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.     

Characterization of suppressor T cells for antibody production by chicken spleen cells. II. Comparison of CT8+ cells from concanavalin A-injected normal and bursa cell-injected agammaglobulinaemic chickens.

The phenotypes of two different types of suppressor T cells in the chicken, both capable of inhibiting secondary antibody responses in vitro, were determined. The first of these, induced by injection of concanavalin A (Con A) into normal chickens, was CT8+, TcR2+ (alpha beta), CT4-, TcR1- (gamma delta). These cells appeared to exhibit histamine type 2 (H2) receptors, as they adhered to cimetidine-BSA-coated dishes. Moreover, cimetidine added to the medium at 2 x 10(-4) M completely prevented the suppression induced by these suppressor cells. The second type of 'suppressor' T-cell studied, induced in agammaglobulinaemic (A gamma) chickens by injection of bursa cells, exhibited the same phenotype, but was insensitive to cimetidine and did not adhere to cimetidine-BSA-coated dishes, indicating heterogeneity with respect to H2 receptor expression on CT8+ chicken T cells with suppressor activity. The results also showed that a relatively larger proportion of CT8+ than of CT4+ cells adhered to cimetidine-BSA-coated dishes and thus appeared to be H2 receptor positive. TcR1 (gamma delta) cells did not contribute significantly to the antigen non-specific suppressor effects examined in this study.     

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.     

Comparison of serological and virological findings from subgroup J avian leukosis virus-infected neoplastic and non-neoplastic flocks in Israel

Blood samples from nine broiler breeder flocks comprising five flocks clinically affected with myeloid leukosis tumours (ML+) and four tumour-free flocks from the same commercial background (ML?) were compared for avian leukosis virus subgroup J (ALV-J) serum antibodies by enzyme-linked immunosorbent assay (ELISA), for antigenemia (group-specific antigen) by antigen-trapping ELISA and for viremia. Group-specific antigen was detected in the sera of 58.1% of ML+ birds and 46.4% of the ML? birds (P=not significant), while 45.5% of ML+ birds and 24.1% of the ML? birds had ALV-J antibodies (P=0.065). In inoculated cell culture, 64.1% of the ML+ sera were viremic compared with 16.7% of the ML? sera (P=0.001). Similar significant differences were found between the two groups of flocks when ALV-J viremia was detected by immunofluorescence using a monoclonal env antibody (P=0.004), and for proviral DNA by polymerase chain reaction using two different sets of env-gene primers, H5?H7 (P=0.001) and R5–F5 (P=0.001). Using the primer pair R5–F5 the product size was approximately 1 kbp, while some heterogeneity in size among isolates was discernable. Our results indicate that a combination of diagnostic tests should be adopted in routine examination of tumour material in order to rule out false-negative findings.     

Correlation between neutralizing antibody titre and protection from tenosynovitis in avian reovirus infections

The relationship between neutralizing antibody titre and protection from tenosynovitis caused by avian reovirus infection was examined using actively or passively immunized chickens. Actively immunized groups which developed geometric mean neutralizing antibody titres (GMT) of 1:238 or higher exhibited good protection (more than 71%) against challenge with avian reovirus strain 58-132 via the footpad. On the other hand, 87% of passively immunized chicks, which received chicken serum hyperimmune to avian reovirus in the yolk sac and possessed GMT of 1:100 upon hatching, were protected against oral challenge. Given the age-related resistance to reoviral tenosynovitis and the half-life of maternal antibody, chicks should ideally have 1:1600 or higher maternal antibody titre at the time of hatching to be protected against oral infection until 3 weeks of age.     

Distribution of viral antigen gp85 and provirus in various tissues from commercial meat-type and experimental White Leghorn Line 0 chickens with different subgroup J avian leukosis virus infection profiles.

