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.

Tumor latency in avian lymphoid leukosis.

Transfer of bursa cells from older chicken infected with lymphoid leukosis (LL) virus into young recipient chicks depleted of bursal lymphocytes by cyclophosphamide shortened the latency period for lymphoma development in recipient chickens by an amount equivalent to the age of the donor. Chickens were crosses between males of the Regional Poultry Research Laboratory, East Lansing, Michigan, inbred line 151, subline 5, and females of inbred line 7, subline 2. In a further experiment with intact infected chicks, artificial metastasis at various ages by surgical bursectomy and inoculation of bursa cells into the bloodstream of the host did not shorten the latency period. These results suggest that latency in avian LL is a property of target B-cells and is unrelated to maturational events of the host physiology.

Viral protein sythesis by tissues from avian leukosis virus-infected chickens. II. Effect of passive neutralizing antibody in normal and agammaglobulinaemic chickens.

Synthesis in vitro of avian leukosis virus (ALV) group (gs proteins, p27 and p12, by various tissues from chickens infected within a few days after hatching was studied by means of autoradiography of immunoelectrophoretic patterns. Viral protein was synthesized in all tissues of chicks examined between days 18 and 50 of age after which time liver, kidney, bursa, thymus, and spleen became negative. The lung and genital organs of the chicks, however, continued to synthesize viral protein up to 100 days of age, when the experiment was ended. Repeated injections of neutralizing chicken antibody to ALV (Ab) starting on day 26 or 37 caused gs protein to decrease in spleen, liver, and thymus within 5 days but not in bursa, lung, and genital organs. Agammaglobulinaemic (Agamma) chickens showed prolonged persistence of gs protein synthesis in the spleen and liver; this synthesis was abrogated by passive Ab. Liver from Agamma chickens, however, also became negative without Ab treatment. The relative roles of antibody and cellular immunity in influencing ALV replication during the initial phase of infection before lymphoma development are discussed.

Are we ready for a National Salmonella Control Program?

Control of contamination of meat and poultry with Salmonella is difficult because of the complexity of the paths of transmission of the organism, the large number of sources of the organism, and the large number of persons, groups, and agencies involved in the production of animal feed and food and in the regulation of these industries. Furthermore, there is a gap between the basic technology available to destroy or prevent contamination with Salmonella and the technology available that is acceptable, inexpensive, and applicable on a large scale. Support of research for the development of new technology amounts only to about $2.7 million. There are programs for the control of Salmonella in certain feeds and in the production, processing, further processing, and cooking of food. With current technology, an eradication program would likely cost far more to the consumer than could be justified by the benefits the consumer would derive from such eradication. Nevertheless, practical control of salmonella contamination can be achieved through progressive application of new technology developed through research.

Prevention of avian lymphoid leukosis by induction of bursal atrophy with infectious bursal disease viruses.

Five groups of genetically susceptible chickens were inoculated at hatching with lymphoid leukosis virus; four of these were given infectious bursal viruses of varying virulence at 14 days of age and one group was not inoculated (control). All chickens in the control group developed evidence of lymphoid leukosis by 180 days. Two groups given relatively virulent bursal disease viruses, which destroyed bursal lymphoid cells, did not develop lymphoid leukosis. Treatment with avirulent vaccines had no visible effect on bursal morphology and did not significantly alter the incidence of lymphoid leukosis in two other groups, although the time of development was delayed. Results of our study show that viral-induced destruction of the bursa of Fabricius eliminates the development of lymphoid leukosis but that infection without bursal destruction has little effect on lymphoid leukosis.

The prevention of natural and experimental avian lymphoid leukosis with the androgen analogue mibolerone.

The development of lymphoid leukosis tumours induced by RAV-1, RAV-2, and field lymphoid leukosis viruses was prevented in chickens by feeding a diet containing the androgen analogue mibolerone (17beta-hydroxy-7a, 17-dimethylestr-4-en-3-one) at low levels during the first 7 weeks of life. Chickens fed mibolerone developed neutralising antibodies after inoculation with RAV-1, RAV-2, and after contact exposure to viraemic-tolerant chickens. Mibolerone did not affect the immunological tolerance status of chickens which were viraemic when hatched. Moreover, mibolerone did not seem to change the pattern of shedding of lymphoid leukosis viruses or group specific antigen in unincubated chicken eggs from infected hens. Mibolerone, thus, prevents the development of lymphoid leukosis tumours without interfering with the cycle of infection of lymphoid leukosis viruses.

