Influence of Marek's disease virus strain AC-1 on cellular immunity in birds carrying endogenous viral genes.

The effects of Marek's disease virus (MDV) strain AC-1 on humoral and cellular immune responses were investigated in two lines of chickens segregating for the endogenous viral (ev) genes ev-6 and ev-12. All birds were vaccinated at 14 days of age against Newcastle disease virus (NDV). At 3 wk of age, 48 birds per line received an intraperitoneal injection of MDV (AC-1 isolate), and 24 were injected with saline. Birds of each group were killed on days 5, 7, 12, and 14 postinfection. Data were first analyzed for each day of testing. Results showed that, for variable measured, treatment effects were similar on days 5 and 7, and on days 12 and 14. Therefore, day 5 and day 7 data, and day 12 and day 14 data were pooled and analyzed. In MDV-infected chickens, proliferative lymphocyte responses to mitogens were suppressed (P < 0.001) after the first and second week of infection, whereas responses to NDV antigen were enhanced (P < 0.001) after the first week and then reduced (P < 0.01) by the end of the second week when compared to uninfected birds. The percentage of CD4+ T cells was higher (P < 0.01), and the percentage of cells expressing major histocompatibility complex (MHC) class II antigens was lower (P < 0.001), in MDV-infected chickens than in uninfected birds. The cytotoxic activity of natural killer (NK) cells was also enhanced (P < 0.001) in MDV-infected birds when compared to uninfected birds. Antibody responses to NDV were not different among groups, and the presence of ev-6 or ev-12 genes did not influence the immune response parameters measured in both infected and uninfected chickens. In conclusion, a marked increase in the CD4+ T lymphocyte population occurred in the early stage of MDV infection in all chickens regardless of the presence of ev genes, whereas the number of cells expressing MHC class II antigen was severely reduced.

Cell-mediated and humoral immune responses in chickens infected with Salmonella typhimurium.

The cellular immune responses of leghorn and New Hampshire chickens following oral challenge at 4 weeks of age with Salmonella typhimurium were measured using assays for lymphocyte proliferative responses to mitogens and cytotoxic activity of natural killer (NK) cells. In addition, the humoral immune response to Newcastle disease virus (NDV) vaccine was assessed in S. typhimurium-infected chickens. At 8 and 20 days after infection with S. typhimurium, lymphocyte proliferative responses to various mitogens were significantly higher in S. typhimurium-infected chickens than in uninfected birds of the same breeds. Cytotoxic activity of NK cells was also significantly increased in infected birds on these days. Thirteen days after S. typhimurium infection, infected NDV-vaccinated chickens had higher antibody titers in response to NDV vaccination than uninfected vaccinated birds. NDV titers did not differ among groups at any other time of testing. These results show that both cellular and humoral immune functions are activated in the first 3 weeks following infection of 4-week-old chickens with S. typhimurium.

Evaluation of lymphocyte stimulation tests for diagnosis of bovine tuberculosis in elk (Cervus elaphus).

Lymphocyte stimulation tests (LST), performed using 6 antigen preparations, were compared individually and in pairs. The tests were performed on 433 blood samples collected from elk in Mycobacterium bovis-infected herds. These elk were killed as part of Agriculture and Agri-Food Canada's bovine tuberculosis eradication policy, and mycobacterial culture results were obtained from tissues of each animal. The LST, which had the highest total sum of sensitivity and specificity, was a comparative test that used M bovis purified protein derivative (PPD) and M paratuberculosis (johnin) PPD. This test had a sensitivity of 76%, with confidence limits (CL) of 63 to 85% for this estimate, and specificity of 77% (CL, 72 to 81%). The LST, using only M bovis PPD antigen, had a sensitivity of 70% (CL, 57 to 80%) and specificity of 74% (CL, 69 to 79%); when it was compared with culture results, using the kappa statistic, agreement was only 32%. This indicated that the LST identified different elk than did M bovis isolation tests.

Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle.

