house, and using genetics to select for multiple traits to increase productivity. These factors have become even more significant after concerns of antibiotic resistance has eliminated the use of in-feed antimicrobials, allowing for reemergence of diseases that were suppressed. Therefore, the objective of this dissertation was to utilize each of these management strategies to determine how a common disease to the poultry industry affects the chicken intestine, how the environment influences the occurrence of this disease, and how genetic selection impacts the early development of chicks that may contribute to how they handle incidences of disease. Chapter 2 investigated how the bacteria Clostridium perfringens and parasite Eimeria maxima that are commonly seen together in the industry impact the intestinal function, structure, and how the chicken's immune systems respond to invasion by these pathogens. The major finds of this chapter included an increased inflammatory response after E. maxima infection that damaged intestinal structures. These pathogens also decreased the expression of a gene involved in absorptive cell formation that contributes to the inflammatory response. In Chapter 3, environment was investigated to determine if poultry houses that harbored more C. perfringens spores, which are resistant to sanitary treatments, predispose chickens to disease. On average, houses with more spores were correlated with increased disease occurrence. The method developed to determine isolate C. perfringens spores can also be used to monitor the abundance in poultry litter and used as a management tool to prevent or diagnose disease outbreaks. In Chapter 4, the influence of genetic selection on early intestinal development was studied using a modern line of broiler chicks compared to chicks selected for low or high body weights. This study gave insight into how intestinal development is mostly conserved after selecting for multiple genetic traits or a single trait (growth). The main differences were greater body weight in the modern line and high weight selected chicks compared to low weight selected chicks and an increase in gene expression of a peptide transporter in low weight chicks and a decrease in secretory cell expression in high weight chicks. These projects investigated multiple management strategies to address intestinal development and response to pathogens, disease occurrence, and genetic selection as tools to shape intestinal structure and composition.

Necrotic enteritis (NE), a devastating enteric disease caused by Clostridium perfringens type A, contributes to the losses of 6 billion dollars worldwide per year and is currently being considered as a major global threat to the poultry industry. In past decades, it has been well-controlled by ineed antimicrobial growth promoters (AGPs). The withdrawal of AGPs due to antibiotic-resistance concerns resulted in a spike in NE incidence and led to the re-emergence of NE in the modern broiler production system. To unveil the association of toxin genes of C. perfringens, particularly for netB, with clinical NE, a self-designed qPCR primer set targeting netB was developed to qualify and quantify netB in NE-producing and non-NE-producing isolates. The netB was demonstrated to exist in the majority of C. perfringens type A isolates. The presence and the amount of netB were not significantly different between two types of isolate, indicating that those indicators are insufficient to predict an association with the pathogenicity of NE. The virulence of netB is suggested to be expressed or triggered under certain conditions, further promoting NE. A side by side trial was implemented with different combinations of netB-positive C. perfringens (CP1) and two predisposing factors to assess their role in NE development. Both CP1 and predisposing factor(s) are required for consistent NE reproduction, and particularly, Eimeria exerts significant effects on NE induction. The use of CP1 without a predisposing factor failed to induce NE. The severity and incidence of NE were positively correlated with the number of predisposing factors given in the NE induction. Analyzing gut microbiota in chickens challenged with CP1 and/or Eimeria by metagenomic sequencing, significant overgrowth of Clostridium sensu stricto 1, the genus contains C. perfringens, was associated with NE. Eimeria infection precedent to CP1 challenge had a synergistic effect on the overrepresentation. In addition to C. perfringens, the other member under Clostridium sensu stricto 1 was found to participate in NE development. Given supplementary dose of 0.4 kg/ton in feed, lauric acid neither exerted the inhibitory effect against proliferation of Clostridium sensu stricto 1 and C. perfringens nor reduced the incidence and severity of NE.

No description available.