Clonal anergy of CD117+chB6+ B cell progenitors induced by avian leukosis virus subgroup J is associated with immunological tolerance.

BACKGROUND: The pathogenesis of immunological tolerance caused by avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, is largely unknown. RESULTS: In this study, the development, differentiation, and immunological capability of B cells and their progenitors infected with ALV-J were studied both morphologically and functionally by using a model of ALV-J congenital infection. Compared with posthatch infection, congenital infection of ALV-J resulted in severe immunological tolerance, which was identified as the absence of detectable specific antivirus antibodies. In congenitally infected chickens, immune organs, particularly the bursa of Fabricius, were poorly developed. Moreover, IgM-and IgG-positive cells and total immunoglobulin levels were significantly decreased in these chickens. Large numbers of bursa follicles with no differentiation into cortex and medulla indicated that B cell development was arrested at the early stage. Flow cytometry analysis further confirmed that ALV-J blocked the differentiation of CD117+chB6+ B cell progenitors in the bursa of Fabricius. Furthermore, both the humoral immunity and the immunological capability of B cells and their progenitors were significantly suppressed, as assessed by (a) the antibody titres against sheep red blood cells and the Marek's disease virus attenuated serotype 1 vaccine; (b) the proliferative response of B cells against thymus-independent antigen lipopolysaccharide (LPS) in the spleen germinal centres; and (c) the capacities for proliferation, differentiation and immunoglobulin gene class-switch recombination of B cell progenitors in response to LPS and interleukin-4(IL-4) in vitro. CONCLUSIONS: These findings suggested that the anergy of B cells in congenitally infected chickens is caused by the developmental arrest and dysfunction of B cell progenitors, which is an important factor for the immunological tolerance induced by ALV-J.

Heterophil function and resistance to staphylococcal challenge in broiler chickens naturally infected with avian leukosis virus subgroup J.

Avian leukosis virus subgroup J has a high tropism for myeloid lineage cells and frequently induces neoplastic transformation of myelocytes. The impact of congenital avian leukosis virus subgroup J infection on the function of circulating heterophils and susceptibility to staphylococcal infection was investigated. Six-week-old broiler chickens negative for exogenous avian leukosis viruses or congenitally infected with avian leukosis virus subgroup J were inoculated intravenously with 10(6) colony-forming units of Staphylococcus aureus, and pre- and postinoculation heterophil function was assessed. All chickens developed a leukocytosis with heterophilia after inoculation, but total leukocyte and heterophil counts were significantly higher in leukosis-negative chickens than in virus-infected chickens. Tenosynovitis was more severe in leukosis-negative chickens, and 2/10 (20%) of the virus-infected chickens had no histologic evidence of tenosynovitis. Osteomyelitis in the tibiotarsus or tarsometatarsus developed in 5/10 (50%) of the chickens in each group. S. aureus was recovered from the hock joint of 6/10 (60%) of the chickens in each group. Heterophils from all chickens exhibited similar phagocytic ability pre- and postinoculation. Heterophils from virus-infected chickens exhibited less bactericidal ability preinoculation than did heterophils from leukosis-negative chickens. However, postinoculation bactericidal ability was similar in both groups. Avian leukosis virus subgroup J provirus was present in heterophils isolated from congenitally infected chickens. Heterophils isolated from broiler chickens congenitally infected with avian leukosis virus subgroup J exhibit no significant functional deficits, and infected and uninfected chickens exhibit similar susceptibility to staphylococcal infection.

A transgene, alv6, that expresses the envelope of subgroup A avian leukosis virus reduces the rate of congenital transmission of a field strain of avian leukosis virus.