Vaccination immunity to selected diseases in chickens fed the androgen analog mibolerone.

Chickens fed the androgen analog mibolerone during the first 7 weeks of life regress their bursa of Fabricius but can be properly immunized by vaccination against avian pathogens of major economic importance such as Newcastle disease virus, infectious laryngotracheitis virus, avian encephalomyelitis virus, infectious bronchitis virus, fowl pox virus, Marek's disease virus, and Pasteurella multocida, the pathogen causing fowl cholera. These findings on immunocompetence to infectious agents are important because we have previously shown that the administration of mibolerone prevents the development of lymphoid leukosis tumors.

Oncogenicity of avian leukosis viruses of different subgroups and of mutants of sarcoma viruses.

Leukosis viruses of seven subgroups were tested for oncogenicity in chickens susceptible to virus infection and to development of lymphoid leukosis (LL) tumors. All subgroup A viruses and the subgroup B virus tested produced a high incidence of LL and other related neoplasms. Viruses of subgroup C and RAV-61 of subgroup F produced a low level of LL. The RAV-50 of subgroup D produced osteopetrosis. In these tests, the viruses of subgroup E and G and one virus of subgroup F were not pathogenic, possibly because infection was not established in the chickens, the chickens were not susceptible to tumor development by these viruses, or the viruses lacked oncogenicity. All temperature-sensitive mutants of Rous sarcoma virus produced sarcomas, but the level varied. One nontransforming mutant produced sarcomas, and the other three tested produced LL. All three mutants that cause cells to grow as colonies in agar produced a high incidence of sarcomas. Thus, sarcoma viruses, by back-mutation, may lose the ability to transform cells in vitro, to make cells grow in agar colonies, or to induce sarcomas in vivo, yet they retain the ability to produce LL. Conversely, it was previously shown that leukosis viruses may be changed into viruses that transform cells in vitro and produce sarcomas in vivo by suitable passage in chicks.

Prevention of Marek's disease: a review.

Marek's disease (MD) is a highly infectious neoplastic condition of chickens caused by a herpesvirus. The virus is cell associated in tumors and in all organs except in the feather follicle where enveloped infectious virions egress from the body. From this source, infection is spread horizontally by the airborne route to the environment and to other chickens. Vertical transmission from dam to offspring does not occur or at best is very rare. The nonpathogenic herpesvirus of turkeys (HTV) is ubiquitous in turkeys and is probably spread horizontally by the airborne route. When chickens are inoculated with this virus, they do not subsequently develop MD even after infection with virulent Marek's disease virus. The Marek's disease virus, not the HVT, will spread horizontally from dually infected birds. The HVT vaccine is safe and highly effective in preventing MD under field conditions, and most chickens throughout the world are vaccinated with this vaccine. Other vaccines that have been used but have disadvantages over HVT include the following: (a) the highly pathogenic HPRS 16 strain of Marek's disease virus was attenuated by passage in cell culture. The attenuated virus protects against MD and does not spread, but "over-attenuated" virus does not protect; (b) naturally apathogenic strains virologically, immunologically, and epizootiologically similar to pathogenic strains will protect when adminstered before infection with the virulent strains; (c) virus preparations that have been chemically treated to inactivate infectivity protect only slightly. When a candidate vaccine virus for the prevention of herpesvirus-induced cancer in humans is developed, the purity of the vaccine preparations will be easily determined by modern techniques. However, measurements of safety and effectiveness are a significant problem. If, analogous to the MD model, the vaccine will have to be administered shortly after birth and the incubation period to development of neoplasms is long, then pathogenicity tests in nonhuman primates and other animals may be of limited valued. However, biochemical demonstration that the segment of the nucleic acid responsible for oncogenesis is absent from the vaccine virus may be the major indication that the vaccine is nonocogenic and therefore safe. Because of the low incidence of neoplasia and long incubation period, the effectiveness of the vaccine will be difficult to test. The vaccine possibly will protect against an acute manifestation of viral infection. Future research on MD will be directed to determining the mechanism of protection against disease, i.e., whether immunity is mediated by thymus- or bursa-dependent systems, and to identifying the protective antigen, i.e., which cell surface or an interior antigen induces the protective immunity. The prevention of MD by vaccination may become a very fruitful area for model studies on prevention of human cancer by vaccination.

