Pathology of chickens infected with avian nephoblastoma virus MAV-2(N)

A neophroblastoma-inducing myeloblastosis-associated virus, MAV-2(N), derived from avian myeloblastosis virus was characterized with respect to biochemical composition and avian pathogenesis. Purified fibroblast-grown virus contained the same size 35S ribonucleic acid and the same relative amounts of viral polypeptides as another myeloblastosis-associated virus inducing predominantly osteopetrosis MAV-2(O). Plaque-purified MAV-2(N) induced a 76 to 93% incidence of nephroblastoma and a 3 to 50% incidence of osteopetrosis in SPAFAS and line 15 x 7 chickens: the oncogenic spectrum and the onset of nephroblastoma varied with the line of chicken and the route of injection. Renal neoplasms were manifest in chickens older than 2 months and grew to a massive size. Furthermore, 29% of control chickens housed with MAV-2(N)-infected chickens demonstrated nephroblastoma. MAV-2(N)-infected chickens had growth rates and blood packed cell volumes comparable to those of uninfected chickens. Infected chickens 2 months of age had increased kidney, liver, and spleen weights; tumor-bearing chickens 3 to 4 months of age had increased liver, lung, brain, pancreas, and bone weights. The concentration of albumin was decreased and the concentration of gamma globulin was increased in the serum of MAV-2(N)-INFECTED CHICKENS. Analysis of the sera of nephroblastoma-bearing chickens for virus and antibody showed that three states existed: (i) high levels of neutralizing antibody, (ii) high levels of virus, and (iii) simultaneous presence of both at low levels. The pathological and virological features of MAV-2(N) which distinguish it from MAV-2(O) are discussed.     

Presence of viral antigens and antibody in the trachea of chickens infected with avian infectious bronchitis virus

Following infection of chickens with infectious bronchitis virus (strain M41) viral antigens were detected by immunofluorescence in the basal layer of the tracheal mucosal epithelium for 44 days. The enzyme-linked immunosorbent assay (ELISA) first detected virus-specific antibody in tracheal washes 7 days after infection and at 10 days these antibodies reached a level that was maintained for at least 44 days, although there was considerable variation between chickens. In contrast a neutralisation test did not detect antibody until day 20 ; titres were in the range 2.2 to 3.2 log2 reaching a maximum at day 27. ELISA detected anti-viral IgA in tracheal washes only on day 7, whereas IgG was detected in all samples containing anti-viral antibody. Serum anti-viral antibodies were detected by ELISA at 7 days and reached peak titres at 10 days, which were maintained. In contrast, serum neutralising antibodies were first detected at 10 days and increased to peak titres on day 24. The implications of the differences between the results of ELISA and neutralisation tests are discussed.     

Karyotype analysis of the acute fibrosarcoma from chickens infected with subgroup J avian leukosis virus associated with v-src oncogene

To understand the cytogenetic characteristics of acute fibrosarcoma in chickens infected with the subgroup J avian leukosis virus associated with the v-src oncogene, we performed a karyotype analysis of fibrosarcoma cell cultures. Twenty-nine of 50 qualified cell culture spreads demonstrated polyploidy of some macrochromosomes, 21 of which were trisomic for chromosome 7, and others were trisomic for chromosomes 3, 4, 5 (sex chromosome w), and 10. In addition, one of them was trisomic for both chromosome 7 and the sex chromosome 5 (w). In contrast, no aneuploidy was found for 10 macrochromosomes of 12 spreads of normal chicken embryo fibroblast cells, although aneuploidy for some microchromosomes was demonstrated in five of the 12 spreads. The cytogenetic mosaicism or polymorphism of the aneuploidy in the acute fibrosarcoma described in this study suggests that the analysed cells are polyclonal.     

Single and concurrent avian leukosis virus infections with avian leukosis virus-J and avian leukosis virus-A in Australian meat-type chickens.

Australian broiler breeders were screened for avian leukosis viruses (ALVs) (May 2001 to December 2003) as surveillance of measures to reduce the prevalence of ALV-J. Samples of blood (4233), albumen (1122), meconium (99) and tumours (16) were obtained from 93 flocks in six Australian states. Virus isolation was performed in C/O chick embryo fibroblast cultures, which were initially screened by group-specific antigen enzyme-linked immunosorbent assay, with follow-up confirmation using polymerase chain reaction. The chronology of isolations reveals the circulation of both ALV-J and ALV-A during this period. On 16 occasions single isolations were found to contain both ALV-A and ALV-J. This is the first report of dual infections with two subgroups of ALV occurring in the same chicken. The effectiveness of ALV-J eradication measures is indicated by the absence of any ALV-J isolations in late 2003. ALV-A however, continued to be isolated from the broiler population. The detection of dual infections, as well as the ongoing occurrence of ALV-A in meat-type birds, is discussed in the context of ongoing potential for recombinations and the associated threat for the emergence of avian leukosis virus with changes in host range and pathogenicity.     

