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.

Two serological types of Marek's disease virus and a herpesvirus of turkeys have been differentiated by indirect immunofluorescence tests as (1) pathogenic strains of Marek's disease virus (MDV) and their attenuated variants: HPRS-16, HPRS-16/att, HPRS-B14, JM, JM/att, GA, VC and 'Oldenburg', a recent field isolate; (2) apathogenic strains HPRS-24 and HPRS-27 of MDV; (3) herpesvirus of turkeys strain FC126 and its HVT(A-) variant. Virus strains could not be distinguished on the basis of qualitative differences in immunofluorescent staining of intracellular virus-induced antigens. Results were similar whether chicken kidney, chicken embryo fibroblast or duck embryo fibroblast cell cultures were used. Fluorescence of virus-induced antigens was stronger with homologous than with heterologous antisera. Using the direct immunofluorescence technique Marek's disease virus and turkey herpesvirus infections could be distinguished. There were never any significant differences in the appearance and distribution of antigen in infected cells treated with homologous or heterologous antisera at dilutions of comparable activity using the indirect immunofluorescence technique. Antibody titres of antisera were 4 to 8-fold higher in the indirect immunofluorescence test against the homologous virus-induced antigens than against heterologous antigens. Cross-reactions between the 3 serological types could be prevented by absorption of antisera with the appropriate antigens. Cross-reactions could also be prevented by the appropriate dilution of antisera before use in the indirect immunofluorescence test.

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.

SUMMARYCertain properties of four strains of reticuloendotheliosis virus (REV), namely REV-F, REV-S, REV-C and chick syncytial virus (CSV) were studied in vivo and in vitro. In chicken embryo fibroblast (CEF) cultures these viruses caused a chronic infection which could be detected by indirect immunofluorescence (FA) tests. Precipitating antigens were present in infected cultured cells as well as in culture supernatants. Antibodies in sera of REV-inoculated chickens were demonstrated by FA tests and agar gel precipitin (AGP) tests. No antigenic differences between the four reference virus strains could be detected by either method. The immune response of chickens was shown to be a sensitive indicator of REV infection. AGP tests proved to be as suitable as FA tests provided that the serum samples were taken not later than 3 weeks after inoculation, since antibody titres decreased afterwards. Infectivity titres of the apathogenic REV-C strain proved to be the same in CEF cultures and in chickens assayed by this method.The immunogenicity of REV was unaffected by the presence of HVT. The humoral immune response to HVT, however, was drastically reduced if REV was present in the inoculum. These results suggest that even minor contaminations with REV can markedly reduce the efficacy of Marek's disease vaccines, and that such REV contaminations can effectively be controlled by tests using chickens.

A total of 639 flock serum samples, consisting of 10 up to 24 individual chicken sera each, from specific pathogen-free (SPF) flocks and from field flocks were examined by indirect fluorescent antibody (FA) tests for the presence or absence of antibodies to chicken anaemia agent (CAA). Comparisons between results of FA tests and serum neutralization (SN) tests included 284 flock samples where individual sera from a flock were pooled to perform SN tests.The SN test for detection of anti-CAA antibody proved to be about 20 % more sensitive than the indirect FA test. Results of SN tests were considered to be always specific, partly confirmed by CAA challenge of day-old chicks from serologically tested flocks. However, the evaluation of results revealed that the indirect FA test was not sufficiently specific. The major problem with SPF flock samples was false positive results (24/43 = 55.8%) of FA tests, and false negative results (7/32 = 21.9 YO) with field flock samples. The causes of non-specific immunofluorescent reactions resembling CAA-specific fluorescence could not yet be elucidated.