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.

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