Two strains of avian sarcoma virus newly isolated from chick fibrosarcomas induced by lymphatic leukemia virus subgroup A in two lines of chickens.

Two strains of avian sarcoma virus, designated S1 and S2, have been newly isolated from avian lymphatic leukemia virus (LLV) subgroup A [LLV(A)]-induced fibrosarcomas in 2 chickens from 2 White Leghorn flocks of lines 151 and BK. Each stock of S1 and S2 contained 2-3 X 10(3) focus-forming units of virus/ml and 10(6) tissue culture infective dose of LLV(A)/ml. The focus-forming titer of S1 and S2 estimated in chick embryo fibroblast cultures was almost consistent with that estimated by tumor (fibrosarcoma) formation in the chicks. All of the chicks bearing wingweb tumors (fibrosarcomas at the site of inoculation) had macroscopically neoplastic lesions (fibrosarcomas) in a number of visceral organs, such as the lung, liver, and spleen. Each stock of S1 and S2 was cloned by picking a single focus, and each focus was examined for the production of focus-forming virus and LLV. The results suggest that S1 and S2 are heterogenous stocks of sarcoma viruses that are replication-defective to various degrees. The low titer of focus-forming virus in the stocks of S1 and S2 was considered to be due to the small amounts of avian sarcoma virus that could be complemented by LLV.

Avian erythroblastosis virus isolated from chick erythroblastosis induced by lymphatic leukemia virus subgroup A.

A new strain of avian erythroblastosis virus (AEV), designated "AEV-H," was established by serial passages of a field isolate of avian lymphatic leukemia virus subgroup A [LLV(A)] in chicks from a White Leghorn flock of line 151 chicks. One stock of AEV-H contained 10(4) focus-forming units/ml virus and 10(9) tissue culture infective dose/ml LLV(A). All of the chicks that received ip inoculations of 0.2 ml AEV-H developed erythroblastosis complicated by fibrosarcoma 16-24 days (approximately equal to 17.7 days) after inoculation. Pathologic changes of erythroblastosis were macroscopically observed, mainly in such visceral organs as the liver, spleen, and bone marrow. In addition, microscopic changes were observed in the lung, kidney, ovary, and heart. Pathologic changes of fibrosarcoma were so conspicuous that they were recognizable by the naked eye in the pancreas and in the serous membrane of the intestine.

Virus distribution and histopathologic changes in organs of chickens inoculated with Newcastle disease virus (avian paramyxovirus-1) isolated from racing pigeons.

The virus distribution and histopathologic changes in organs of 1-week-old chickens inoculated with three representative isolates of Newcastle disease virus isolated from racing pigeons in Japan were examined. All three isolates were recovered from various organs, including brain, for several days, but not from the blood. Results were highly correlated with their high intracerebral pathogenicity indices (ICPI), in spite of their long mean death time of minimum lethal dose (MDT/MLD).

Improved preparation of ELISA antigen of infectious bursal disease virus.

Serotype 1 of infectious bursal disease virus (IBDV) adapted to chicken embryo fibroblasts (CEF) was used for the preparation of enzyme-linked immunosorbent assay (ELISA) antigen. After several passages of diluted viruses in CEF cultures, the titer of seed virus increased to 1.2 x 10(8) plaque-forming units/ml. Purified virus prepared from this seed virus had high titers of antigen and was less nonspecific than that from low titer of seed virus in an ELISA. The nonspecific reaction of purified virus decreased further after treatment with Triton X-100. When the specificity of this treated antigen was examined with specific-pathogen-free chicken sera before and during lay and with 14 antisera to some major avian viruses, this ELISA antigen had no nonspecific reaction and was specific to antibodies to serotypes 1 and 2 of IBDV.

Conditions for successful cultivation of tumor cells from chickens with avian lymphoid leukosis.

To determine a suitable source of tumor cells that would readily adapt to in vitro growth, attempts were made to culture lymphoid tumor cells in soft agar from several neoplastic organs from chickens with lymphoid leukosis. Of 10 tumors from 7 chickens, cells from only two--both from enlarged bursae--were grown successfully. The remaining 8 tumors failed to propagate. The two cell lines thus established have been propagated by serial passage for more than two years in continuous culture. The two cell lines grow as single, free-floating cells in suspension. Morphologically they are usually round, about 8-10 microns in diameter, and have the characteristics of lymphoblastoid cells. They propagate to a maximum concentration of about 1-2 X 10(6)/ml and release infective virus of subgroup A to titers as high as 10(7) TCID/ml. Their leukosis/sarcoma virus susceptibility phenotype is C/E. Lymphoid tumors developed on chorioallantoic membranes (CAM) of 10-day-old embryonated eggs of the BK line 7 days after inoculation, whereas no tumors developed on CAMs from the 15I line.

