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.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1975. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 116-125.

Proefschrift Universiteit Maastricht. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-18Bitstream added on 2014-06-13T18:31:18Z : No. of bitstreams: 1 rosa_acg_me_araca.pdf: 260018 bytes, checksum: 83d376eb61dc20b26646b886718270e8 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo descreve o desenvolvimento e a aplicação da reação de transcrição reversa in situ em cadeia da polimerase (RT-PCR in situ), para detectar a co-infecção experimental de perus de 1-dia de idade com Coronavirus (TCoV) e Astrovirus (TAstV-2) isolados de casos clínicos no Brasil. A primeira etapa da reação consistiu na preparação específica de sondas de DNA biotiniladas homólogas ao gene da polimerase viral do TAstV-2 e da região 3'UTR do TCoV. Foram utilizados cortes histológicos de intestino correspondendo ás regiões do íleo, junção íleo-ceco e ceco para avaliar a reação de RT-PCR in situ. Para permeabilização tecidual uma digestão foi aplicada com 10 μg/μl proteinase K por 30 min. Na etapa de hibridização, as sondas de DNA homólogo às regiões genômicas virais ligadas à biotina foram diluídas na concentração de 2μg/μl na solução de hibridização e incubadas overnight à 42ºC. Em seguida, uma diluição ótima do anticorpo monoclonal anti-biotina acoplado a fosfatase alcalina e a peroxidase foram aplicados, para TAstV-2 e TCoV, respectivamente. O substratos diaminobenzidina 3,3 (DAB) e FastRed Ò foram utilizados para identificar a hibridização das regiões homólogas correspondentes ao TCoV e TAstV-2, respectivamente. A reação positiva foi visualizada por deposição de pigmentos vermelhos (TAstV-2) e marrom acastanhado (TCoV). Em relação à localização dos genes virais amplificados, foram confirmados nas células da base (células caliciformes) e ao longo das vilosidades intestinais principalmente no citoplasma dos enterócitos de forma difusa para ambos os vírus. Marcações positivas também foram evidenciadas na submucosa próximas às regiões com intensa congestão vascular... / This study describes the development and application of the reaction in situ reverse transcription polymerase chain reaction (RT-PCR in situ) to detect co-infection of turkeys to experimental 1-day-old with Coronavirus (TCoV) and Astrovirus ( TAstV-2) isolated from clinical cases in Brazil. The first step of the reaction has been to prepare specific biotinylated DNA probes homologous to the viral polymerase gene of TAstV-2 and the 3'UTR region of TCoV. We used histological sections of intestine corresponding to the regions of the ileum, ileum-cecum junction and cecum to evaluate the reaction of RT-PCR in situ. For permeabilization tissue digestion was applied with 10 g / uL proteinase K for 30 min. In step hybridization, DNA probes homologous to the viral genomic regions linked to biotin were diluted to the concentration of 2μg/μl in hybridization solution and incubated overnight to 42 ° C. Then, an optimal dilution of monoclonal anti-biotin coupled to alkaline phosphatase and peroxidase were applied to TAstV-2 and TCoV, respectively. 3.3 The substrate were used to identify the hybridization ofÒdiaminobenzidine (DAB) and FastRed homologous regions corresponding to TCoV and TAstV-2, respectively. The positive reaction was visualized by deposition of red pigment (TAstV-2) and brown brown (TCoV). Concerning the location of the amplified viral genes was confirmed by the base cells (goblet cells) and along the intestinal villi in the cytoplasm of enterocytes diffusely to both viruses. Tags positive were also demonstrated in the submucosa close to the areas with intense vascular congestion. In conclusion, the RT-PCR in situ standard in this study showed good ability to detect viral RNA promoting a desirable... (Complete abstract click electronic access below)

