<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

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.

<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

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A proper vaccination program can play a critical role in prevention and control of avian influenza (AI) in commercial poultry. Low pathogenic avian influenza viruses (LPAIV) of H5 and H7 AI subtypes cause serious economic losses to the poultry industry and have the potential to mutate to highly pathogenic AI (HPAI) strains. Due to trade implications, differentiation of infected from vaccinated animals (DIVA) is an important issue in the control of AI. Therefore, the development and characterization of vaccine candidates with DIVA properties is critical in improving vaccination programs. Keeping these aspects in mind, we investigated the role of an NS1 mutant virus as a potential live attenuated DIVA vaccine. The NS1 protein of influenza virus plays a major role in blocking the host's antiviral response. Using an eight-plasmid reverse genetics system, we recovered the low pathogenic parental (H5N3) and NS1 mutant (H5N3/NS1/144) viruses. H5N3/NS1/144 expresses only the first 144 amino acids of the NS1 protein compared to the 230 of the parental H5N3. The growth properties of H5N3 and H5N3/NS1/144 were compared in cell culture and in different age embryonated chicken eggs. Our results confirmed that NS1 is involved in down regulation of interferon as shown by IFN-beta mRNA expression analysis and by the inability of H5N3/NS1-144 to efficiently grow in older age, interferon competent, chicken embryos. However with regards to safety the virus reverted to virulence within five back passages in chickens and was therefore not a safe vaccine candidate. However the killed form of H5N3/NS1-144 was a safer alternative and it also induced antibody titers and protection not significantly different from the parental H5N3 as vaccine. To further understand the reversion of H5N3/NS1/144 to virulence, we carried out 3 independent serial passages of H5N3/NS1/144 in increasing age of embryonated chicken eggs and examined the NS1 gene for presence of mutations. RT-PCR and sequence analysis of the NS gene in all three lineages showed the presence of a 54 amino acid deletion resulting in the generation of a 87 amino acids long NS1 ORF with a point mutation (L80V) at the site of deletion. In addition, the NS1 ORF in lineages L2 and L3 presented two additional point mutations in the RNA binding domain (Q40R and T73M). To determine if these mutations played a role in increased virulence, recombinant viruses expressing these mutant NS1 proteins in the background of parental virus were generated by reverse genetics and their replication properties and pathogenicity was examined in vitro, in ovo and in vivo systems. Our results showed that the 87 amino acid long NS1 protein clearly increased virus replication and virulence specifically in interferon competent systems. In addition, the two point mutations in the RNA binding domain of NS1 ORF expressing 87 a protein slightly increased the virus virulence. Overall this study reinforces the role of NS1 in influenza virus pathogenicity and supports the use of killed inactivated NS1 mutant virus vaccines as potential DIVA vaccines.

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.

Thesis (M.S.)--California Polytechnic State University, 2009. / Title from PDF title page; viewed on January 6, 2010. Major professor: Brooke D. Humphrey, Ph.D. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Agriculture, with Specialization in Animal Science." "October 2009." Includes bibliographical references (p. 102).

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains x, 157 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.F.A.)--Kent State University, 2009. / Title from PDF t.p. (viewed Jan. 21, 2010). Advisor: Kirk Mangus. Keywords: Chickenware, ceramics, craft, pottery, Kevin, McCreary. Includes bibliographical references (p. 15).