Efficacy of a combination vaccine containing MDV CVI 988 strain and HVT against challenge with very virulent MDV.

With the emergence of very virulent Marek's disease virus (MDV) strains, vaccines based on herpesvirus of turkeys (HVT) appear to be not powerful enough to confer full protection, whereas in chicken flocks vaccinated with MDV CVI 988 strain protective immunity sometimes is generated not early enough for full protection. For this reason combination vaccines containing HVT as well as CVI 988 have been developed. In this paper the beneficial effect of combining both types of virus strains in one vaccine for early protection is shown in a vaccination challenge experiment, in which one-day-old chickens were vaccinated with suboptimal dosages of the monovalent vaccines and the same dosages in a combination vaccine. After 5 days the chickens were challenged with a very virulent MDV strain and subsequently observed for a period of approx. 50 days. It appeared that the combination vaccine provided better early protection than the monovalent vaccines. In addition, the combination vaccine was tested as vaccine administered in ovo. It appeared that after in ovo vaccination the vaccine conferred adequate protection against challenge with a very virulent MDV strain, 5 days after hatch, and that protection after in ovo vaccination was similar to that obtained after subcutaneous vaccination with the same combination vaccine.

Enhanced expression of cytokine genes in spleen macrophages during acute infection with infectious bursal disease virus in chickens.

We examined the effects of infectious bursal disease virus (IBDV) on splenic T cells and macrophages. In acute IBDV infection, splenocytes responded poorly to Con A stimulation. However, when T cells were isolated from whole spleen cells, purified T cells responded normally to Con A. This result indicated that functional T cells were present in the spleen but mitogen-induced proliferation of T cells was being suppressed by other cells. Previous studies indicated that soluble factors from suppressor cells may mediate this inhibition of T cell mitogenesis. We thus examined the effects of IBDV on spleen adherent cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to quantitate the expression of several cytokine genes in splenic macrophages. In acute IBDV infection, splenic macrophages exhibited enhanced gene expression of type I interferon (IFN), chicken myelomonocytic growth factor (cMGF), an avian homolog of mammalian IL-6, and 9E3/CEF4, an avian homolog of mammalian IL-8. Mitogen-stimulated spleen cell cultures also produced elevated levels of nitric oxide. The elevation of cytokine gene expression by macrophages occurred transiently during the acute phase of viral infection and coincided with in vitro inhibition of T cell mitogenic response of spleen cells.

Suppressor macrophages mediate depressed lymphoproliferation in chickens infected with avian reovirus.

A previous study indicated that spleens from reovirus-infected chickens contained macrophages that were primed to produce nitric oxide (NO). The presence of these primed macrophages correlated with depressed in vitro T cell mitogenesis. The current studies indicated that splenic adherent macrophages from virus-exposed chickens inhibited concanavalin A (ConA) induced proliferation of normal spleen cells. ConA-stimulated spleen cells from uninfected chickens, but not virus-exposed chickens, produced large quantities of interleukin-2 (IL-2) and a factor that induced NO production. This factor was tentatively named NO inducing factor (NOIF). The removal of macrophages from the spleens of virus-exposed chickens by plastic adherence resulted in partial recovery of ConA-induced proliferation and the production of normal levels of IL-2 and increased levels of NOIF, although these remained below normal. However, nonadherent spleen cells produced substantial quantities of NO, which indicated an incomplete removal of macrophages. Because removal by plastic adherence did not result in the depletion of all macrophages, spleen cells were panned with anti-CD3 antibody to obtain an almost pure population of T cells. Fractionated T cells from virus-exposed chickens proliferated vigorously to ConA and produced normal levels of IL-2 and NOIF. When splenic adherent cells from virus-exposed chickens were added to purified T cells, the T cells failed to respond to ConA. Addition of splenic adherent cells from virus-free chickens did not induce mitogenic inhibition. Further, the addition of purified T cells from the spleens of reovirus-infected chickens to T cells from virus-free birds did not adversely affect T cell mitogenesis. These data indicated that reovirus infection in chickens does not compromise the functional capabilities of T cells but induces suppressor macrophages that inhibit T cell functions.

Immunohistochemical detection of lymphocyte subpopulations in the tarsal joints of chickens with experimental viral arthritis.

