Early replication in pulmonary B cells after infection with Marek's disease herpesvirus by the respiratory route.

Abstract Natural infection with Marek's disease virus occurs through the respiratory mucosa after chickens inhale dander shed from infected chickens. The early events in the lung following exposure to the feather and squamous epithelial cell debris containing the viral particles remain unclear. In order to elucidate the virological and immunological consequences of MDV infection for the respiratory tract, chickens were infected by intratracheal administration of infective dander. Differences between susceptible and resistant chickens were immediately apparent, with delayed viral replication and earlier onset of interferon (IFN)-gamma production in the latter. CD4(+) and CD8(+) T cells surrounded infected cells in the lung. Although viral replication was evident in macrophages, pulmonary B cells were the main target cell type in susceptible chickens following intratracheal infection with MDV. In accordance, depletion of B cells curtailed viremia and substantially affected pathogenesis in susceptible chickens. Together the data described here demonstrate the role of pulmonary B cells as the primary and predominant target cells and their importance for MDV pathogenesis.

In ovo DNA immunisation followed by a recombinant fowlpox boost is fully protective to challenge with virulent IBDV.

The aim of this study was to investigate the potential use of DNA vaccination delivered in ovo for protecting against challenge with infectious bursal disease virus (IBDV). Using a plasmid expressing the beta-galactosidase gene, DNA was successfully delivered to the embryo after in ovo injection and localises to the proventriculus and thymus. The coding sequence for the immunogenic IBDV protein, VP2, was cloned into pCI-neo, creating pCI-Vp2. Complete protection against IBDV was obtained by priming in ovo with pCI-Vp2, followed by boosting with the fowlpox recombinant, fpIBD1, also expressing the VP2 gene. This complete protection was not evident with either of the experimental vaccines on their own. An antibody response was not detected after the prime-boost vaccination, even after chicks had been challenged with IBDV, implying that the DNA prime delivered in ovo stimulated a protective cellular immune response.

Enhanced immunopathology induced by very virulent infectious bursal disease virus.

Immunohistochemical and flow cytometric analyses of the bursa, spleen and thymus following infection with the very virulent infectious bursal disease virus (vvIBDV) strain UK661 revealed discrete differences from classical virulent infectious bursal disease virus strains. Bu-1+, immunoglobulin (Ig)M+ and IgG+ cells were all depleted from the bursa, spleen and thymus, suggesting loss of both immature and mature B lymphocytes. Small numbers of Bu-1+ cells repopulated the bursa 14 days post-infection but few of these expressed IgM or IgG. A transient increase in macrophages at 3 to 5 days post-infection was followed by a later influx of CD4+ and CD8+ T cells into the bursa. Loss of cortical thymocytes during the acute phase of infection suggested disruption of the T-cell system. The results showed that vvIBDV strain UK661 caused earlier and more severe pathology than classical virulent strains of infectious bursal disease virus. The marked influx of T cells into the infected bursa indicates that cell-mediated immunity is likely to be important in the clearance of vvIBDV.

Marek's disease is a natural model for lymphomas overexpressing Hodgkin's disease antigen (CD30).

Animal models are essential for elucidating the molecular mechanisms of carcinogenesis. Hodgkin's and many diverse non-Hodgkin's lymphomas overexpress the Hodgkin's disease antigen CD30 (CD30(hi)), a tumor necrosis factor receptor II family member. Here we show that chicken Marek's disease (MD) lymphoma cells are also CD30(hi) and are a unique natural model for CD30(hi) lymphoma. Chicken CD30 resembles an ancestral form, and we identify a previously undescribed potential cytoplasmic signaling domain conserved in chicken, human, and mouse CD30. Our phylogeneic analysis defines a relationship between the structures of human and mouse CD30 and confirms that mouse CD30 represents the ancestral mammalian gene structure. CD30 expression by MD virus (MDV)-transformed lymphocytes correlates with expression of the MDV Meq putative oncogene (a c-Jun homologue) in vivo. The chicken CD30 promoter has 15 predicted high-stringency Meq-binding transcription factor recognition motifs, and Meq enhances transcription from the CD30 promoter in vitro. Plasma proteomics identified a soluble form of CD30. CD30 overexpression is evolutionarily conserved and defines one class of neoplastic transformation events, regardless of etiology. We propose that CD30 is a component of a critical intracellular signaling pathway perturbed in neoplastic transformation. Specific anti-CD30 Igs occurred after infection of genetically MD-resistant chickens with oncogenic MDV, suggesting immunity to CD30 could play a role in MD lymphoma regression.