Immunohistochemistry and polymerase chain reaction (PCR) were used to test for the presence of avian leukosis virus (ALV) J viral antigen gp85 and proviral DNA, respectively, in various tissues (adrenal gland, bone marrow, gonad, heart, kidney, liver, lung, pancreas, proventriculus, sciatic nerve, spleen, and thymus). Tissues were collected from 32-week-old commercial meat-type and Avian Disease and Oncology Laboratory experimental White Leghorn Line 0 chickens with the following different infection profiles: tV + A-, included in ovo-tolerized viraemic chickens with no neutralizing antibodies (NAbs) on any sampling; ntV + A-, included chickens that were viraemic and NAb-negative at the time of termination at 32 weeks post hatch, but had NAbs on up to two occasions; V+ A+, included chickens that were viraemic and NAb-positive at the time of termination at 32 weeks post hatch, and had NAbs on more than two occasions; V - A+, included chickens that were negative for viraemia and NAb-positive at the time of termination at 32 weeks post hatch, and had antibody on more than two occasions; V - A-, included chickens that were never exposed to ALV J virus. There was a direct correlation between viraemia and tissue distribution of gp85, regardless of the NAb status and strain of chickens, as expression of ALV J gp85 was noted in only viraemic chickens (tV + A-, ntV + A-, V+ A+), but not in non-viraemic seroconverted chickens (V - A+). Of the four oligonucleotide primers pairs used in PCR to identify ALV J provirus, only one primer set termed H5/H7 was useful in demonstrating ALV J proviral DNA in the majority of the tissues tested from non-viraemic, antibody-positive chickens (V - A+). The results suggest that PCR using primer pair H5/H7 is more sensitive than immunohistochemistry in identifying ALV J in chickens that have been exposed to virus, but are not actively viraemic.     

Epidemiological and pathological studies of subgroup J avian leukosis virus infections in Chinese local "yellow" chickens.

Epidemiological, pathological and molecular studies indicate that subgroup J avian leukosis virus (ALV-J) infections are widely spread in "yellow chickens" of local breeds in China. ALV-J induced tumour mortality and the serological conversion rates to ALV-J were very high in some breeder flocks. Typical myelocytomatosis was demonstrated not only in livers, spleens, kidneys, and sternums, as in white meat-type chickens, but also in thymuses and the bursa of Fabricius. Especially, severe myeloid cell infiltration was found throughout the whole enlarged thymuses of some birds. ALV-J was isolated at high positive rates from both liver tumour samples and embryos collected from breeder flocks with tumours. At the same time, reticuloendotheliosis virus was also co-isolated with ALV-J in some tumour samples and embryos. Sequence analysis of env genes demonstrated that the gp85 and gp37 among six ALV-J isolates from "yellow chickens" of Chinese local breeds varied as highly as among ALV-J strains isolated from white meat-type chickens worldwide. But strain GD0512 isolated in 2005 from a "yellow chicken" farm in southern China had high identity of 95.1% for gp85 or 99.5% for gp37 to strain HN0001 isolated in 2000 from a white meat-type breeder farm in northern China, a much higher identity than to other yellow chicken and white chicken strains. This is the first report of the isolation and identification of ALV-J from yellow chickens of Chinese local breeds and also the first report of vertical co-infection of ALV-J and reticuloendotheliosis virus. The significance of co-infection of ALV-J and reticuloendotheliosis virus in pathogenesis is discussed.     

Synergistic pathogenic effects of co-infection of subgroup J avian leukosis virus and reticuloendotheliosis virus in broiler chickens

To study interactions between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) and the effects of co-infection on pathogenicity of these viruses, 1-day-old broiler chicks were infected with ALV-J, REV or both ALV-J and REV. The results indicated that co-infection of ALV-J and REV induced more growth retardation and higher mortality rate than ALV-J or REV single infection (P < 0.05). Chickens co-infected with ALV-J and REV also showed more severe immunosuppression than those with a single infection. This was manifested by significantly lower bursa of Fabricius and thymus to body weight ratios and lower antibody responses to Newcastle disease virus and H9-avian influenza virus (P < 0.05). Perihepatitis and pericarditis related to severe infection with Escherichia coli were found in many of the dead birds. E. coli was isolated from each case of perihepatitis and pericarditis. The mortality associated with E. coli infection in the co-infection groups was significantly higher than in the other groups (P < 0.05). Among 516 tested E. coli isolates from 58 dead birds, 12 serotypes of the O-antigen were identified in two experiments. Different serotypes of E. coli strains were even isolated from the same organ of the same bird. Diversification of O-serotypes suggested that perihepatitis and pericarditis associated with E. coli infection was the most frequent secondary infection following the immunosuppression induced by ALV-J and REV co-infection. These results suggested that the co-infection of ALV-J and REV caused more serious synergistic pathogenic effects, growth retardation, immunosuppression, and secondary E. coli infection in broiler chickens.     