We characterized the lymphocytes in the tarsal joint synovium of chickens inoculated with an arthrotropic strain of avian reovirus. Cryostat sections of whole joints taken from 2 days to 35 days postinoculation were analyzed using monoclonal antibodies directed against B lymphocytes, T lymphocytes, and chicken Ia antigen. Plasma cells were morphologically identified using stained sections of whole joints. Time-dependent changes were found in the type and number of positively staining cells. Synoviocytes and cells with a dendritic morphology stained positive for Ia in normal joint sections. T cells, mostly CD8 positive, were present in low numbers in acute phase arthritis (2-6 days postinfection) in the perivascular and superficial regions of the synovium. Subacute arthritis (8-14 days postinfection) was characterized by increased numbers of CD4 and Cd8 T cells in the perivascular and superficial regions. The perivascular T cells began to organize into aggregates, with IgM-positive B cells and plasma cells on the periphery of these aggregates. Some CD8-positive cells were detected on the surface of the articular cartilage. Cells staining positively for Ia were not lymphocytes. Chronic arthritis ( > 14 days postinfection) was characterized by large numbers of T cells in the perivascular and superficial regions, with the CD4-positive T cells found primarily in the lymphoid aggregates of the perivascular regions. IgM-positive B cells were fewer, but more plasma cells, few of which stained positive for IgM, were present. Lymphocytes in chronic arthritis stained positively for Ia. These data suggest that the types, numbers, and activation level of lymphocytes present in the tarsal joints are similar but not identical to those seen in rheumatoid arthritis.

An antiviral effect of nitric oxide: inhibition of reovirus replication.

We have previously shown that macrophages from chickens infected with avian reovirus are primed to produce nitric oxide (NO) in response to T cell cytokines and bacterial lipopolysaccharide (LPS). We now show that NO exerts potent antireovirus effects. Reovirus replication was substantially reduced in a chicken macrophage cell line, HD11, induced to make NO by stimulation with LPS or conditioned medium from concanavalin A-stimulated spleen cells. The use of a competitive inhibitor of nitric oxide synthase, NG-monomethyl-L-arginine, reduced the antiviral effect of LPS-stimulated HD11 cells. Cytostatic effects were concurrent with the observed antiviral effects of NO. Among these cytostatic effects were reduction in DNA synthesis, protein synthesis, and mitochondrial metabolism. These results indicated that a potential consequence of macrophage priming following virus infection is the protection of cells against virus-induced replication and cytopathic effects, and this protection may be mediated by the cytostatic effects of NO on the host cell.

Reovirus infection in chickens primes splenic adherent macrophages to produce nitric oxide in response to T cell-produced factors.

In this study, we examined the mechanisms by which avian reovirus infection of chickens depresses in vitro proliferative responses of spleen cells to T cell mitogens. We showed an enhanced production of nitric oxide (NO) by phytohemagglutinin (PHA)-stimulated spleen cells from reovirus-infected birds but not from virus-free birds. Since macrophages are a primary source of NO, we compared splenic adherent macrophages from virus-free and virus-exposed chickens. There was a fourfold increase in the number of adherent macrophages from the spleens of virus-exposed chickens. Production of NO by macrophages from virus-exposed chickens required T-cell-produced factors and was not due to direct stimulation of macrophages by PHA. Although T cell products were needed for NO production by macrophages, in an apparent paradox, we found significantly reduced levels of NO-inducing activity in the supernatants of PHA-stimulated spleen cells from virus-exposed chickens than in supernatants from PHA-stimulated normal spleen cells. Cocultures of adherent cells from infected chickens with normal spleen cells indicated that although macrophages secreted NO following PHA stimulation, macrophages ultimately suppressed the continued production of NO-inducing factors by normal spleen cells. We further showed in experiments utilizing NG-monomethyl-L-arginine, an NO synthesis inhibitor, that NO was not responsible for the mitogenic inhibition of spleen cells from virus-exposed chickens. In summary, our results indicated that following reovirus infection, macrophages are primed in vivo and activated in vitro by T-cell-produced factors. Despite the requirement of T cell cytokines for NO production, T cells did not proliferate to mitogenic stimuli, which indicated that the early events (i.e., cytokine secretion) but not the late events (i.e., proliferation) of the T cell activation cascade were functional. Macrophage priming following reovirus infection may have important implications for impaired T cell responsiveness.