广州地区禽H9N2亚型流感病毒的发现及感染人调查

目的 了解广州地区禽流感病毒在家禽中的流行及感染人的情况，防止香港H5N1禽流感在广州地区流行。方法 对广州地区的主要鸡场和农贸市场的家禽和密切接触家禽的职业人群进行病原学和血清学的检测。病毒分离同时采用MDCK细胞和鸡胚双腔接种法；采用微量血凝抑制半致敏法进行血清学检测。结果 从54份鸡咽拭液中分离到1株H9N2亚型流感病毒；鸡及职业人群血对分离的H9N2毒株的血抑抗体阳性率分别为12．8％和15．1％。结论 广州地区鸡群中有H9N2，亚型流感病毒存在，禽H9N2亚型流感病毒能感染人。     

Highly pathogenic avian influenza viruses with low virulence for chickens in in vivo tests.

Four avian influenza viruses have been recognized that have genetic coding for highly pathogenic avian influenza viruses, but do not show virulence for chickens. The two different mechanisms that prevent this potential being expressed have been determined for A/chicken/Pennsylvania/1/83 (H5N2) and A/goose/Guandong/2/96 (H5N1), but neither of these applies to A/turkey/England/87-92BFC/91 (H5N1) or A/chicken/Texas/298313/04 (H5N2).     

Age related resistance to avian leukosis virus. III. Infectious virus, neutralising antibody and tumours in chickens inoculated at various ages

Viraemia and neutralising antibodies were determined in chickens of six age-groups following inoculation with leukosis virus of subgroups A and B at the age of 1 day, and 2, 4, 6, 8 and 10 weeks respectively. The birds were kept in a filtered air positive pressure (FAPP) house. A seventh age-group, accommodated in a separate FAPP-house, was used as an untreated control. Serum samples, received at biweekly intervals between 1-17 weeks post-inoculation, from birds of the groups inoculated at 4, 6, 8 and 10 weeks of age, showed at 1 week post-inoculation a transient viraemia followed by neutralising antibodies at the later sampling times. Neutralising antibody to subgroup A virus was detected in nearly all birds tested; this was not so for antibody to subgroup B. In all four groups the average titre of the former antibody was higher than that of the latter. Midway through the laying period birds of each group inoculated with leukosis virus, and some of the uninoculated controls, were challenged by infection with either subgroup A or B virus. At termination of the experiment survivors from each group were tested for the presence of leukosis virus. The virus recovery was performed with plasma samples, white blood cell preparations and explant cultures of various organs. The plasma samples were all negative; the great majority of blood cell specimens received from birds inoculated early with leukosis virus were positive, whereas the majority of the preparations from the birds inoculated later remained negative. The organ explants from the two youngest age groups were mostly leukosis virus-positive, from the birds inoculated at 4 weeks of age the spleen and kidney explants contained leukosis virus whereas in the groups inoculated at 6, 8 and 10 weeks of age only the spleen explants of birds challenged with subgroup A virus In a subsidiary experiment, started 4 months after the challenge infection, four birds from each group (two challenged with leukosis virus of subgroup A and two with subgroup B) were accommodated in isolators. The birds were challenged again, this time with Rous sarcoma virus (RSV) of the homologous subgroup used for the previous challenge. The tests for virus just prior to the challenge showed leukosis virus only in the white blood cell preparations from the birds in the three youngest age groups; the birds from the older groups were virus-negative. The serological tests after challenge showed neutralising antibodies to both subgroups in birds of nearly all groups. Tumour formation at the site of injection was mainly observed in the chickens challenged with RSV of subgroup B. The virological and serological results as well as the tumour response show that the immune system of birds between 0-4 weeks of age is insufficiently developed to cope with a controlled exposure with leukosis virus, whereas in birds of 4-10 weeks of age an adequate immunological response has developed. The significance of the presence of leukosis virus in sera, plasma, white blood cell preparations and organ explant cultures is mentioned. In programmes for the control of lymphoid leukosis in reproductive stock the use of information on virus and neutralising antibodies is recommended.     

Analyses of the spleen proteome of chickens infected with Marek's disease virus

Marek's disease virus (MDV), which causes a lymphoproliferative disease in chickens, is known to induce host responses leading to protection against disease in a manner dependent on genetic background of chickens and virulence of the virus. In the present study, changes in the spleen proteome at 7, 14 and 21 days post-infection in response to MDV infection were studied using two-dimensional polyacrylamide gel electrophoresis. Differentially expressed proteins were identified using one-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (1D LC ESI MS/MS). Comparative analysis of multiple gels revealed that the majority of changes had occurred at early stages of the disease. In total, 61 protein spots representing 48 host proteins were detected as either quantitatively (false discovery rate (FDR) ≤ 0.05 and fold change ≥ 2) or qualitatively differentially expressed at least once during different sampling points. Overall, the proteins identified in the present study are involved in a variety of cellular processes such as the antigen processing and presentation, ubiquitin–proteasome protein degradation (UPP), formation of the cytoskeleton, cellular metabolism, signal transduction and regulation of translation. Notably, early stages of the disease were characterized by changes in the UPP, and antigen presentation. Furthermore, changes indicative of active cell proliferation as well as apoptosis together with significant changes in cytoskeletal components that were observed throughout the experimental period suggested the complexity of the pathogenesis. The present findings provide a basis for further studies aimed at elucidation of the role of these proteins in MDV interactions with its host.     