Study of host-pathogen interactions to identify sustainable vaccine strategies to Marek's disease.

Marek's disease virus is a highly cell-associated, lymphotropic alpha-herpesvirus that causes paralysis and neoplastic disease in chickens. The disease has been contained by vaccination with attenuated viruses and provides the first evidence for a malignant cancer being controlled by an antiviral vaccine. Marek's disease pathogenesis is complex, involving cytolytic and latent infection of lymphoid cells and oncogenic transformation of CD4+ T cells in susceptible chickens. Innate and adaptive immune responses develop in response to infection, but infection of lymphocytes results in immunosuppressive effects. The remarkable ability of MDV to escape immune responses by interacting with, and down-regulating, some key aspects of the immune system will be discussed in the context of genetic resistance. Resistance conferred by vaccination and the implications of targeting replicative stages of the virus will also be examined.

Protection from infectious bursal disease virus (IBDV)-induced immunosuppression by immunization with a fowlpox recombinant containing IBDV-VP2.

Immunosuppression resulting from infectious bursal disease virus (IBDV) infection has critical health and welfare implications for birds, yet it is incompletely understood and largely overlooked as a measure of vaccine efficacy. The ability of a fowlpoxvirus recombinant (fpIBD1) containing the VP2 protein of IBDV to protect against IBDV-induced immunosuppression was investigated by measuring the convalescent chicken's ability to mount antibody responses to IBDV infection, and to inactivated IBDV and salmonella vaccines. An immunoglobulin (Ig)M response, but no IgG response, occurred after IBDV infection. Uninfected chickens produced a sustained IgM response and some IgG response to inactivated IBDV vaccine, while in previously infected birds only a transient IgM response was detected. A moderate suppression of the response to a commercial salmonella vaccine was evident after IBDV infection, which was largely prevented by immunization with fpIBD1. These results indicate that measurement of immunosuppression could be a useful strategy for assessing the efficacy of vaccines to protect against the consequences of IBDV infection.

The immunologists' debt to the chicken.

The immune system of the chicken is an invaluable model for studying basic immunology and has made seminal contributions to fundamental immunological principles. Graft versus host responses and the key role of lymphocytes in adaptive immunity were first described in work with chicken embryos and chickens. 2. Most notably, the bursa of Fabricius provided the first substantive evidence that there are two major lineages of lymphocytes. Bursa-derived lymphocytes, or B cells, make antibodies while thymus-derived, or T cells, are involved in cell-mediated immune responses. 3. Gene conversion, the mechanism used by the chicken to produce its antibody repertoire, was first described in the chicken and requires the unique environment of the bursa. Subsequently it has been shown that some mammals also use gene conversion. 4. The chicken's Major Histocompatibility Complex (MHC), the first non-mammalian MHC to be sequenced, is minimal, compact and some 20-fold smaller than that of mammals. Uniquely, the chicken MHC is strongly associated with resistance to infectious diseases. 5. The first attenuated vaccine was developed by Louis Pasteur against a chicken pathogen, fowl cholera, and the first vaccine against a natural occurring cancer agent, Marek's disease virus, was developed for the chicken. 6. Vaccination of chick embryos on the 18th d of incubation, another breakthrough using chickens, provides protection early after hatching. In ovo vaccination now is widely practised by the poultry industry. 7. Evidence that widespread and intensive vaccination can lead to increased virulence with some pathogens, such as Marek's disease virus and infectious bursal disease virus, was first described with chicken populations. It warns of the need to develop mo resustainable vaccination strategies in future and provides useful lessons for other species, including in the human population. 8. Recombinant DNA technologies now provide the opportunity for the rational design of new vaccines. Such vaccines could contain the protective immunogenic elements from several pathogens and immunomodulatory molecules to direct and enhance immune responses so providing improved protection. The important thing will be to design vaccines that are sustainable and do not drive pathogens to ever-increasing virulence.