Hemorrhagic enteritis is a disease of turkeys caused by virulent strains of Turkey Hemorrhagic Enteritis Virus (THEV) resulting in depression, splenomegaly, intestinal hemorrhage, immunosuppression, and mortality. Avirulent strains that do not produce intestinal lesions and mortality are used in live-virus vaccines that protect turkeys from virulent field challenge. The cause for the difference in phenotype between virulent and avirulent strains is unknown. The full-length genome of the Virginia Avirulent Strain (VAS) of THEV was sequenced and compared to the genome sequence of a virulent field isolate from Israel. Genetic differences were found in seven viral genes. Further sequencing narrowed the focus from seven genes to three: ORF1, E3, and Fiber. Consistent variation in these genes between strains of THEV with different phenotypes strongly indicates these genes as key factors affecting virulence. THEV is an officially recognized member of the viral family Adenoviridae, genus Siadenovirus. The genomes of the members of the genus, THEV and Frog Adenovirus 1, are not well-characterized. The genome sequences of both members were compared for the prediction of genetic and structural elements. Common features were found that distinguish this genus from all other adenoviruses, and differences were found that possibly contribute to host specificity of the members. The VAS is known to stimulate a life-long protective antibody response, though viral replication is only of short duration. Several studies were undertaken to determine changes in virus location and serology over time. Viral DNA was detected in various tissues through 15 weeks post-infection in the presence of high antibody titers. THEV infection was found to be similar to the non-lytic persistent infections seen with human adenoviruses. Regardless of the mechanism involved in the persistent stimulation of antibodies in infected turkeys, the VAS was shown to be an ideal vector for use in a recombinant live-virus vaccine. The next step in THEV research should be the creation of a full-length infectious DNA clone, which could be used in the creation of a recombinant vaccine. The infectious clone would also allow for the systematic testing of genes that are suspected to be involved in virulence. / Ph. D.

Hemorrhagic Enteritis (HE) vaccine is perhaps the most commonly used vaccine in the turkey industry. Although it provides protection against clinical disease, the vaccine is still thought to produce transient immunosuppression. In the field, HE still remains a significant concern for turkey producers. Research conducted over the years has shown that management stressors such as movement of turkeys from brooding to finishing environments and the timing of these stressors may influence the short-term response to vaccination. Strategic stress application may be of benefit in the optimization of protective responses and the development of vaccination protocols without detrimental effects on performance. Ascorbic acid may also have important implications on social stress and may play a role in immunity and response to HE vaccination in turkeys. Trials were conducted to examine the interrelationship among social stress, nutrition (vitamin C), immunity and their influence on response to hemorrhagic enteritis virus (HEV) vaccination. Stress is unavoidable, however if it is managed properly, it can be beneficial. In this dissertation, it was first demonstrated that stress in the form of social disruption can have negative physiological and immunological effects on turkey poults and that these effects can be alleviated with the addition of 300mg/kg vitamin C to the diet. Secondly, it was also demonstrated that when stress is applied on the day of vaccination, response to HEV vaccination can be improved. Thirdly, vitamin C supplementation at 300mg/kg can improve responses to HEV vaccination. However, it was concluded that vitamin C supplementation during periods of simultaneous stress application and vaccination does not provide benefit to response to vaccination. / Ph. D.

Turkey hemorrhagic enteritis virus (THEV) is responsible for the hemorrhagic enteritis (HE) disease in commercial turkeys through infections by its virulent strains. HE is an acute condition characterized by depression, immunosuppression, bloody droppings, intestinal hemorrhage, and death. THEV (also known as turkey adenovirus 3) is an official member of the family Adenoviridae, genus Siadenovirus, species Turkey siadenovirus A. Two main types of live vaccines are currently used for the protection of turkeys against HE; a crude splenic vaccine propagated in live turkeys, and a cell culture-based vaccine generated in RP19 cells. The only laboratory-adapted tests for assessing the titers of these vaccines are agar gel immunodiffusion test and cell culture endpoint dilution, respectively. The assays suffer from low sensitivity, inaccuracy, and time consumption. A SYBR Green-based real time PCR assay for determining the genomic titer of THEV through the quantification of its hexon gene was developed. The assay was applied as a quality control for the titration of splenic vaccines and was found useful in distinguishing the differences in virus titer among many vaccine batches. Additionally, using the qPCR assay along with a cell culture system, a novel infectivity assay was developed for the titration of THEV, as an alternative for the endpoint dilution assay. Applying the assay on nine batches of commercial HE cell culture vaccines, high variations in infectious virus titers were detected. The new method is rapid, sensitive, and very accurate. A strong correlation was found between the genomic titer and qPCR infectious titer in HE cell culture vaccines. Moreover, the qPCR infectivity assay proved as an instrumental research tool. It was used to measure the effect of several treatments of RP19 cells on virus infection. The main target cell type for THEV infection and replication is B-lymphocytes, which are represented in vitro by the B lymphoblastoid, RP19 cells. The cellular surface components used by the virus to gain entry into cells are unknown. As an adenovirus, we hypothesized that THEV uses two different molecules on RP19 cells for the attachment and internalization. A recent study has shown that the synthesized THEV fiber knob domain binds to sialyllactose, based on a glycan array analysis. In our studies, the treatment of RP19 cells with neuraminidases and lectins resulted in high reduction of virus entry, which provide a strong evidence of the utilization of cell surface sialic acids as attachment receptor for THEV. Destruction of surface carbohydrates and proteins on RP19 cells also reduced virus entry, indicating that these components are part of the THEV receptor. Using virus overlay protein blot assay, THEV was found to specifically bind to two RP19 surface membrane proteins, most likely, representing primary and secondary receptors for virus entry. Further studies are required to identify these proteins and verify their role in THEV endocytosis in host cells. / Ph. D.