Wounding acts as a tumor promoter in chickens inoculated with avian sarcoma virus 17.

Avian sarcoma virus 17 (ASV17) is an acutely transforming retrovirus which carries the oncogene v-jun. The virus induces fibrosarcomas in chickens at the site of inoculation. Here we describe wound-related tumor formation in 77% of chickens inoculated with ASV17 in one wing and wounded by metal clip insertion in the opposite wing. Tumors from both wound-related and inoculation-related sites were histologically diagnosed as fibrosarcomas. Tissues cultured from both tumor sites produced infectious virus in culture and expressed high levels of the v-Jun oncoprotein detectable by immunofluorescent staining. By varying the time of wounding relative to virus inoculation we defined the early stages of wound healing (2-7 days postinoculation) as favoring wound-related tumor formation. Three other acutely transforming retroviruses containing oncogenes coding for nonreceptor protein tyrosine kinases (v-src, v-yes, and v-fps), inoculated in the same manner, induced wound-related tumors in all cases. We conclude that in chickens, ASV17 collaborates with wound healing to promote tumorigenesis by a process which may relate either to a biochemical function of Jun or to a more general, shared characteristic of transforming retroviruses.     

Pathogenicity of avian nephritis virus in chicks previously infected with infectious bursal disease virus

Pathogenicity of avian nephritis virus (ANV) was studied in 7‐day‐old chicks previously infected with infectious bursal disease virus (IBDV) at 1 day of age. Three IBDV+ANV infected chicks were found dead with no apparent clinical signs within 10 days of inoculation with ANV and they had marked urate deposits throughout the body. All of the surviving IBDV+ANV infected chicks had reduced body weight gain, unlike the ANV‐only infected, IBDV‐only infected and non‐infected control chicks.

In a chronological study, all of the ANV‐infected chicks revealed interstitial nephritis accompanied by tubular cell degeneration and lymphoid cell infiltration. Avian nephritis virus antigen was detected with equal sensitivity by fluorescent antibody and immunoperoxidase techniques and was widely distributed in the degenerated epithelial cells. The concentration of serum uric acid in IBDV+ANV infected chicks was higher than that in ANV‐only infected chicks. The antibody against ANV in ANV‐only infected chicks was first detected 8 dpi and its titre increased gradually. In contrast, the antibody in the IBDV+ANV infected chicks was very low and was detected in only three out of seven chicks 16 dpi; the titre was 1:4 in one chick and 1:8 in the other 2 chicks. Coincident with serological results, the number of lgG‐ and IgM‐containing cells in the kidneys and spleen were less in IBDV + ANV‐infected chicks than in ANV‐only infected chicks. These finding suggested that IBDV infection in chicks might influence the immunological response and susceptibility to subsequent ANV infection.     

Leukocyte subpopulations in kidney and trachea of chickens infected with infectious bronchitis virus

Using immunohistochemical methods, we studied the nephropathogenicity of the infectious bronchitis virus (IBV)-strain V1648- and the leukocyte phenorypes in the pathological lesions in the kidneys and the trachea formed after inoculation with this virus strain. One-day-old WLA chickens were intravenously inoculated, and after 5, 7 and 11 days their kidneys, trachea and lungs were removed. Monoclonal antibodies were used to detect viral antigen, and lymphoid and non-lymphoid cell populations. In serial sections, the detection of the viral antigen was correlated to the phenotypes of the cells. At days 5 and 7 after inoculation, viral antigen was detected in the epithelium and the interstitium of the kidney tubuli and in the epithelium of the trachea. The infiltrated cells in these tissues were mainly of the T cell phenotype. The cellular immune reaction was correlated with the detection of viral antigen.     

Apoptosis and CD8-down-regulation in the thymus of chickens infected with Marek's disease virus

Summary Marek's disease virus (MDV)-infected chickens show thymic atrophy during the acute phase of infection. We examined whether the thymic atrophy by MDV-infection was mediated by apoptosis. Apoptosis-specific DNA ladderings were clearly observed in thymocytes one week after MDV-infection. Histological and flow cytometry studies revealed that immature CD4⁺CD8⁺ thymocytes underwent apototic cell death. In addition, the expression level of CD8 molecules on both CD4^–CD8⁺ and CD4⁺CD8⁺ thymocyte populations was down-regulated in the infected chickens. These thymic changes might be involved in the pathogenesis of Marek's disease.     

Suppression and enhancement of mitogen response in chickens infected with Marek''s disease virus and the herpesvirus of turkeys.

The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 7(2) chickens and in HVT-inoculated line 7(2) chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response.