W. Okazaki

Protection against Marek's disease by vaccination with a herpesvirus of turkeys.

SUMMARY Studies were conducted to determine whether a herpesvirus isolated from turkeys (HVT) would protect chickens against subsequent challenge with the virulent JM strain of Mareks disease herpesvirus (MDHV). HVT administered intra-abdominally at doses as low as 600 plaque-forming units per one-day-old chick gave protection against Mareks disease (MD). The virus would protect when birds were challenged with MDHV by intra-abdominal inoculation at 3 weeks or by contact exposure as early as 2 weeks postvaccination. Chickens inoculated with HVT and observed for 17 to 20 weeks did not develop lesions. Birds produced antibody and infection persisted throughout this period, however, as indicated by reisolation of the virus. Furthermore, HVT did not spread to chickens in direct contact with vaccinated birds. Thus HVT gives substantial protection against the development of MD yet is nonpathogenic and noncontagious, all of which are important characteristics of vaccine viruses.

An Enzyme-Linked Immunosorbent Assay For Detecting Avian Leukosis-Sarcoma Viruses

Immunoglobulins from antiserum raised against chromatographically purified avian myeloblastosis virus (AMV) group-specific (gs) antigens were used in enzyme-linked immunosorbent assay (ELISA). Readily discernible color was produced with 2--3 ng of AMV protein in microplate wells coated with 4 micrograms of salt-precipitated immunoglobulins. When a biological assay, i.e., phenotypic mixing (PM), was the criterion for the infectious status of specimens, the ELISA consistently identified a greater percentage of virus-positive specimens than direct complement-fixation (DCF) tests. Over 95% concordance was obtained between the ELISA and PM bioassays when meconia and whole-blood samples were tested. Moreover, three DCF(-) egg albumens from one virus shedder hen were positive by the direct ELISA. Complete agreement was found between a biological assay for endogenous virus and the ELISA when blood and albumens from inbred chickens were tested. The ELISA is a rapid and convenient alternative to the DCF test for identifying infected chickens in eradication programs, because virus-rich sources such as meconia and blood that are unsuitable for DCF can be tested directly.

Phenotypic mixing test to detect and assay avian leukosis viruses.

A phenotypic mixing (PM) test for detecting and assaying avian leukosis viruses (ALV) of the A, B, C, and D subgroups is described. An ALV and Rous sarcoma virus RSV-0) are phenotypically mixed by co-cultivating on C/O (cells susceptible to all subgroups of ALV) cells for a certain period. Then the RSV with the new virus property is assayed on C/E cells (cells resistant to infection with subgroup E leukosis/sarcoma viruses). The test is relatively simple and rapid, and its results are unequivocal. It is as sensitive as the more lengthy complement-fixation test (COFAL). The system is suitable for detecting avian leukosis viruses in samples such as heparinized blood, plasma, and embryo extracts.

Long-Term Field Trials with the Herpesvirus of Turkeys Vaccine against Marek's Disease

In long-term field trials lasting approximately 18 months, the herpesvirus of turkeys proved safe and highly effective in preventing Mareks disease (MD). The average MD mortality was reduced from 19.2 to 2.8%, and the degree of protection averaged 80.8% in the 11 experiments in which detailed necropsies were performed. Protection lasted the entire experimental period and there was a reduction in leukosis condemnation at slaughter. The degree of protection varied from flock to flock but was greatest in flocks that had the highest incidence of MD in the unvaccinated controls. There was no apparent difference in protection between the low, intermediate, or high (respectively about 500, 2,000, or 10,000 PFU per chick) doses of vaccine. Vaccination reduced the number of birds from which MD virus could be isolated and also reduced the level of virus in the positive birds. Lymphoid leukosis mortality exceeded 10% on 2 of the farms and the occurrence of this disease was not affected by the vaccine. The incidence of other neoplasms is recorded. Vaccinated birds produced 4.0 and 14.7% more eggs than unvaccinated birds on a hen-day and hen-housed basis, respectively. In addition, vaccinated birds reached 50% egg production 2 to 8 days earlier than unvaccinated controls.