A major mode of transmission of avian leukosis virus (ALV) is from a dam that is viremic with and immunologically tolerant to ALV, through the egg to the progeny. The authors have produced a line of chickens transgenic for a defective ALV provirus that expresses envelope glycoprotein, but not infectious virus, and is very resistant to infection with Subgroup A ALV. In the present experiment the authors sought to prevent or reduce congenital transmission by mating viremic-tolerant hens to males carrying the inserted provirus, thus introducing a gene for resistance into the progeny. Mature viremic females were mated with males hemizygous for the transgene to produce over 80 progeny each with and without the transgene. The chicks were hatched and maintained for 36 wk and observed for viremia, antibody, and the incidence of bursal lymphomas. Over 90% of the transgene-negative controls remained viremic through 36 wk of age and 51% developed bursal lymphomas. In contrast, 27% of the transgene-positive birds remained viremic and 18% died with bursal lymphomas. Thus, expression of Subgroup A envelope protein in the developing embryo reduced but did not eliminate congenital infection.

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.

Influence of maternal antibody on avian leukosis virus infection in White Leghorn chickens harboring endogenous virus-21 (EV21).

Slow-feathering (SF) white leghorn dams harboring the endogenous viral gene ev21, which encodes for complete endogenous virus-21 (EV21), and rapid-feathering (RF) dams lacking EV21 were immunized with a live field strain of avian leukosis virus (ALV) subgroup A. One group of SF dams and one group of RF dams were not immunized and were maintained to produce chicks lacking maternal ALV antibody. When the SF dams were crossed with line 15B1 males, the resulting male progeny were SF, EV21-positive, and the females were RF, lacking EV21 or congenitally infected with EV21. EV21-positive and -negative progeny of immunized and unimmunized SF and RF dams were exposed to ALV at hatching. Viremia, antibody development, cloacal shedding, and tumors in chickens lacking EV21 were compared with those in chickens with EV21. Congenital transmission of EV21 from SF dams to RF female chicks was significantly higher in immunized dams than in unimmunized dams. Maternal ALV antibody delayed infection with ALV and reduced viremia and cloacal shedding of virus in progeny. The effect of maternal antibody on ALV infection was much more pronounced in progeny lacking EV21 than in progeny harboring EV21. The data suggest that the development of ALV infection and tumors may be influenced by status of infection with EV21 and by the immune status of dams.

Congenital transmission of avian leukosis virus in the absence of detectable shedding of group specific antigen.

Congenital transmission of avian leukosis virus (ALV) in the absence of detectable amounts of group specific (gs) antigen in egg albumen was found to occur in one commercial and one specific pathogen-free (SPF) flock. The prevalence of congenitally transmitting hens which did not excrete gs antigen was particularly high in a commercial flock where 26/27 hens transmitted ALV. Some of the ALV-transmitting hens in the commercial flock had virus in vaginal swabs thus enabling infection to be detected. The reasons for such a high proportion of congenitally transmitting hens which did not shed detectable amounts of gs antigen in the commercial flock was not determined. In the SPF flock, 2/15 hens congenitally transmitted ALV although virus could not be detected in vaginal swabs, whole blood or egg albumen and antibodies to subgroups A or B were not present. This form of ALV-infection persisted in two successive generations. These results indicate the necessity of testing for infectious ALV in embryos, in order to ascertain that a flock is genuinely free of ALV.

Genetic cellular resistance to subgroup E avian leukosis virus in slow-feathering dams reduces congenital transmission of an endogenous retrovirus encoded at locus ev21.

The gene ev21, which encodes an infectious endogenous subgroup E avian leukosis virus (ALV-E), designated EV21, is closely linked to the sex-linked, slow-feathering (SF) gene K. To address the relationship between congenital transmission of EV21 and host susceptibility to ALV-E infection, SF roosters that were heterozygous for the dominant gene for susceptibility to ALV-E were mated with ev-negative rapid-feathering (RF), subgroup E-resistant dams to produce SF and RF progeny. The SF female progeny that were heterozygous ALV-E susceptible were viremic at hatch and, when mature, consistently shed the ALV group-specific antigen, p27, in the egg albumen they produced. In contrast, SF females that were homozygous ALV-E resistant were neither viremic at hatch nor shedders of ALV p27. Infectious ALV-E assays of progeny from susceptible and resistant SF dams confirmed that maternal cellular resistance to ALV-E limited congenital transmission of EV21. It is proposed that, in progeny, immunologic tolerance toward exogenous ALV infection due to congenital transmission of antigenically related endogenous viruses may be abrogated by selection for cellular resistance to ALV-E in SF lines used as female parents of feather-sexed crosses.