Resistance to Marek's disease herpesvirus-induced lymphoma is multiphasic and dependent on host genotype.

Genotype-dependent differences in Marek's disease (MD) susceptibility were identified using 14-day-old line N and 6(1) (resistant) and 151 and 7(2) (susceptible) inbred chickens infected with HPRS-16 MD virus (MDV). All line 72 chickens developed progressive MD. Line 15I had fluctuating MD-specific clinical signs and individuals recovered. A novel histologic scoring system enabled indices to be calculated for lymphocyte infiltration into nonlymphoid organs. All genotypes had increased mean lesion scores (MLSs) and mean total lesion scores after MDV infection. These differed quantitatively and qualitatively between the genotypes. Lines 6(1) and 7(2) had a similar MLS distribution in the cytolytic phase, although scores were greater in line 7(2). At the time lymphomas were visible in line 7(2), histologic lesions in line 6(1) were regressing. AV37+ cells were present in similar numbers in all genotypes in the cytolytic phase, suggesting that neoplastically transformed cells were present in all genotypes regardless of MD susceptibility. After the cytolytic phase, AV37+ cell numbers increased in lines 7(2) and 15I but decreased in lines 6(1) and N. In the cytolytic and latent phases, in all genotypes, most infiltrating cells were CD4+. After this time, line 7(2) and 15I lesions increased in size and most cells were CD4+; line 6(1) and N lesions decreased in size and most cells were CD8+. In all genotypes, AV37 immunostaining was weak in lesions with many CD8+ cells, suggesting that AV37 antigen expression or AV37+ cells were controlled by CD8+ cells. The rank order, determined by clinical signs and pathology, for MD susceptibility (highest to lowest) was 7(2) > 15I > 6(1) > N.

Protection from IBDV-induced bursal damage by a recombinant fowlpox vaccine, fpIBD1, is dependent on the titre of challenge virus and chicken genotype.

Expression of the VP2 capsid protein of infectious bursal disease virus (IBDV) in an vaccine strain of fowlpox has produced an experimental recombinant vaccine, fpIBD1. Successful vaccination with fpIBD1 was dependent on the titre of challenge virus for high titres of challenge virus were able to overcome protection induced by fpIBD1 whereas challenge with a low titre of virus did not. The genotype of chicken also has an important effect on the outcome of challenge possibly as a result of the major histocompatability complex and its ability to present VP2-derived peptides to the immune system. It was not possible to protect the inbred white leghorn chicken strain, line 15I, from IBDV-induced bursal damage by vaccination with fpIBD1 even at the lowest titre of challenge virus used. All other inbred white leghorn chickens examined (line 6(1), C. B4 and C.B12) and outbred Rhode Island Red chickens were protected by fpIBD1. Protection by the fpIBD1 vaccine is induced in the absence of detectable serum antibodies, suggesting the possibility of a significant role for cell-mediated immunity in protection from IBDV challenge.

A quantitative duplex PCR technique for measuring amounts of cell-associated Marek's disease virus: differences in two populations of lymphoma cells.