Outbreak of comb necrosis in layer breeder chickens.

Comb necrosis with leg weakness was seen in 41-day-old female layer breeder chickens. This disease occurred in three flocks at a breeder farm, but not in other flocks of growing chickens and broiler breeder hens at the same farm. The disease started in 35-day-old chicks in three flocks. The morbidity of comb necrosis was 10% and that of leg weakness was 3%. Characteristic gross lesions of affected chickens were swelling and necrosis of the whole comb. Histologically, liquefactive necrosis of epidermal epithelial cells with hyperplasia, vesicle formation in the epidermis, congestion, and hemorrhages with fibrinous thrombi of underlying dermis in the comb were noted. In mature comb lesions, the epidermis showed eosinophilic necrosis (scab formation). In the livers, multiple fibrinous thrombi were present in the sinusoids and there was necrosis of hepatic cells. Staphylococcus aureus and Pasturella spp. were isolated from comb lesions. There were no significant lesions causing leg weakness.

Efficacy of three live vaccines against highly virulent infectious bursal disease virus in chickens with or without maternal antibodies.

Since 1990, highly virulent infectious bursal disease virus (IBDV), which induces high mortality, has been infecting even vaccinated flocks in Japan. We report the efficacy of three live vaccines that are available in Japan. Two mildly attenuated strains (A and B) and one intermediate strain (C) were each tested both in specific-pathogen-free (SPF) chickens and in commercial chickens that have maternal antibodies against IBDV. Chickens were vaccinated at 20 days old and challenged with highly virulent IBDV 10 days post-vaccination. Protection was determined 7 days after challenge by measuring bursa/body weight ratios, histopathological lesions, and antibody responses to IBDV. All three lie vaccines conferred protection to SPF chickens. However, only vaccine C protected 100% of vaccinated commercial chickens against highly virulent IBDV; Vaccines A and B respectively protected three-fourths and none of vaccinated commercial chickens from severe bursal lesions. Vaccines A, B, and C and highly virulent IBDV induced bursal lesions in 3%, 0%, 23%, and 61% of inoculated commercial chickens, respectively. These results suggest that serological determination of the optimum vaccination time for each flock is required to effectively control highly virulent IBDV in the field. The optimum vaccination timing could be approximated by titrating the maternal IBDV antibodies of 1-day-old chicks by an enzyme-linked immunosorbent assay or by an agar gel precipitin test.

Lesions of bone and bone marrow in myeloid leukosis occurring naturally in adult broiler breeders.

Lesions of bone and bone marrow in myeloid leukosis (ML) occurring naturally in adult broiler breeders were investigated pathologically. During gross examination, nodules and protrusions were commonly observed on the surface of the sternum, ribs, vertebrae, and synsacrum. The bone marrow of all the bones of the body was pale in color. Histologically, granulated myelocytes proliferated in the bone marrow of various bones and in the periosteum of the sternum, ribs, vertebrae, and synsacrum. The first proliferation of tumor cells occurred in the bone marrow of epiphysis. The myelocytes invaded through haversian and Volkmann's canals from the bone marrow to periosteal areas. Hematopoiesis was suppressed by marked proliferation of tumor cells in the bone marrow of the whole bone. Atrophy was also seen in the bones, including medullary bones of the chickens suffering from ML. Proliferation of myelocytes was seen in the bone marrow and periosteum of ossified cartilaginous rings of the trachea and larynx. Marked proliferation of myelocytes was seen in the dura mater of spinal cords, and it subsequently depressed the spinal cords. Bone formation with cartilage was seen in the periosteum of the sternum having marked proliferation of myelocytes in the bone marrow and periosteum. Ultrastructurally, tumor cells showed large nuclei and cytoplasm with large round electron-dense lysosomes. The virus particles were rarely detected in the cytoplasm of tumor cells. The polymerase chain reaction test of tumor samples showed positive for subgroup J avian leukosis virus. This study indicates that the myelocytes can invade through the compact bones to the periosteum in the sternum, ribs, vertebrae, synsarcum, and ossified cartilage of trachea and larynx having thinner compact bones. In addition, the periosteal osteogenesis with cartilage in the sternum may be reactive change against the bone atrophy because of the marked proliferation of myelocytes.

Experimental infection in specific-pathogen-free chicks with avian reovirus and avian nephritis virus isolated from broiler chicks showing runting syndrome.