Influence of congenital transmission of endogenous virus-21 on the immune response to avian leukosis virus infection and the incidence of tumors in chickens.

After contact exposure to Strain RPL-40 avian leukosis virus-infected hatchmates, a dilatory neutralizing antibody response and prolonged RPL-40 viremia was found among most pullets that were congenitally infected with endogenous virus 21 (EV21). Conversely, most of the hatchmates that were not congenitally infected seroconverted within 10 wk after exposure to Strain RPL-40 virus. Compared with noncongenitally infected hatchmates, EV-21 infection-induced tolerance to pathogenic avian leukosis viruses was reflected in a significantly higher incidence of lymphomas in congenitally infected hens. The rate of seroconversion and the incidence of RPL-40 virus-induced tumors among noncongenitally infected daughters from slow-feathering dams homozygous resistant to EV were similar to those found among daughters of rapid-feathering dams that lacked genetic locus ev21. Results suggest that selection for resistance to EV may eliminate tolerance toward oncogenic field strains of avian leukosis viruses and may improve the performance of progeny from a feather-sex cross.

New findings on the congenital transmission of avian leukosis viruses.

RAV-0, an endogenous avian leukosis virus, does not undergo congenital transmission in infected K28 chickens. In contrast, avian leukosis viruses of exogenous origin undergo highly efficient congenital transmission. The relative abilities of endogenous and exogenous viruses to undergo congenital transmission appear to be determined by the p27 capsid proteins of these viruses.

Studies of avian leukosis virus infection in chickens from a commercial breeder flock.

Progeny chicks from a commercial breeder flock with a high rate of avian leukosis virus (ALV) infection were classified as congenitally or contact infected with ALV on the basis of virus tests on their meconia at hatching and the presence or absence of antibodies at 20 weeks of age. The ALV infection profile, shedding, tumor mortality, and egg production in the congenitally-infected chickens were compared with those in the contact-infected hatchmates. Results show that viremic-tolerant hens classed as congenitally infected consistently shed virus into albumen of their eggs and progeny and 18% died from tumors. In contrast, hens classified as contact infected had a transient viremia; most of them developed antibody to ALV, and only 10% shed virus into albumen and progeny. No mortality from tumors occurred in this group of chickens during the 48-week experimental period, and egg production was significantly higher than in the group classified as congenitally infected. In addition, progeny chickens hatched from the contact-infected group maintained a low rate (2.9 to 3.8%) of ALV infection for the next two generations without further selection. These data further document principles and procedures for reduction of ALV infection in commercial flocks and the considerable economic benefits to be derived therefrom.

Hatchery-related contact transmission and short- term small-group-rearing as related to lymphoid-leukosis-virus-eradication programs.

Pathogen-free chickens highly susceptible to lymphoid leukosis virus (LLV) were used to study the role of hatchery-related contact transmission of LLV. In one experiment, the efficiency of short-term small-group-rearing in conventional-type rearing units to prevent contact transmission of LLV from infected to uninfected groups was evaluated. Results obtained from these experiments suggest that LLV is not transmitted by congenitally infected chicks to uninfected hatchmates in the hatcher. However, LLV can be transmitted by congenitally infected chicks to uninfected hatchmates during standard manual vent sexing. Rate of contact transmission was lower in chickens reared on wire-mesh floors than in chickens reared on solid floors. Short-term procedures in which chicks were reared in colony cages and brooder batteries effectively reduced the transmission of LLV by infected groups of hatching chicks to uninfected groups. All these procedures should facilitate the rearing of small groups of chicks with little risk of infection for a period sufficient to conduct virus assays, an essential step in LLV eradication.

A reexamination of humoral tolerance in chickens congenitally infected with an avian leukosis virus.