A duplex polymerase chain reaction (PCR) was developed to measure Marek's disease virus (MDV) load in two subpopulations of Marek's disease (MD) lymphoma cells from chickens. PCR primers were designed using the sequence of the MDV-ICP4 gene and the chicken IFNgamma gene. Each set of primers was present in the same reaction tube so that the 327 bp ICP4 product and the 420 bp IFNgamma product were co-amplified. Two different fluorescent dyes were used to 5'-end label one PCR primer of each pair to distinguish the IFNgamma and ICP4 products by colour. The IFNgamma PCR product was used as an internal standard enabling comparisons of MDV-ICP4 products between different samples. Neither duplex PCR product was preferentially amplified and both reactions were in their exponential phases when stopped. The products could be distinguished by both size and colour. MD lymphoma cells were taken ex vivo and separated on the basis of expressing a novel host surface antigen recognised by the monoclonal antibody AV37. AV37 + lymphoma cells had greater MDV-loads than AV37 lymphoma cells. The principles used here should be applicable to any cell phenotype and/or cell-associated DNA virus.

Counting absolute numbers of specific leukocyte subpopulations in avian whole blood using a single-step flow cytometric technique: comparison of two inbred lines of chickens.

A novel flow cytometric technique was developed to determine the absolute numbers of leukocytes of specific phenotypes in whole blood from two lines of inbred chickens (line 7(2) and line 6(1)). This single step method is rapid, accurate, repeatable, can be used in the presence of nucleated erythrocytes and addresses the problems encountered when electronically counting the numbers of leukocytes in specific subpopulations in the blood of non-mammalian species. It is superior to previous methods in that (1) peripheral blood leukocytes (PBL) do not need to be separated by density gradient centrifugation, (2) erythrocyte lysis is not necessary and (3) absolute numbers of specific phenotypes of cells are determined directly. A standard volume of diluted whole blood was added to a standard number of fluorescent beads before incubation with fluorescently-conjugated monoclonal antibodies recognising specific PBL surface antigens. Samples were analysed by flow cytometry and electronic gates were set to count a standard number of beads and the concomitant fluorescently-labelled cells. Absolute numbers of B, CD4+ and CD8+ PBL were determined. Since the bead fluorescence is constant, it was also possible to measure relative MHC class I expression using fluorescence intensity. In both lines of chickens absolute numbers of all of the phenotypes of PBL measured increased with age. Although line 7(2) chickens had greater numbers of B, CD4+, and CD8+ PBL than line 6(1) chickens, there was no significant difference in the CD4+:CD8+ PBL ratios, the T:B PBL ratios or relative MHC class I expression between the two lines. Relative MHC class I expression increased with age in both lines.

Development and composition of lymphoid lesions in the spleens of Marek's disease virus-infected chickens: Association with virus spread and the pathogenesis of Marek's disease.

Changes in lymphocyte distribution in spleens of Marek's disease virus (MDV) infected White Leghorn chickens of line 72 (MD susceptible) and line 61 (MD resistant) were studied by immunocytochemistry. Lymphocytes expressing the MDV antigen pp38 (predominantly B cells) were detected from 4 to 6 days post-inoculation (d.p.i.) but not at or after 8 d.p.i., and were more numerous in line 72. In line 61, infection resulted in depletion of B lymphocytes and an increase in T lymphocytes from 3 to 6 d.p.i., but no change in distribution of these cells. From 8 d.p.i., the B-dependent tissue began to recover and the T cells decreased in number. In line 72, infection caused a dramatic change in lymphocyte distribution, with formation of 'lymphoid lesions'. Diffuse, irregular patches of B lymphocytes, around the capillaries, became surrounded by large aggregates of TCRαß1(+) CD8(+) and CD4(+) lymphocytes, bordered by a band of TCRγδ(+) lymphocytes. From 8 d.p.i., the B-dependent areas partially recovered, while TCRαß1(+) CD4(+) and CD8(+) lymphocytes, potentially transformed, became extensively scattered throughout the spleen. We conclude that in line 72, replication and spread of MDV is more efficient and T cell responses in early infection are greater, favouring the tumour stage of the disease.