Avian reovirus (ARV) and avian nephritis virus (ANV) were individually isolated from runty 10-day-old broiler chicks. The ARV isolate, IR-R, the ANV isolate, IR-N, and the reference strain of ANV, G-4260, were inoculated orally into 1-day-old chicks of two specific-pathogen-free (SPF) chicken lines, 151 and PDL-1. Growth retardation without the presence of gross lesions was clearly observed at 7 and 14 days postinoculation (PI) in chicks of both lines inoculated with the IR-R strain. On the other hand, in chicks inoculated with IR-N strain, growth retardation was observed only in the chicks of line 151 at 7 and 14 days PI. Microscopically, nephritis was observed in both chicken lines at 7 and 14 days PI. When chicks that were inoculated with the IR-N strain at 1 day of age were inoculated with the IR-R strain at 3 days of age, growth retardation was observed in the chicks of line PDL-1 at 10 and 17 days PI. However, the growth retardation was less severe than in the group receiving a single inoculation of the IR-R strain.

Persistent infection with chicken anaemia virus and some effects of highly virulent infectious bursal disease virus infection on its persistency.

Chicken anaemia virus (CAV) infectivity and the effect of highly virulent infectious bursal disease virus (hv IBDV) infection on CAV's infectivity were examined in chickens inoculated with CAV or inoculated dually with CAV and hv IBDV. Five chickens inoculated dually with hv IBDV at 35 days old and then with CAV at 40 days old exhibited no clinical signs of disease, but showed atrophic bursae of Fabricius when necropsied 4 weeks later. Upon examining the chickens at 7 days postinoculation (dpi) with CAV, it was found that hv IBDV infection had inhibited production of virus neutralising (VN) antibody to CAV, and that it was possible to recover CAV from plasma of these chickens. Although VN antibody to CAV appeared after 14 dpi, CAV was recovered from blood cells (BC s) at high titres ranging from 10(2.5)to 10(5.5)TCID(50)/0.1 ml, 7 to 28 dpi in IBDV -induced immunosuppressed chickens. In addition, CAV was sporadically recovered, using rectal swabs, from the dually inoculated chickens at low titers, ranging from 10(1.0)to 10(2. 0)TCID(50)/0.1 ml). In contrast, although CAV was recovered from BC s in most of the chickens inoculated with CAV alone, the titers were lower (10(1.0)to 10(2.5)TCID(50)/0.1 ml). No CAV was detected from the rectal swabs of these chickens. The results of virus recovery were confirmed by polymerase chain reaction. This study first examined the persistency of CAV in BC s and the effective enhancement of primary CAV infection as a result of immunosuppression caused by hv IBDV infection.

A long term observation of antibody status to chicken anaemia virus in individual chickens of breeder flocks.

The antibody status to chicken anaemia virus (CAV) in four layer breeder flocks was evaluated. Sera were periodically collected from the same 17 to 20 individual chickens of each flock ranging in age from 10 to 63 weeks old. The neutralising and fluorescence antibody were detectable in individual chickens during the observation periods ranging from 13 to 44 weeks. A high prevalence of both neutralising and fluorescence antibodies was observed; however, the prevalence of fluorescence antibody in older chickens was lower than that of neutralising antibody. The geometric mean (GM) of neutralising antibody titres, after all the chickens examined had seroconverted in flocks 1, 2 and 4, ranged from 373.2 to 2940.6. In flock 1, the GM titre at 63 weeks old was significantly lower than that at 37 and 52 weeks old. In flock 4, the GM titre at 48 weeks old was significantly lower than that at 24 and 35 weeks old. In flock 2, the GM titre at more than 31 weeks old significantly increased compared with that at 25 weeks old; this tendency was not seen in the GM of the fluorescence antibody titres. The results indicate that immunity to CAV can last a long time in naturally infected individual chickens.

Ultrastructural cytochemical characterization of alpha-naphthyl acetate esterase in chicken monocytic cell lines.

In order to study the differentiation level of two chicken monocytic cell lines, IN24 and LSCC-NP1 cells, alpha-naphthyl acetate esterase (ANAE) was examined by ultrastructural cytochemical technique. Morphologically and functionally, IN24 cells showed immature character compared to LSCC-NP1 cells. In IN24 and LSCC-NP1 cells, ANAE activity was localized on the external face of the plasma membrane and coated vesicles, but not in lysosomes and phagocytic vacuoles. ANAE reactivity showed a dense linear reaction product on the entire cell surface in IN24 cells and granular reaction product in LSCC-NP1 cells. On treatment with interferon-gamma (IFN-gamma), ANAE reactivity of IN24 cells became similar to that of LSCC-NP1 cells. IN24 cells treated with lipopolysaccharide (LPS) had less amounts of ANAE surface product, and reactivity was partially dispersed on the cell surface.