Immunogenicity of E. coli-expressed sigma3 protein of avian reovirus in chickens.

An E. coli-expressed fusion protein was used to study the immunogenicity of avian reovirus sigma3 protein in chickens. The protein induced the production of specific antibodies detectable by immunofluorescence, ELISA and immunoblot. The antibody titre was low as determined by ELISA and was negative by virus neutralization test. However, when tested in passive immunization studies, the chicken antiserum specific to this protein and control antiserum from chickens vaccinated with avian reovirus SI 133 strain showed some protection against virus-induced mortality in 1-day-old chicks. The results thus confirm the importance of humoral immunity against avian reovirus infection and indicate that sigma3 protein may play a role in the induction of protective antibodies.     

Viral spread in the presence of neutralizing antibody: mechanisms of persistence in foamy virus infection.

Several viruses were categorized on the basis of their ability to spread from cell to contiguous cell and form plaques in the presence of antiviral antibody. Herpes simplex virus, cytomegalovirus, and vaccinia, measles, and foamy viruses were able to spread in the presence of neutralizing antibody, whereas coxsackievirus, encephalomyocarditis virus, vesicular stomatitis virus, mumps virus, and simian virus 5 failed to spread. A detailed study of one of these virus groups (simian foamy viruses) suggested that the ability of these viruses to spread from cell to cell in the presence of antiviral antibody, the failure of antiviral antibody and complement to lyse infected cells, and the poor induction and relative resistance of these viruses to the antiviral action of interferon contribute to the persistent nature of this infection.     

Measurement of endogenous leukosis virus nucleotide sequences in the DNA of normal avian embryos by RNA-DNA hybridization

Rous associated virus type O (RAV-O) is a subgroup E avian leukosis virus selected as an example of “endogenous” leukosis viruses which are associated with normal avian embryo cells. DNA sequences complementary to RAV-O 60–70S RNA genome were detected and quantitated in DNA from normal avian embryos by the technique of RNA-DNA hybridization with DNA excess. DNA from “leukosis free” white leghorn chicken embryos, as well as from line 7 chicken embryos, contained sequences related to at least 70%, if not all, of RAV-O RNA sequences. A tentative estimate of gene frequency is low, in the range of two to four copies of RAV-O per cell genome. Some difficulties in precisely calculating gene frequency by this method are discussed. Embryos of ring-necked pheasant contain only about 10% of the RAV-O genome. These findings are not affected by the presence or absence of viral group specific (gs) antigen in chicken or pheasant embryos. Embryos of Japanese quail, a species which lacks a known leukosis virus and does not synthesize avian gs antigen, contained DNA sequences specifically related to no more than 4% of the RAV-O genome. The data document the specificity and sensitivity of this hybridization technique and provide additional firm support for the proposition that normal avian cells carry endogenous avian leukosis virus in the form of DNA proviruses.     

Rapid quantitation of cytomegalovirus and assay of neutralizing antibody by using monoclonal antibody to the major immediate-early viral protein.

An overnight assay, based on staining cytomegalovirus-infected cells with monoclonal antibody to the 72,000-molecular-weight major immediate-early viral protein, was compared with a conventional 14-day plaque assay for quantitation of cell-free stocks of cytomegalovirus laboratory strain AD-169 and 20 other clinical strains. Viral titers were quantitatively similar when determined by either method, but centrifugation of monolayers during inoculation enhanced viral infectivity an average of 4.1-fold. When used for scoring neutralizing antibody assays, monoclonal antibody staining yielded titers within one dilution of 14-day plaque-reduction assays in 54 of 56 titrations. Of 21 cytomegalovirus strains, 2 were not recognized by the monoclonal antibody used. Assay with monoclonal antibody offers a rapid and accurate alternative to plaque assays for quantitation or neutralization of cytomegalovirus.     

Antibody for detecting p53 protein by immunohistochemistry in normal tissues.