The immune response to bovine herpesvirus 1 (BHV-1) infection can protect cattle from subsequent challenge with the virus. This protection involves a variety of defensive strategies, and the activation of most of these defenses requires the recognition of viral proteins by the cellular immune system. To identify some of the BHV-1 proteins recognized by T lymphocytes, we measured in vitro proliferative responses to individual proteins. Viral proteins were separated by gel electrophoresis followed by Western immunoblotting, and immunoblots were evaluated for serological reactions. Unstained blotted fractions were processed into antigen-bearing particles for analysis in blastogenesis assays. Purified BHV-1 proteins obtained by immunoadsorbent chromatography were processed and included for comparison in both enzyme-linked immunosorbent and proliferation assays. The tegument protein VP8 and the glycoprotein gIV appeared to be the antigens which most consistently stimulated the proliferation of lymphocytes from BHV-1-immunized animals. Positive blastogenic responses were also detected to gI, gIII, and to one or more uncharacterized, low-molecular-weight proteins in some of the cattle tested. These results indicate that T-lymphocyte proliferative responses to BHV-1 proteins are detectable in immune cattle and may be important in protection from BHV-1 infection.

Inhibition of antigen-induced and interleukin-2-induced proliferation of bovine peripheral blood leukocytes by inactivated bovine herpes virus 1.

The mechanism by which bovine herpesvirus 1 (BHV-1) predisposes cattle to bacterial pneumonia was investigated by using an in vitro system to demonstrate immunosuppression. At a multiplicity of infection of 0.001, live or inactivated BHV-1 induced a 50% inhibition of the proliferative response of peripheral blood mononuclear leukocytes to antigen (vaccinia virus in vaccinia virus-immunized cattle which were BHV-1 negative) or interleukin-2. At this same multiplicity of infection, the mitogen-induced proliferation of peripheral blood mononuclear leukocytes was unaffected. This inhibition of antigen and interleukin-2-induced proliferative responses could not be reversed by the addition of excess amounts of interleukin-2 and could not be prevented by the addition of indomethacin to block prostaglandin production. Antibodies to BHV-1, especially those specific for glycoproteins gI and gIV, were able to block the inhibitory effect of BHV-1 in these in vitro assays. These results showed that antibody to BHV-1 blocks the immunosuppressive effect of the virus in vitro and suggested that an appropriate antibody response to BHV-1 could protect cattle from virus-induced immunosuppression leading to secondary bacterial pneumonia.

Generation of IL-2 dependent bovine cytotoxic T lymphocyte clones reactive against BHV-1 infected target cells: loss of genetic restriction and virus specificity.

To gain insight into the mechanisms of immunity to bovine herpesvirus type-1 (BHV-1) in particular the importance of the T-cell response, we attempted to clone bovine cytotoxic T lymphocytes that were specific for BHV-1 infected autologous target cells. A number of bovine T cell clones were generated by limiting dilution in the presence of bovine recombinant IL-2 and BHV-1 infected target cells as feeder layers. These clones were maintained in culture on crude IL-2 containing supernatants. In functional studies, 4 of the 16 T cell clones were shown to have high levels of cytotoxic activity specific for autolgous BHV-1 infected target cells with significantly lower cytotoxic activity against uninfected target cells and heterologous BHV-1 infected target cells. Continuous culturing of these 4 T cell clones, using either the crude IL-2 or high concentrations of recombinant bovine IL-2, resulted in the loss of both MHC restricted and BHV-1 specific cytotoxic activity. These clones now exhibit promiscuous type cytotoxic activity with the ability to lyse a variety of target cells. Using flow cytometric analysis, the phenotype of the T cell clones were shown to have bovine T lymphocyte characteristics including expression of the BoT8 marker. This is the first report of cloned bovine cytotoxic T lymphocytes reactive against BHV-1 and the generation from these clones of promiscuous cytotoxic activity against both virus-infected and non-infected bovine target cells.

A mail survey of factors associated with morbidity and mortality in feedlot calves in southwestern Ontario.

The design and results of a mail survey of a simple random sample of southwestern Ontario feedlot owners are presented. The survey provided general data about management of feedlot calves and the association between a number of factors and disease and/or death rates. The number of calves purchased was related positively, in a linear manner, to mortality and morbidity rates. Increased levels of morbidity and mortality were noted when the ration was changed to corn silage from dry-hay within the first month after arrival. However, it was not clear whether the ration changes preceded or followed increased rates of morbidity and mortality. Prophylactic levels of antimicrobials in the water supply were associated with increased death losses. Shipping cattle by truck, rather than train, was associated with decreased rates of disease. Processing factors, including using vaccines against respiratory disease, were not associated significantly with mortality or morbidity. It was concluded that reducing the number of calves, to approximately 100 per group, not changing the ration to silage within the first month and not using antibiotics in the water supply on arrival could significantly reduce disease and death losses.