Detection of avian leukosis virus antigens by the ELISA and its use for detecting infectious virus after cultivation of samples and partial characterization of specific pathogen-free chicken lines maintained in this laboratory.

An enzyme-linked immunosorbent assay (ELISA) for detecting avian leukosis virus (ALV) antigens was developed with rabbit anti-ALV serum. The ELISA detected purified ALV of subgroups A and B at a concentration of 0.4 ng/well and about 10(3) infectious units/well estimated by a resistance-inducing factor (RIF) test, and antigens in culture fluids from chicken embryo fibroblasts infected with subgroups A, B or E of ALV. These results showed that common antigens among the subgroups were detected by the ELISA. When virus titration was performed, virus infectivity could be determined by the ELISA within 7 days after cultivation. The titer was similar to that obtained by the RIF test on 19 days after 3 subcultures. These results indicate that the ALV-isolation test by the ELISA was superior to the RIF test in rapidity and applicability to large-scale field trials. Four specific pathogen-free (SPF) chicken lines maintained in this laboratory were examined for endogenous ALV antigens by the ELISA. Sera from laying hens had considerably high absorbance (A) values, whereas albumen samples showed low A values except for some samples (7/40 hens). Although most of sera from 1-day-old SPF chicks showed lower A values than those from laying hens, some sera showed A values as high as those from viremic chicks in 2 lines. Endogenous ALV was isolated from sera from laying hens (6/40) and their albumens (4/7) with high A values. Two SPF chicken lines were found to produce endogenous virus at a high frequency.

Rearrangement of c-myc gene in rapidly induced avian lymphoid leukosis tumors.

Southern blot hybridization of DNA samples from 9 primary tumors of avian lymphoid leukosis (LL) rapidly induced by ALV infection 27-74 days post inoculation was carried out to search for rearrangement of the c-myc gene with human c-myc gene exon III as a probe. Rearrangement of the c-myc gene was detected by appearance of new EcoRI fragments in 7 out of 9 tumors examined. The size of the fragments ranged from 3.1 to 4.0 kilobases (kb). In addition to these fragments, two fragments (9.0 kb and 13 kb) were observed in one tumor, and a faint fragment (3.5 kb) was observed in another tumor. Rearrangement of the c-myc gene was not detected in the remaining two tumors kept in unsuitable condition. These results suggest that rearrangement of c-myc gene was induced even in rapidly induced LL as well as that induced after long incubation period. This is the first report of involvement of c-myc gene in rapidly induced B-cell lymphoma (LL).

Isolation of serotype 2 Marek's disease virus from a cell line of avian lymphoid leukosis.

To determine a serotype of Marek's disease virus (MDV) persistently infected in a lymphoid leukosis (LL)-cell line, LSCC-BK3, clone A (BK3A), and to examine the pathogenicity of the virus, an attempt was made to isolate MDV from culture fluid of the LL-cell line, using chick embryo fibroblast cultures resistant to infection with subgroup A avian leukosis virus (ALV) to eliminate subgroup A ALV. The MDV isolate, serologically identified as serotype 2 MDV and designated as the 2H strain, was free from ALV and reticuloendotheliosis virus. A serotype 2-specific antigen of MDV was detected in 44% of BK3A, but antigens of serotypes 1 and 3 were not detected in this cell line, by the indirect immunofluorescent antibody test using serotype-specific monoclonal antibodies. MDV antigens were undetectable in LSCC-BK3, clone 2C; both cell lines were established from the same chicken. Neither clinical signs nor macroscopic lesions were observed in any of 19 chicks inoculated with the 2H strain. Three out of 19 chicks histopathologically examined had no lesions. These results suggest that serotype 2 MDV can persist in B cells transformed by ALV without cytopathic effect at a high rate, and the isolate may become a candidate for MD vaccine strains.

Identification and characterization of hens transmitting avian leukosis virus (ALV) to their embryos by ELISAs for detecting infectious ALV, ALV antigens and antibodies to ALV.