AIMS--To establish whether PAb248 recognises human p53 as well as murine p53 and if so, to determine its distribution in normal tissues. METHODS--The ability of PAb248 to recognise human p53 was established by analysis of the human osteosarcoma derived Saos-2 cell line, which lacks the p53 gene, before and after transfection with p53 cDNA, using western blotting and immunoprecipitation. Immunostaining on normal tissues and cell lines was carried out using an immunoperoxidase technique. The two anti-p53 antibodies PAb 240 and DO-7 were used as controls. RESULTS--The anti-p53 PAb248 monoclonal antibody stained the Saos-2 cell line after, but not before, transfection with p53 cDNA. Both western blots and immunoprecipitations performed with this antibody revealed a 53,000 molecular weight band. With immunostaining, this antibody detects p53 protein in most lymphoid and human epithelial cells in a cytoplasmic-perinuclear localisation that has not been described before. In the same tissues nuclear staining could be seen in a few scattered cells using the PAb240 antibody. The topographical distribution of wild type p53 was not related to proliferating areas but, rather, to short-lived populations of cells. CONCLUSIONS--Immunostaining of wild type p53 is demonstrable not only in its nuclear form using antibody PAb240 but also in it common cytoplasmic-perinuclear localisation in normal tissues using the PAb248 monoclonal antibody. This opens up new possibilities for its study in both physiological and pathological conditions.     

Reduction of horizontal transmission of avian leukosis virus subgroup J in broiler breeder chickens hatched and reared in small groups

Transmission of avian leukosis virus, subgroup J (ALV-J), from donor chickens inoculated as embryos to simulate congenital infection to uninfected hatchmates was studied in two strains of commercial broiler breeder chickens. Chicks of two commercial lines free of ALV-J became infected when hatched (1/2 lots positive) or reared (8/8 lots positive) in direct physical contact with ALV-J-infected donors. Infection also occurred when chicks were exposed in the hatchery to ALV-J-infected donors by cloacal swab transfer (2/2 lots positive), needle transfer during subcutaneous inoculation (2/2 lots positive), or ingestion of infected meconium (2/2 lots positive). However, transmission was delayed or prevented by wire partitions in the hatcher and rearing of small groups in cubicles, and rarely (1/10 lots positive) resulted from short-term direct or indirect contact. In a simulated field test, a flock of 503 broiler breeder chickens with an initial embryo infection rate of 4.6% was hatched and reared as 48 small groups to 4 weeks of age. Groups were tested at hatch and at 3 weeks, and 14 infected groups were eliminated. This flock tested negative for ALV-J infection from 4 to 32 weeks and did not transmit infection to progeny or develop tumours. A control group of 377 chickens with a similar initial infection rate was hatched and reared as a single group. This control flock transmitted virus to 5.7% of its progeny and about 5% of the hens developed tumours. The small-group hatching and rearing practices employed in these studies allowed for the accurate identification and removal of groups containing chickens infected prior to hatching and prevented horizontal transmission of ALV-J between uninfected and infected groups for at least 4 weeks. More importantly, application of these procedures successfully eradicated ALV-J in a single generation under laboratory conditions. This suggests that similar procedures could be a valuable adjunct to virus eradication programmes in the field.     

HHF35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive, and neoplastic human tissues.

Monoclonal antibody HHF35 has previously been characterized biochemically as recognizing isotypes of actin (alpha and gamma) which are specific to muscle cells. In this study, the authors have investigated the normal and pathologic tissue distribution of HHF35-positive cells using the avidin-biotin immunoperoxidase method on methacarn-fixed, paraffin-embedded sections of human tissue. In addition to muscle tissues (smooth, skeletal, and cardiac) the antibody localizes to myoepithelium, as well as most of the capsular cells of several parenchymal organs, including liver, kidney, and spleen, with extension of the latter cells into the splenic trabeculaes. In pathologic tissues, the antibody localizes to cells, identified by some investigators as "myofibroblasts," in the stroma of certain tumors, within hyperplastic fibrous tissue responses ("fibromatoses") such as Dupuytren's contracture, and within fibrotic lung tissue. HHF35 also localizes to cells that proliferate within the intima in lesions of atherosclerosis and to a unique population of reactive mesothelial and submesothelial cells. Among tumors, it is positive only on leiomyomas, leiomyosarcomas, and rhabdomyosarcomas, and negative on all nonmuscle sarcomas. This antibody thus shows great potential utility as a diagnostic reagent in various pathologic conditions, most especially in the diagnosis of tumors of muscle origin.     