Lymphoid Leukosis: Interrelations among Virus Infections in Hens, Eggs, Embryos, and Chicks

Hens from a commercial source were selected because they were infected with lymphoid leukosis virus (LLV). LLV was detected in vaginal swabs from 17 viremic hens and from 27 of 44 hens that were not viremic. All hens that were positive on the vaginal swab test (VST) produced one or more eggs with virus in albumen or in embryos, whereas in comparable tests, virus was detected only in eggs from 5 of 17 hens that were negative on VST. Congenital transmission of LLV was erratic and neither the VST nor tests for virus in egg albumen prior to incubating eggs identified all hens that transmitted infection. For example, 14 hens negative on VST produced 50 eggs negative for virus in albumen and yet one of the embryos from these eggs was infected. Eggs from other hens had infectious virus in albumen and about half of the embryos from these were infected. Tests for virus in cloacal swabs from one-day-old chicks were as sensitive as tests on embryos for detecting congenital transmission. Titers of LLV in the meconium of congenitally infected chicks were as high as 10(7) infectious units per ml. The cloacal swab test should be a valuable adjunct to the VST and tests on egg albumen in programs designed to eradicate lymphoid leukosis from chickens.

Pathogenicity of Avian Leukosis Viruses

Three methods were used in attempts to obtain non-oncogenic avian leukosis virus for possible use as an immunoprophylactic agent for the control of lymphoid leukosis in chickens. These were: 1) isolate a nononcogenic virus from commercial breeder flocks experiencing very little or no lymphoid leukosis; 2) obtain a non-oncogenic recombinant from mixed infection of a strain with low oncogenicity, Rous-associated virus-60 (RAV-60), with RAV-1 or RAV-2 in cell culture; and 3) attempt to attenuate subgroup A avian leukosis virus by serial passage in avian cell culture. Of 43 isolates obtained from field sources, all were pathogenic except one, and its pathogenicity was questionable because of the low amount of virus tested. All 42 clones from mixed infection of highly oncogenic and poorly oncogenic virus and all clones passaged serially in cell culture were oncogenic.

The temporal relationship between vaccination with the herpesvirus of turkeys and challenge with virulent Marek's disease virus.

Experiments were designed to determine when to vaccinate chicks with the herpesvirus of turkeys (HVT) to obtain maximum protection against Mareks disease (MD). Vaccination one or more weeks before challenge with MD virus gave protection, vaccination after challenge gave no protection, and simultaneous vaccination and challenge gave partial protection. In commercial situations, where the time of infection with MD virus is unknown, birds should be vaccinated as early as possible, preferably on the day they hatch.

An Evaluation of Methods for Eradication of Avian Leukosis Virus from a Commercial Breeder Flock

Two trials were conducted to determine whether lymphoid leukosis virus (LLV) could be eradicated from chicken breeder stocks in one generation. Dams were selected as potentially virus-free parents on the basis of negative tests for virus in progeny embryos (trial 1) or in vaginal-cloacal swabs (VCS) (trial 2) of the dams. In trial 1, 8 of 12 groups of chickens hatched from selected breeders remained free of LLV infection through 36 weeks of life. In trial 2, VCS appeared to be more efficient in detecting possible shedder dams, and only 1 of 72 groups of chickens showed evidence of infection at 14 weeks after hatching. Within that positive group, a single chicken was shown to be the possible cause of the infection. The results show that eradication of LLV can be accomplished in one generation by: 1) virological examination of dams for shedding; 2) elimination of the shedder dams; and 3) small-group rearing of the progeny chicks.

Phenotypic mixing between reticuloendotheliosis virus and avian sarcoma viruses

Abstract Coinfection of chicken embryo fibroblasts with reticuloendotheliosis virus (REV) and avian sarcoma virus leads to the formation of avian sarcoma viral pseudotypes which carry envelope determinants of REV. These pseudotypes can be neutralized by REV antiserum, have a host range which is different from that of any known avian sarcoma virus, and are unable to form foci in cells preinfected with REV. The REV stocks used in these experiments were plaque-purified. They were free of avian leukosis virus detectable in the COFAL tests, and their ability to form pseudotypes with avian sarcoma virus was neutralized with specific REV antiserum.

The NP activation test for assay of avian leukosis/sarcoma viruses.

SUMMARY Procedures are described for routine isolation and production of nonproducing Rous sarcoma cells (NP cells) and for use of these cells in the NP activation test for assay of avian leukosis/sarcoma viruses. The NP activation test was found to be sensitive, reproducible, easy to perform, and highly reliable. It required only 5-7 days of cultivation for the activation phase, and 8 days for the RSV assay phase. The sensitivity of the NP activation test for detection of avian leukosis viruses appeared comparable to the COFAL and RIF tests. Because of its relative simplicity and shorter culture periods, a far greater number of samples can be tested in the same period.