Immunohistochemical detection of scrapie prion proteins in clinically normal sheep in Pennsylvania.

Following diagnosis of scrapie in a clinically suspect Suffolk sheep, 7 clinically normal flockmates were purchased by the Pennsylvania Department of Agriculture to determine their scrapie status using an immunohistochemical procedure. Two of the 7 euthanized healthy sheep had positive immunohistochemical staining of the prion protein of scrapie (PrP-Sc) in their brains, nictitating membranes, and tonsils. The PrP-Sc was localized in the areas of the brain where, histopathologically, there was neurodegeneration and astrocytosis. The PrP-Sc occurred within germinal centers of the affected nictitating membranes and tonsils and was located in the cytoplasm of the dendrite-like cells, lymphoid cells, and macrophages. These results confirm that immunohistochemical examination of the nictitating membrane can be used as a screen for the presence of scrapie infection in clinically normal sheep at a capable veterinary diagnostic laboratory. In sheep with a PrP-Sc-positive nictitating membrane, the diagnosis of scrapie should be confirmed by histopathology and immunohistochemical examination of the brain following necropsy. Following full validation, immunohistochemistry assays for detection of PrP-Sc in nictitating membrane lymphoid tissues can improve the effectiveness of the scrapie control and eradication program by allowing diagnosis of the disease in sheep before the appearance of clinical signs.

Immunohistochemical study of constitutive neuronal and inducible nitric oxide synthase in the central nervous system of goat with natural listeriosis.

The expression of both constitutive and inducible forms of nitric oxide synthase (NOS) was investigated by immunohistochemical staining of formalin-fixed paraffin-embedded sections in normal and Listeria monocytogenes-infected brains of goats. In normal control goats, a small number of neurons showed immunoreactivity of both iNOS and nNOS, and the number of iNOS-positive neurons was higher than the number of nNOS-positive neurons. In natural listeriosis, listeria antigens were easily immunostained in the inflammatory cells of microabscesses. In this lesion, the immunoreactivity of iNOS in neurons was more intense than the control, but nNOS was not. In microabscesses, nNOS was weakly visualized in macrophages and neutrophils, while iNOS was expressed in macrophages, but not in neutrophils. These findings suggest that normal caprine brain cells, including neurons, constitutively express iNOS and nNOS, and the expressions of these molecules is increased in Listeria monocytogenes infections. Furthermore, inflammatory cells, including macrophages, expressing both nNOS and iNOS may play important roles in the pathogenesis of bacterial meningoencephalitis in goat.

Practical application of nucleic acid techniques to avian disease problems.

A workshop in which 17 practicing scientists participated was intended to address primarily people who use or could use biotechnology in their work and was confined to five techniques. Endonuclease fingerprinting and mapping involved cleaving nucleic acid with a specific restriction enzyme and separating the nucleic acid fragments by electrophoresis. Field and vaccine isolates of Pasteurella multocida could be distinguished; Salmonella enteritidis could be divided into three groups; chlamydia could be grouped into seven groups; and vaccinia, quail pox, and fowl pox could be clearly distinguished. Preparation of nucleic acid probes involved producing large amounts of labeled oligonucleotides, usually of unknown sequence. Successful probes had been made for infectious bursal disease virus, avian influenza virus, Newcastle disease virus, and infectious bronchitis virus. In Southern, Northern, and dot blotting, either DNA or RNA fragments were placed on or transferred to a solid substrate and probed. The procedure was able to detect infectious bursal disease virus, infectious bronchitis virus, Mycoplasma gallisepticum, and Marek's disease virus. In situ hybridization involved applying a labeled probe to frozen or fixed sections or to intact cells. In Polymerase chain reaction, two primers, some distance apart, were annealed to a denatured target DNA. Repeated cycles of DNA synthesis with a thermostable polymerase, denaturing, and reannealing resulted in great amplification of a rare sequence. After 30 cycles, a rare gene sequence could be amplified more than 10(6) times. It was used successfully to detect minute quantities of influenza virus and infectious bursal disease virus, and the process was used to facilitate DNA sequencing of coccidiosis gene segments.

Future applications of biotechnology in poultry.

The major biotechnological advances that can be applied in the poultry industry will include molecular genetics, molecular immunology, and solid-state reactions. The elucidation of the genetic code and the development of techniques to manipulate genes offer new opportunities for changing pathogenic agents and changing chickens to reduce the effect of disease and improve productivity. The monoclonal antibody technique and the discovery that cells of the immune response communicate with one another through peptide factors will permit improved diagnostic techniques and enhanced immune responses to vaccines. Immunologic and biochemical reactions that occur on a solid substrate can be used to simplify and accelerate diagnostic tests and to purify antigens and antibodies. These advances will lead to improvements in diagnosis, disease resistance, and productivity of poultry.

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.

Absence of B stem cells in spleens of chickens fed the androgen analog mibolerone.

Bursa-derived stem cells in the spleens of chickens treated with the androgen analog mibolerone or with cyclophosphamide and in the spleens of untreated controls were examined by a transplantation technique. Spleen cells from syngeneic donors 2-20 weeks old were transferred to syngeneic cyclophosphamide-treated recipients, and the immunocompetence of the recipients was tested 5 weeks after the transfer by antigenic stimuli with sheep erythrocytes and Brucella abortus. Spleens of mibolerone-treated chickens failed to reconstitute the immunologic capability of cyclophosphamide-treated recipients in a manner similar to spleen cells from normal donors. Spleens of cyclophosphamide-treated donors did not contain stem cells. Stem cells were detected in the spleens of untreated controls as young as 2 weeks old; activity was highest after the 13th week of age.

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.

Immune responses of chickens fed the androgen analog mibolerone.

Mibolerone, an androgen analog (17 beta-hydroxy-7-alpha, 17-dimethylestr-4-en-3-one), induces a slow but progressive involution of the bursa of Fabricius when fed to chickens at microng levels during the first 7 weeks of life. Chickens receiving mibolerone remained immunologically competent, evidenced by: 1) their antibody response to nonreplicating antigens and infectious antigens; 2) the number of antibody-producing cells in their spleens; 3) the stimulation of their peripheral leukocytes with the plant mitogen phytohemagglutinin; and 4) their capacity to resist challenge with Marek's disease virus and Newcastle disease virus after vaccinations with turkey herpes-virus and the B-1 LaSota strain. This, coupled with the fact that it prevents experimental lymphoid leukosis, makes mibolerone a potential agent to be used under field conditions for the control of lymphoid leukosis.