A total of 72 White Leghorn grandparent hens was examined by ELISA for avian leukosis virus (ALV), ALV antigens and anti-ALV antibodies to identify and characterize the hens transmitting ALV to their embryos (transmitters) by using fertilized eggs. These hens were divided into 3 groups as no antibody and non-viremic (NANV) (49 hens), antibody-positive and non-viremic (APNV) (21 hens) and no antibody and viremic (NAV) (2 hens) by testing the sera for the presence of ALV and anti-ALV antibody. Egg albumen and embryos were tested for the presence of ALV and ALV antigens. As a result, no ALV was detected in both albumen and embryos in the NANV group. On the other hand, all albumen samples collected repeatedly from 3 hens of the APNV group and 2 hens of the NAV group contained infectious ALV, although the infectivity differed with the individual. Also, these 5 hens produced infected embryos at varying frequencies. However, on AP hen which shed neither ALV nor ALV antigens into the albumen produced an infected embryo at a lower rate. These results indicate that testing for infectious ALV in albumen from a newly laid egg per hen is effective to identify the transmitters to some extent. When virus titers in each of 8 tissue samples from the 6 transmitting hens were determined, the highest virus titers were found in washing from the ampulla of the oviducts in most of the shedders, suggesting that embryo infection is closely correlated with ALV produced at the oviduct, but not with ALV transferred from the other parts of the body.

Sporadic congenital transmission of avian leukosis virus in hens discharging the virus into the oviducts.

The efficacy of the albumen test for infectious avian leukosis virus (ALV) was examined in detecting congenitally transmitting hens. Seventy-three White Leghorn non-viremic hens with antibody to ALV were used. Eleven of the hens shed infectious ALV into their egg albumen, whereas only 7 of the 11 ALV-positive hens shed ALV antigens. The egg albumen test for infectious ALV was shown to be more effective in detecting the congenitally transmitting hens than that for ALV antigens. Then, twenty of the 62 hens which shed no infectious ALV into the albumen were studied for transmission of ALV to their embryos and for discharging ALV into the oviduct and vagina. Six of the 50 embryos from 4 hens were found to be infected with ALV but all of the 227 embryos from remaining 16 hens were free from the infection. Discharge of the virus into the oviduct and vagina was found both in the 4 transmitting hens and in 6 of the 16 non-transmitting hens. These results suggest that the hens discharging ALV into the oviduct, even though they do not shed ALV into egg albumen, may transmit the virus sporadically to their embryos.

Rapid induction of lymphoid leukosis and ascites by avian leukosis virus from a lymphoid leukosis cell line.

To examine whether a lymphoid leukosis (LL) cell line releases an LL-specific avian leukosis virus (ALV) or not, two viral materials, culture fluid and a concentrated viral material from an LL-cell line, were inoculated into a total of 74 day-old chicks of line 15I in 5 experiments. Spectrum of diseases induced, their incidence and incubation periods to onset were examined. Fifteen chicks were inoculated with the culture fluid and 9 (60%) developed ascites [59-119 days post inoculation (dpi); geometric mean (GM) of dpi, GM: 89.6)], but LL was not induced in any chicks inoculated. Fifty-nine chicks were inoculated with the concentrated viral material and LL was recognized in 13 (22.0%) (27-74 dpi; GM: 48.4), ascites with LL in 11 (18.6%) (34-75 dpi; GM: 41.3), ascites alone in 21 (35.6%) (32-83 dpi; GM: 48.2), erythroblastosis in 2 (3.4%) (70-102 dpi; GM: 84.5), and other diseases in 12 (20.3%) (43-102 dpi; GM: 61.8). LL lesions were frequently observed in the liver, spleen, kidneys, bursa of Fabricius (bursa), bone marrow and gonads. Mild lymphocytic foci in some visceral organs and perivascular cuffing in the central nervous system were observed mainly in several chicks diagnosed as having complication of ascites with LL or other diseases. In addition to these lesions, atrophy of bursa and thymuses was recognized in them. No antibodies against Marek's disease virus (MDV) and reticuloendotheliosis virus were detected in 36 sera taken from the chicks inoculated with the concentrated viral material. Serotype 2 MDV was isolated from the buffy coat of some inoculated chicks. These results suggest that the properties of ALV inoculated and immunosuppression caused by inoculation with high doses of ALV are involved in rapid induction of LL and expression of pathogenicity of serotype 2 MDV released from the LL cell line and included in the viral inoculum. This is the first report describing the rapid induction of LL and ascites in chicks.

Visceral urate deposits in chicks inoculated with avian nephritis virus.

Avian nephritis virus, G-4260 strain, was inoculated orally into one-day-old specific-pathogen-free chicks of two lines. Approximately 20 per cent of the chicks of both lines died with visceral urate deposits from eight to 12 days after infection, and the virus was isolated from the kidneys of the dead chicks. At 14 or 15 days of age the mean liveweight of the surviving infected chicks was approximately 16 per cent less than that of the uninfected control chicks.