Effect of an in ovo infection with a Dutch avian leukosis virus subgroup J isolate on the growth and immunological performance of SPF broiler chickens

The effect of an in ovo infection with a Dutch isolate of avian leukosis virus subgroup J (ALV-J) on the growth of specific pathogen free (SPF) broiler chickens was analysed. During this study, possible immune suppressive effects of ALV-J were assessed by measuring delayed-type hypersensitivity with keyhole limpet haemocyanin (KLH), natural killer (NK) cell activity, the production of radicals of nitric oxide (NO) by macrophages, humoral immune response against Newcastle and infectious bursal disease vaccine viruses, and automated total and differential leukocyte counts. In an attempt to elucidate the underlying causal mechanisms of the induced growth retardation, 3,3',5-triiodothyronine (T3) concentrations in serum were measured. Four experiments were conducted. In experiment 1, ALV-J-injected birds were compared with ALV subgroup A (ALV-A)-injected and negative control chickens. In experiment 2, ALV-J-injected birds were only compared with negative controls. Finally, in experiments 3a and 3b, ALV-J-injected chickens were compared with negative controls and a group of chickens in which only 10% of birds had been injected with ALV-J. Birds were injected in ovo at day 7 of incubation with 10 4 median tissue culture infectious dose (TCID 50 ) ALV-J or ALV-A, except in experiment 3a where 10 2 TCID 50 ALV-J was injected. Significant growth suppression was found in all 100% of ALV-J-infected groups. The average growth retardation of ALV-J-infected birds compared with negative controls at 6 weeks of age was approximately 8, 11, 2.5 and 6% for the four successive experiments performed. The delayed-type hypersensitivity test against KLH of ALV-J-infected birds showed a tendency towards lower wattle thickness; however, the difference with controls was not significant ( P > 0.05). The same was true for NK cell activity and NO production by macrophages, although the difference was not significant. The total and differential leukocyte counts performed on blood samples from birds at 3, 4 and 6 weeks of age as well as the humoral immune response against Newcastle and infectious bursal disease vaccine viruses did not show significant differences between treatment groups either. Only the number of basophils were significantly higher ( P = 0.02) in ALV-J-infected birds at 3 weeks of age. No significant lower T 3 levels were found in ALV-J-infected birds in weeks 2 and 3 (experiment 2) and weeks 3 and 5 (experiment 3b); however, at 4 weeks (experiment 2) and 6 weeks (experiment 3b) of age, T 3 levels were significantly lower suggesting mild hypothyroidism in these broilers. In conclusion, the present experiments show the occurrence of significant growth retardation in SPF broilers after an ALV-J in ovo infection. The various studies performed to assess the immune competence of ALV-J-infected chickens did not show significant differences in immune responsiveness. The assays on cellular immunity showed a tendency to a lower response in ALV-J-infected birds, but these differences were not statistically significant.     

Detection of avian leukosis virus subgroup J in chicken flocks from Malaysia and their molecular characterization.

Previously we have shown that avian leukosis virus subgroup J (ALV-J) might be present in chicken flocks from Malaysia based on serological study and also on detection of tissue samples with myelocytic infiltration. In this study, the polymerase chain reaction was used to detect ALV-J sequences from archived frozen samples. Out of 21 tissue samples examined, 16 samples were positive for proviral DNA and four samples for ALV-J RNA. However, only nine samples were found positive for myelocytic infiltration. A total of 465 base pairs equivalent to positions 5305 to 5769 of HPRS-103 from each of the viral RNA positive samples were characterized. Sequence analysis indicated that the samples showed high identity (95.9 to 98.2%) and were close to HPRS-103 with identities between 97.4 and 99.3%. This study indicates that ALV-J-specific sequences can be detected by polymerase chain reaction from frozen tissue samples with and without myelocytic infiltration.