Differential susceptibility to Marek's disease is associated with differences in number, but not phenotype or location, of pp38+ lymphocytes.

Flow cytometric and immunocytochemical techniques were used to quantify, identify and locate Marek's disease herpesvirus (MDV)-infected lymphocytes in lymphoid organs of infected chickens, by expression of the virus antigen pp38. Two closely related lines of chicken, one susceptible to Marek's disease (line 7(2)) and another resistant (line 6(1)), were infected at 2 weeks of age and compared at 10 sampling times between 0 and 50 days post-infection. In both lines 6(1) and 7(2), pp38+ lymphocytes were detected at 4-6 days in the spleen, thymus and bursa. pp38+ cells could not be detected from day 8 onwards. In both lines, pp38+ lymphocytes were located in the peri-ellipsoidal area of the spleen, the medulla of the thymic lobes and the medulla of the bursal follicles. In both lines, pp38+ cells were predominantly B lymphocytes, but CD4+ and CD8+ TCR alphabeta+ T lymphocytes were also detected in the thymus and spleen. For each organ, the mean number of pp38+ lymphocytes was greater in line 7(2) than in line 6(1). pp38+ lymphocytes were not detected in the peripheral blood at any time. The data show that the differential susceptibility of lines 6(1) and 7(2) to the development of Marek's disease lymphoma is not attributable to differences in phenotype or location of pp38+ lymphocytes, or the time of expression of pp38. However, susceptibility is associated with greater numbers of pp38+ lymphocytes.

Marek's disease virus EcoRI-Q gene (meq) and a small RNA antisense to ICP4 are abundantly expressed in CD4+ cells and cells carrying a novel lymphoid marker, AV37, in Marek's disease lymphomas.

Mature lymphomas produced in Rhode Island Red (RIR) chickens infected with the RB1B strain of Marek's disease virus (MDV) were examined for the presence of viral DNA and RNA and expression of viral antigens. In situ hybridization showed that all tumours examined contained viral DNA in areas of lymphoid infiltration. In 3/5 tumours, there was a correlation between the number and distribution of cells expressing the Marek's disease EcoRI-Q gene (meq) and those that carried the lymphoid cell marker AV37. Expression of the MDV-specific phosphoprotein pp38 was infrequent in lymphomas but abundant in a splenic tumour which also expressed the viral glycoprotein gB. Northern blot analysis of lymphocyte fractions purified by immunoaffinity showed that CD4+ and AV37+ fractions from lymphomas expressed meq and the small RNA antisense to ICP4 (SAR). The results are consistent with the notion that transformed cells are CD4+ cells, carrying the AV37 marker and expressing meq and SAR.

Monoclonal and polyclonal antibodies to chicken immunoglobulin isotypes specifically detect turkey immunoglobulin isotypes.

Turkey immunoglobulin (Ig) isotypes IgG and IgM were isolated from blood and IgA was isolated from bile. Isolation was accomplished by gel filtration of the ammonium sulphate cut on Sephacryl S-200. Using immunoelectrophoresis and indirect ELISA, the cross-reactivity between antibodies, of monoclonal and polyclonal origin, specific for the Ig isotypes of chicken, and the purified turkey Ig isotypes was evaluated. Commercially available polyclonal antibodies, anti-chicken/IgA (alpha-chain specific, affinity purified), anti-chicken/IgG (Fc-fragment specific) and anti-chicken/IgM (mu-chain specific) showed an interspecies cross-reactivity with the corresponding turkey Ig isotypes. The monoclonal antibody (MAb) AV-G3 specifically detected turkey IgG, whereas MAb M1 reacted exclusively with turkey IgM. This panel of anti-immunoglobulins represents a useful tool for examining the humoral immune responses of turkeys.

Comparison of the in situ changes in lymphoid cells during infection with infectious bursal disease virus in chickens of different ages.