Lymphoid leukosis in chickens chemically bursectomized and subsequently inoculated with bursa cells.

Lymphoid leukosis (LL), a neoplasm of the bursa-dependent lymphoid cells of the chicken, was induced by Rous-associated virus-1 in susceptible chickens. Cyclophosphamide (CY), which destroyed the lymphoid elements of the bursa of Fabricius and abrogated humoral immunity, prevented LL. Concomitantly, osteopetorosis and other neoplasms increased. Transfer of bursa cells from chickens into CY-treated hatchmates restored immune competence. Birds whose B-cell functions were reconstituted died of LL and were less likely to die of osteopetrosis and other neoplasms than were CY-treated chicks. These results suggested that the bursa cell transferred into the CY-treated chicks were the target cells for lymphoid leukosis transformation.

Low oncogenic potential of avian endogenous RNA tumor virus infection or expression.

Of chickens either spontaneously producing or exogenously infected in ovo with Rous-associated virus, type O (RAV-O), an endogenous virus of the chicken, only 1 died with lymphoid leukosis (LL), the most common neoplasm associated with the leukosis-sarcoma virus group. Because the chickens were not kept in strict isolation, it could not be assumed that the one LL was induced by RAV-O. In contrast, RAV-1-infected chickens from the same lines had a high incidence of LL and other neoplasms. Over 800 chickens of several inbred lines were maintained in plastic isolators free of exogenous avian leukosis-sarcoma virus infection for from 500 to nearly 1,000 days of age. No LL was observed, even though some lines are known to produce RAV-O spontaneously or to express inherited gs antigen. Three neoplasms of unknown etiology were observed, but none generally associated with leukosis virus infection. We concluded that avian endogenous virus expression had little, if any, oncogenic potential, and that exogenous avian leukosis viruses were responsible for most naturally occurring neoplasms.

Oncogenesis by Marek's disease herpesvirus in chickens lacking expression of endogenous (gs, chick helper factor, Rous-associated virus-O) and exogenous avian RNA tumor viruses.

Chickens free of exogenous avian leukosis virus (ALV) infection, replicating endogenous ALV (Rous-associated virus-O), gs antigen, and chick helper factor were fully susceptible to induction of Marek's disease (MD) by ALV-free MD viruses. Dual infection with Rous-associated virus-2 and MD virus did not significantly alter the character of the MD lesions. Thus exogenous ALV infection was not requisite for MD virus-induced oncogenesis. Although participation of endogenous RNA tumor virus genes in MD lesion induction could not be excluded, expression of such genes in MD tumors as gs antigen was not established.

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.

Chicken leukosis virus genome sequences in DNA from normal chick cells and virus-induced bursal lymphomas.

Genome sequences of two recent field isolates of avian leukosis viruses in the DNA of normal and neoplastic chicken cells were studied by DNA-RNA hybridization under conditions of DNA excess. Comparisons were made between 60-70S RNA from these viruses and that of a chicken endogenous type C virus (RAV-0), and of a series of "laboratory" leukosis and sarcoma viruses, by competitive hybridization analysis. A minimum of 18% of the genome sequences of both ALV isolates detected in DNA from lymphomas they induced were not detected in normal chicken DNA. The vast majority of the fraction of RNA sequences from ALV which do form hybrids with normal chick DNA appear to be reacting with the endogenous provirus of RAV-0. The genomic representation of a variety of avian leukosis and sarcoma viruses in normal chicken cells could not be distinguished by these methods (except that 13% of the RAV-0 genome was not shared with any of the other viruses). In contrast, the portion of the ALV genome exogenous to the normal chicken geome showed significant divergence from that of two sarcoma viruses (Pr RSV-C and B-77). The increased hybridization of ALV RNA with lymphoma DNA was used to detect the appearance of ALV specific sequences in the bursa of Fabricius following infection.increased hybridization was correlated with both the time after infection and the extent of replacement of the bursa by lymphoma. About one half of the increase in hybridization preceded histologic evidence of transformation.