[Vaccination against infectious anemia of poultry (CAA)--results of field studies]. / Impfungen gegen die Infektiöse Anämie des Geflügels (CAA)--Ergebnisse von Feldversuchen.

Infectious anemia of poultry is a disease of high economical significance. Connatal infection of chicks with the chicken anemia agent (CAA) via the embryonated egg causes anemia along with severe immunosuppression, thus rendering the chicken susceptible for secondary infections. In order to prevent infection of young chicks, it is necessary to induce immunity against CAA in parent flocks, with the aim to prevent connatal spread of the infection and provide maternal protection for baby chicks. In this publication, the efficacy and use of a live CAA vaccine is reported. From autumn 1986 until summer 1990, 3 experimental vaccine charges were applied in 85 broiler parent flocks with totally 3.1 million chickens. In addition, totally 293,000 broiler breeder and 171,000 layer breeder chicken were vaccinated in 1989/90. The vaccine was administered between the 13th and 19th week of life by drinking water without adverse effect to the birds. Chicken anemia symptoms were observed only at the begin of laying period in two parent flocks. These flocks had been vaccinated in the 17th and 19th week, respectively. The offsprings of all other vaccinated parent flocks remained free of chicken anemia. Day-old chicks derived from vaccinated parent flocks were protected against CAA challenge infection. It is emphasized, that vaccination should be performed within the 13th to 15th week of life, because according to our observations, this will lead to an immediate seroconversion.

Morphological characterization of chicken anaemia agent (CAA).

Chicken anaemia agent (CAA) was characterized as a virion with 25 nm in diameter, with a buoyant density in CsCl of 1.36-1.37 g/cm3, and containing a circular, single-stranded DNA genome. The virus is composed of 32 hollow morphological units representing a regular T = 3 icosahedron.

Development of optimal conditions for lymphokine production by chicken lymphocytes.

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine-containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50% cytostasis-inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)-stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41 degrees C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 micrograms ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 micrograms and 45 micrograms ConA/ml.

Detection of different macrophage-activating factor and interferon activities in supernatants of chicken lymphocyte cultures.

Chicken spleen lymphocytes were cultivated under various conditions in order to produce and characterise functionally avian lymphokines. Biological properties of lymphokine-containing culture supernatants were evaluated with regard to their antiviral activity and their effects on cultured bone marrow-derived chicken macrophages, including induction of cytostatic activity, enhancement of phagocytic activity towards vital Candida albicans, and giant cell formation. Optimal doses of concanavalin A (ConA) for stimulation of lymphocytes varied between 2.5 and 40 microg/ml, depending on the cell concentration and presence or absence of serum. Lymphokine production occurred even without exogenous mitogen stimulation when cells were cultured at sufficiently high concentrations (1 to 2 x 10(7) cells/ml). Cytostasis-inducing capacity of lymphokine preparations against lymphoblastoid MDCC-RP1 cells was always combined with the presence of antiviral activity. Experimental results suggested that these two activities had to be attributed to at least two distinct lymphokines, i.e. macrophage-activating factor and interferon (IFN). ConA stimulation of lymphocytes from a single donor appeared to be the appropriate signal for production of IFN-gamma. Endogenous stimulation in mixed lymphocyte cultures more appropriately triggered production of IFN-alpha or IFN-ss, although production of IFN-gamma was not completely suppressed. Phagocytic activity of macrophages could also be increased by a cytokine present in conditioned media from confluent cultures of chicken embryo fibroblasts, chicken kidney cells, or bone marrow-derived chicken macrophages. In lymphokine preparations, this mediator may-even be a cofactor necessary for induction of multinucleated giant cell formation of cultured macrophages.

Growth inhibition of Marek's disease T-lymphoblastoid cell lines by chicken bone-marrow-derived macrophages activated in vitro.

Studies have been performed on the induction of cytostatic activity of cultured chicken bone-marrow-derived macrophages. Cultured macrophages were exposed to supernatants of ConA-stimulated spleen cell cultures (lymphokines), lipopolysaccharide (LPS), ConA or infectious bursal disease virus (IBDV). Cytostatic activity of macrophages was examined by testing their growth-inhibiting effect on T-lymphoblastoid Marek's disease cell lines RP-1, HP-2 and MSB-1 as target cells. Lymphokines, LPS and IBDV caused considerable enlargement of adherent macrophages. Results of cytostasis assays suggested that morphological signs of macrophage activation were not correlated with the cytostatic potency of activated macrophages. Lymphokine-activated macrophages caused at least an 80% growth inhibition of target cells, whereas LPS, ConA or IBDV-activated macrophages exhibited only a marginal cytostatic effect in the range of 20%. Attempts to detect soluble cytostatic factors in supernatants of activated macrophage cultures failed or had equivocal results. Untreated macrophages were rarely cytostatic at an effector:target cell ratio of I:1 to 4:1, and they stimulated RP-1 cell growth if these cells were seeded at suboptimal concentrations. The results suggest that the suppressor activity of macrophages from Marek's disease virus-infected chickens, as demonstrated by other authors in vitro, is probably a result of nonspecific immunomodulation in vivo where lymphokines may also play a major role.

Effects of avian viruses on cultured chicken bone-marrow-derived macrophages.

Cultured chicken bone-marrow-derived macrophages have been assayed for their susceptibility to infection with various avian viruses. Three criteria of infection were employed: (1) Virus-induced alterations in cell morphology ; (2) presence of intracellular viral antigens detectable by immunofluorescence; (3) kinetics of virus release by infected macrophages. Macrophages proved to be resistant to Marek's disease virus (MDV), herpesvirus of turkeys (HVT-FC126), infectious bronchitis virus (IBV) and reticuloendotheliosis virus (REV). MDV included the pathogenic HPRS-16 strain prepared from feather follicles, and the apathogenic HPRS-24 strain adapted to growth in chick embryo fibroblast cultures. IBV included both embryo-propagated and tissue culture-adapted variants of the apathogenic Beaudette strain and a pathogenic Massachusetts-type strain. REV comprised the strains REV-C, CSV and oncogenic virus of the REV-F strain. Adenovirus, infectious laryngotracheitis (ILT) virus, reovirus, infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV) replicated in macrophages causing different but characteristic cytopathic effects, or alterations in cell morphology associated with macrophage activation. The most prominent effect of IBDV and lentogenic NDV infection were morphological signs of macrophage activation, i.e. enlargement or 'transformation' of cells which tended to survive in infected cultures and were usually free of detectable amounts of immunofluorescent viral antigens. Macrophage cultures were less susceptible to infection with adenovirus (OTE strain), pathogenic ILT virus and lentogenic NDV (B1 strain) than permissive chicken kidney cell (CKC) cultures. In contrast, macrophage cultures were significantly more susceptible to infection with reovirus than CKC cultures, indicating that bone-marrow-derived macrophages might be the major target cells of this virus species. Virus restriction by cultured bone-marrow-derived macrophages was expressed to various degrees among the different avian virus species and among different strains of the same virus species, however, it was not generally correlated with the pathogenicity of these viruses in chickens.