The application of hybridoma technology to the study of bovine immunoglobulins.

Studies are described in which hybridoma technology is used to produce a variety of reagents for the characterization and manipulation of the bovine humoral immune system. Selected members of a set of murine monoclonal antibodies (MAb) specific for each of four major isotypes of bovine Ig constant regions, one specific for anti-bovine Ig constant regions as well as one specific for anti-bovine light chains are discussed. Interspecific fusion of bovine lymphocytes with the established mouse cell line, SP2/0 was used to produce a collection of stable hybridomas among which were found secretors of bovine IgG1, IgG2, IgM, IgA and bovine light chain. Interspecific fusion of SP2/0 with lymphocytes from a multiparous Holstein four days post immunization with Streptococcus agalactiae yielded MAb with specificity for the immunizing antigen. One of these hybridomas, LHRB 19.17, which displayed a particularly stable secretory phenotype, was used as an immunogen for the production of a library of murine monoclonal anti-idiotype antibodies. Competitive antigen binding analysis showed that 15 of the 24 anti-LHRB 19.17 idiotype antibodies isolated blocked the binding of the idiotype to its nominal antigen and so were candidates for evaluation as antigen mimics. Some of the ways in which monoclonal anti-idiotypes in particular, and monoclonal in general, might be of use in problems of animal disease are discussed.

Instability studies of porcine somatotropin in aqueous solutions and the possible reagents for its stabilization.

The instability of porcine somatotropin (pST) in various solutions and possible stabilization of the hormone by sugars and mild detergents were studied. Aggregation and decomposition of the hormone molecules in various pH solutions and under presence of sugar or detergent were monitored by gel permeation chromatography (GPC) or ultraviolet spectroscopy (UV). The pST is a very unstable hormone in an aqueous environment. It was found in this project that the peptide hormone underwent aggregation or decomposition quickly in acidic and alkaline solutions but slowly in neutral pH solutions. High losses of pST monomers were seen in concentrated solutions of the hormone. On the other hand, pST monomers were stabilized to a certain degree in glucose solutions and at a low concentration of urea. These results should facilitate the development of efficient controlled-release systems which are essential for commercializing porcine somatotropin.

Studies of histocompatibility and immune response of chickens selected for resistance and susceptibility to Marek's disease.

Two sets (four lines) of chickens, lines 7 and 6 and lines P and N, the former of each set susceptible and the latter resistant to Marek's disease, were examined for their relative histocompatibility and immunocompetence. Results from the in vivo graft-versus-host response splenomegaly assay, and graft-versus-host chorioallantoic membrane pock formation assay confirmed the within-line, B-locus homozygosity of chickens of lines 7, 6, and N and the heterozygosity of line-P chickens. These assays further confirmed that line-7 and line-6 chickens share identical alleles at the major histocompatibility locus. The capacity of the lines of chickens to elicit specific cell-mediated immune lysis as measured by the release of chromium 51 generally agreed with the in vivo graft-versus-host responses. These data demonstrate that the 51Cr-release assay is a reliable measure of histocompatibility within the avian system.

Cellular immune response to Marek's disease: listeriosis as a model of study.

The immune response of chickens to Listeria monocytogenes was studied as a potential model for cell-mediated immunocompetence. Chickens genetically resistant and susceptible to Marek's disease (MD) did not differ in their ability to survive Listeria, although during the early stages of infection the bacteria replicated more readily in MD-susceptible chickens. MD-susceptible chickens responded earlier than MD-resistant chickens, and with equal or increased intensity, in assays of various components of the cell-mediated reaction. These assays included T-cell activation, delayed-type hypersensitivity, and macrophage activation. These data indicate that genetic resistance or susceptibility to MD is not wholly dependent on the innate immunocompetence of the host. Co-infection with Listeria was used to measure cellular immunocompetence in MD-infected chickens. MD virus had no effect on the ability of host macrophages to control the growth of Listeria. The cell-mediated response was suppressed in MD-susceptible chickens. The occurrence of spleen cell proliferation, followed by marked suppression of the effector arm of the immune response in susceptible but not resistant chickens, indicated the possibility of an active suppressor-cell population associated with genetic susceptibility to MD.

