Development of a vaccine for the prevention of hemorrhagic enteritis in turkeys

Hemorrhagic enteritis (HE) in turkeys is an acute infectious disease characterized by depression, intestinal bleeding, and death. HE occurs worldwide affecting 6 to 12 week-old turkeys and lasting 4 to 6 days. This economically important disease is caused by hemorrhagic enteritis virus (HEV), a turkey adenovirus which is tentatively classified as a member of the group II avian adenoviruses. Serologically related HEV strains with marked differences in pathogenicity for turkeys have been described. Until recently, only 2 vaccines were available for the prevention of HE in turkeys. Both are live virus vaccines containing avirulent HEV (HEV-A) and both elicit protective immunity in turkeys. However, since the first vaccine is a crude extract prepared from spleens of turkeys infected with HEV-A, and the second vaccine is propagated in a transformed cell line contaminated with Marek's disease virus, their safety features are questionable.

HEV is unique among the adenoviruses because it is not antigenically related with the mammalian or group I avian adenoviruses. Its classification as an adenovirus is based upon common physical, chemical, morphological and structural properties. An adenovirus is composed of 240 hexons and 12 pentons, outer capsid proteins which give the virus its characteristic icosahedral shape, capsid associated proteins, and core proteins associated with the double-stranded linear DNA genome with a molecular weight of 17 - 30 x 10 ⁶. Until recently, HEV and its structural proteins had been poorly characterized due to the lack of a suitable invitro system for virus propagation. In summary, there was a need for an improved vaccine for HE in turkeys, and the development of a such a vaccine would be facilitated by the discovery of a cell type suitable for HEV replication and by a more basic knowledge of the virus itself.

The major goal of the research described in this dissertation was the development and testing of a safe and efficient vaccine for HE in turkeys. In order to achieve this goal, a cell culture system for virus propagation as well as methods to measure virus replication invitro and protection in immunized birds had to be developed. In addition, the knowledge of virus and viral components had to be expanded.

The development and application of sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for the quantitation of HEV antibodies in turkey sera and HEV antigen in tissue extracts is described in Chapter 2. The presence and decline of maternal antibody titers in sera of poults and seroconversion and induction of protective antibody titers in turkeys following immunization with HEV-A were determined by ELISA (Chapters 2 and 6). The ELISA for the titration of antigen was used to monitor protection in turkeys following immunization with HEV-A and challenge with virulent HEV (HEV-V) (Chapter 6). A strong antigenic relationship between HEV-A and HEV-V was measured with both ELISAs.

The characterization of both HEV-A and HEV-V and their structural proteins, purified from spleens of infected turkeys is described in the Chapters 3 and 4. The electron microscopic data on the size (72nm) and structure of the virion and its density in CsCl (ρ= 1.34 g/cm3 ), as well as the profile of the viral polypeptides in polyacrylamide gels showing molecular weights ranging from 96,000 to 9,500, justified the classification of HEV as an adenovirus. The major structural proteins were identified as hexon, penton, penton base, fiber, IIIa, and core proteins based on their structure observed by electron microscopy and/or recognition by specific antibodies. Free hexon and penton proteins, purified by immunoaffinity chromatography using monoclonal antibodies, had identical properties as their counterparts in the virus. The hexon was an important neutralizing antigen. The penton of HEV consisted of a single fiber attached to its penton base, a feature shared with the mammalian adenoviruses and the avian egg drop syndrome 1976 virus, but not with the fowl adenoviruses which have double fibers. In contrast to the many common properties of HEV-A and HEV-V, serological differences between the fibers of and differences in electrophoretic migration between the penton bases of both strains were observed. The IIIa, proteins of HEV and human adenovirus type 2 shared a common epitope. This is the first antigenic relationship detected between avian and mammalian adenoviruses.

The propagation of HEV-A and HEV-V in turkey blood leukocyte cells is escribed in Chapter 5. The presence of HEV in the nuclei of non- adherent as well as in adherent cells was revealed by electron microscopy and by light microscopy, using a fluorescent antibody test. The non-adherent infected cells had the characteristics of immature mononuclear leukocytes while the adherent cells had monocyte-macrophage characteristics. HEV-A could be serially passed in turkey leukokcytes at least seven times. optimum conditions for virus propagation in turkey leukocyte cultures and harvest times were determined. HEV could not be produced in chicken leukocytes.

HEV-A, propagated in turkey leukocyte cell cultures, was tested as a vaccine to prevent HE in turkeys in experimental and field trials (Chapter 6). Immunization of turkeys with live HEV-A resulted in protection against a challenge with HEV-V as measured by the serological response and the absence of clinical disease and HEV antigen in spleens. In the field trials, 19 out of 20 flocks seroconverted within 21 days after vaccination with live HEV-A distributed in the drinking water. The overall immune response of the turkeys in these flocks was 96%. Most importantly, neither clinical nor other adverse effects caused by HEV-A vaccination were observed in any of the vaccinated turkeys in the experimental and field trials. The optimum time of the vaccination of poults was determined in relation to interference with maternal antibodies.