The immunopharmacologic and immunopathologic mechanisms through which hemorrhagic enteritis virus (HEV) induces intestinal lesion formation associated with clinical infection is unknown. These studies were designed to 1) determine differences in age susceptibility to HEV infection and parameters which best indicate severity of virus infection, 2) determine the involvement of specific inflammatory mediators in the formation of intestinal lesions by using anti-inflammatory compounds which directly or indirectly inhibit either the synthesis or the mechanism of action of the mediators, and 3) determine the involvement of immunologically-active cells—basophils, connective tissue mast cells (CTMC), and mucosal mast cells (MMC)—in the formation of HEV-induced lesions. Seven-week-old poults were more susceptible to viral infection when compared to four-week-old poults as judged by HEV antigen presence within the spleen, spleen weight/body weight ratio, heterophil to lymphocyte ratio, and changes in serum lipid and albumin concentration. Of those anti-inflammatory agents used, corticosterone, vitamin E, and indomethacin significantly reduced lesion scores. FPL 55712 and FPL 57231, specific leukotriene receptor blockers, markedly increased lesion scores. Inoculated birds had significantly higher MMC counts than uninoculated birds (120±64 vs. 55±39, respectively). CTMC and basophils were unaffected by viral challenge. In addition to the increase of MMC within the lamina propria of the duodenal villus, there was also a concurrent increase in vascular permeability within the lamina propria which was demonstrated using colloidal carbon and vascular permeability within the lamina propria which was demonstrated using colloidal carbon and ferritin as vascular markers. The results of these studies indicate that vasoactive mediators, such as histamine and the eicosanoids, play a role in lesion formation associated with HEV infection, and that a source of at least some of these compounds appears to be the MMC within the lamina propria of the duodenal villus. Finally, some of the other clinical manifestations of HEV infection, such as decreased serum lipid, protein, and albumin, may be a result of increased vascular permeability which results from vasoactive mediator release and action on the vessels of the lamina propria of the intestinal villus. / Ph. D.

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.

Hemorrhagic enteritis virus (HEV) causes an acute viral disease in turkeys characterized by bloody diarrhea and death. Current live HEV virus vaccines are immunosuppressive and predispose turkeys to secondary bacterial infections. Data indicates that the capsid proteins (fiber, penton base, hexon) of HEV are capable of stimulating protective antibodies against an HEV challenge. Using tobacco as a model, we sought to determine if a plant could be used to synthesize the HEV fiber protein and produce sufficient antigen to stimulate protective antibodies. To introduce the fiber gene into plants, the coding region of the HEV fiber gene was fused to either a constitutive plant promoter (35S) or a wound inducible promoter (hmg2) on plasmids adapted for Agrobacterium-mediated transformation. Approximately sixty transgenic plants of each construct were generated and determined to contain the HEV fiber gene based on amplification of specific HEV DNA sequences by the polymerase chain reaction. Plants were screened by Northern dot blot to identify lines expressing high levels of fiber mRNA. Expression of fiber protein was observed in selected lines of transgenic tobacco by Western blot analysis using turkey anti - HEV serum. The accumulation of fiber protein in leaves of tobacco transformants was quantified by Sandwich ELISA. Fiber protein from these plants has undergoing large - scale purification and concentration for a turkey immunization trials to determine if plant expressed fiber antigen is capable of inducing protective antibodies against HEV in turkeys. / Master of Science