Pathologic changes in pigeons infected with a virulent Trichomonas gallinae strain (Eiberg).

Trichomonas gallinae, Eiberg strain, is a virulent hepatotropic flagellate parasite of pigeons. The parasite initially infects the upper digestive tract, causing the formation of ulcers, which allow it to enter the circulatory system. The trichomonads later gain access to the liver, where they cause the formation of caseous lesions. Vascular congestion and perivascular cuffing in the liver were seen as early as 4 days postinfection (PI). By day 7 PI, a marked reduction in abdominal fat and hepatosplenomegaly was evident. Hepatocytes underwent fatty degeneration (seen by day 7 PI) before total necrosis set in. On day 8 PI, trichomonads could be found among the necrotic hepatocytes in caseous lesions. These lesions were delineated by a wall of leukocytes and occasional giant cells. Nonimmune pigeons died within 14 to 17 days PI of liver dysfunction. Other organs (kidney and genitalia) were also seen to undergo degeneration. These manifestations probably reflect the progression of liver dysfunction.

Detection of equine infectious anemia virus in horse leukocyte cultures derived from horses in various stages of equine infectious anemia viral infection.

The enzyme-linked immunosorbent assay (ELISA) antigen-positive and agar-gel immunodiffusion test (AGID)-negative horses do not have infective equine infectious anemia (EIA) virus. The ELISA testing of horse leukocyte culture (HLC) supernatants did detect EIA virus in a HLC that was infected with the Wyoming strain of EIA virus and in HLC derived from horses in febrile, acute, or subacute stages of EIA infection. In supernatants of HLC derived from chronic and inapparent carrier horses, EIA virus was not detected with ELISA. Direct fluorescent antibody tests detected EIA virus in HLC infected with 10(6)TCID50 of the Wyoming strain of EIA virus and in 50% of the HLC from febrile acute or subacute horses. The direct fluorescent antibody testing of HLC derived from chronic and inapparent carrier horses did not detect cell-associated EIA virus. The pony inoculation test proved to be the most reliable and accurate method for detecting infective EIA virus in horses in various stages of EIA infection and accurately correlated with the AGID test.

The effects of sonication, freezing and lyophilization on JMV leukosis strain.

JMV lymphoblastic leukemic cells were subjected to sonication, followed by freezing and lyophilization in an attempt to learn the tolerance of JMV cells to these treatments. Sonication experiments indicated that a high percentage of cell breakage (greater than 89%) is necessary for any decrease in lethality to be observed. Freezing experiments involving a wide range of cryoprotectors demonstrated 2M glycerol to be the best for JMV preservation. Subsequent freeze-drying of sonicated, frozen JMV preparations, of high titer, consistently resulted in all loss of lethality.

In vitro studies on the role of the macrophages of resistant and susceptible chickens with Marek's disease.

Macrophages, from susceptible S- and resistant K-strain White Leghorn chickens, were studied in vitro to determine their contribution to the dynamics of Marek's disease (MD) and possible role in genetic resistance to this disease. In relation to basic macrophage properties (morphology, cell spreading, enzyme levels, pinocytosis, and phagocytosis) there were no significant differences between S- and K-strain macrophages. Both macrophage strains were observed to respond to JM virus by phagocytic responses, but no viral replication or transference of infectivity was observed within inoculated macrophage cultures. In general, there were no differences between S- and K-strain macrophages in vitro that may account for the genetic resistance or susceptibility to MD.

Antibody response of genetically susceptible and resistant chickens to cell-free and attenuated JM-V leukosis strain and its influence on early type II (Marek's) leukosis infection.

Experiments were carried out to: 1) determine the antibody response of chickens to cell-free and attenuated preparations of JMV leukosis strain, 2) determine the differences in antibody response to these antigens between susceptible (P-line) and resistant (N-line) chickens by means of serum neutralization and indirect fluorescent antibody tests, 3) investigate the influence of maternal (passive) antibody on early (day-old) JMV vaccination and 4) investigate the influence of maternal antibody in chicks naturally exposed continuously to JM virus from day-old to 8 weeks of age on the pathogenesis of Type II leudosis (Marek's) infections and oncogenesis.