In a reexamination of congenital infection of chickens with ALV, one of the classical models of immunologic tolerance, we were unable to demonstrate that there was any evidence suggesting humoral immune reactivity to the infecting virus. In ALV-F42 congenitally infected birds that have a persistent viremia and no neutralizing antibody detectable by conventional means, we could find no evidence of host IgG deposits in the kidneys, nor any suggestion of renal pathology, or damage. In addition, attempts to precipitate any putative circulating infections virus-antibody complexes by treatment with a highly specific anti-globulin were negative nor were there titer differences in the viremic plasmas of bursectomized birds compared to nonbursectomized birds, indicating that the chickens were not making either a masked neutralizing or a nonneutralizing antibody response to the infecting virus. The evidence presented indicates that congenital infection of chickens with ALV does, at least at the humoral level, result in a state of immunologic tolerance to the infecting agent.

Lymphoid leukosis: interrelations among virus infections in hens, eggs, embryos, and chicks.

Hens from a commercial source were selected because they were infected with lymphoid leukosis virus (LLV). LLV was detected in vaginal swabs from 17 viremic hens and from 27 of 44 hens that were not viremic. All hens that were positive on the vaginal swab test (VST) produced one or more eggs with virus in albumen or in embryos, whereas in comparable tests, virus was detected only in eggs from 5 of 17 hens that were negative on VST. Congenital transmission of LLV was erratic and neither the VST nor tests for virus in egg albumen prior to incubating eggs identified all hens that transmitted infection. For example, 14 hens negative on VST produced 50 eggs negative for virus in albumen and yet one of the embryos from these eggs was infected. Eggs from other hens had infectious virus in albumen and about half of the embryos from these were infected. Tests for virus in cloacal swabs from one-day-old chicks were as sensitive as tests on embryos for detecting congenital transmission. Titers of LLV in the meconium of congenitally infected chicks were as high as 10(7) infectious units per ml. The cloacal swab test should be a valuable adjunct to the VST and tests on egg albumen in programs designed to eradicate lymphoid leukosis from chickens.

Tumor growth and antibodies after RSV-challenge in normal chickens and in chickens congenitally infected with avian leukosis virus.

Normal chickens and chickens congenitally infected with an avian leukosis virus (ALV) of antigenic subgroup A were challenged with strains of Rous sarcoma virus (RSV) of two different antigenic subgroups (B and C) and tumor induction and growth as well as humoral antibody to viral envelope antigen (VEA) and tumor-specific surface antigen (TSSA) were measured. There was no effect of congenital ALV infection on RSV tumor incidence or latent period but the growth rate and size of the tumors were much higher in congenitally infected birds as compared to controls. Whereas most tumors in the RSV-challenged normal birds regressed, tumors in ALV-infected birds grew progressively. There were no striking differences in the number of birds in either group in the incidence of anti-TSSA or anti-VEA antibodies nor did the presence of either type of antibody reflect the tumor status of the host.

The efficacy of chemical bursectomy in chickens with congenital leukosis-virus infection.

Since treatment with Freund's complete adjuvant in chickens with congenital avian leukosisvirus infection results in severe hypergammaglobulinaemia, it was of interest to determine how various means of chemical bursectomy affected such animals. Testosterone injected during the embryonic period or cyclophosphamide administered neonatally were, alone, relatively ineffectual in suppressing the response to influenza virus or production of natural rabbit haemagglutinins, nor did these treatments reliably depress IgG or IgM levels. However, the combination of both testosterone and cyclophosphamide completely suppressed antibody responses to the antigens and resulted in animals with serum immunoglobulin levels 10-(4)-10-(5) that of untreated hypergammaglobulinaemic controls.

Hypergammaglobulinemia in chickens congenitally infected with an avian leukosis virus.

Significantly elevated (2- to 5-fold higher than controls) serum levels of IgG were found in chickens congenitally infected with F42 strain of avian leukosis (ALV-F42) a subgroup A avian leukosis virus (ALV). A further increase in IgG levels in congenitally infected birds was found to be induced by injection of influenza virus in complete Freund's adjuvant(CFA). Serum immunoglobulin M (IgM) levels were not significantly elevated in ALV congenitally infected chickens except in those animals that had been injected with influenza virus in CFA. Hypergammaglobulinemia in ALV infected birds resulted only after congenital infection and not after infection of immunologically competent birds. Therefore this phenomenon appeared to have striking parallels with other persistent or chronic viral infections that have been previously described in mammals.