In situ immunocytochemical staining was used to characterise leukocyte changes and determine tropism of infectious bursal disease virus following infection of neonate and 3-week-old chickens. In the bursae of both groups, massive replication of the virus, a rapid depletion of B cells and an influx of CD4(+) TCR-alphabeta(1)(+) and CD8 (+) TCR-alphabeta(1)(+) cells was detected within 4 days post-inoculation. Leukocyte changes in the spleen, thymus and Harderian gland were similar in both groups. From 8 days post-inoculation onwards all the lymphoid organs became repopulated with leukocytes and tissue architecture began to be slowly restored. Virus neutralizing antibodies developed more slowly in neonate birds and at 21 days post-inoculation the titres were much lower compared to older birds. Lack of clinical signs in neonate chickens was neither due to a failure to respond to the virus, to recruit leukocytes to the infected tissues nor to a lack of viral replication.

The effects of cyclosporin A and cyclophosphamide on the populations of B and T cells and virus in the Harderian gland of chickens vaccinated with the Hitchner B1 strain of Newcastle disease virus.

The cellular response to conjunctival vaccination with the Hitchner B1 strain of Newcastle disease virus was studied in the Harderian gland (HG) by immunohistochemistry. Bu-1+ cells and all subpopulations of T cells, (CD3+, CD4+, CD8+, TCR gamma delta, TCR alpha beta 1, and TCR alpha beta 2) were in the interstitial tissue between the ducts and the acini. Plasma cells with cytoplasmic IgM were more dispersed than the other cells and outlined the acini. Bu-1+ cells and all subpopulations of T cells increased at least three-fold after vaccination when compared to uninfected birds on the basis of the average cell counts in sections taken at 3, 5, 7, 10, 14, and 20 days after vaccination. The most marked increase was in the CD8+ cells which increased six-fold. Virus replicated for 10 days in cyclophosphamide (Cy) treated birds and for 7 days in cyclosporin A (CsA) treated birds compared with 5 days in untreated birds. Cy treatment prevented an antibody response to NDV and reduced Bu-1+ and IgM cells in the HG by 20-fold. Cy treatment resulted in a doubling of the number of T cells in the HG but these T cells may have been transiently disabled because it also caused a poor response of the lymphocytes in whole blood to the T cell mitogen concanavalin A (ConA). CsA reduced the T cell numbers in the HG and whole-blood responses to ConA by about 4-fold but T cell numbers rebounded to normal resting values after vaccination with NDV. The clearance time was prolonged either by T cells being less numerous than normal after CsA or being disabled after Cy. T cells, but not B cells, may therefore be essential for virus clearance. CD8+ cells expanded more than CD4+ cells after the vaccination of untreated and CsA-treated birds indicating that CD8+ cells may be key players in vaccinal immunity to NDV.

Identification of chicken Bu-1 alloantigens using the monoclonal antibody AV20.

The genetically polymorphic chicken antigen Bu-1 (chB6) has been identified by alloantisera raised against RPL line 6(3) (Bu-1a) and line 7(2) (Bu-1b) birds and subsequently by monoclonal antibodies (mAbs) which identify individual alloantigens. We have produced a monoclonal antibody, AV20, which recognises a monomorphic determinant on the antigen Bu-1. AV20 identifies a marker on both bursal and peripheral B cells. Staining characteristics on bursa, spleen, thymus and peripheral blood lymphocytes are similar to those of the allotypic antibodies which identify Bu-1a and Bu-1b. However, AV20 identified B cells in partially inbred birds as well as inbred lines including line 6(1) and line 7(2), indicating that it recognises a monomorphic determinant, AV20 immunoprecipitated an antigen with a Mwr of 150 kDa under non-reducing conditions and 70-75 kDa under reducing conditions indicating it is a homodimer. Serial immunoprecipitations or bursal-cell lysates from line 6(1) or line 7(2) confirmed that AV20 recognised the same antigen as mAbs against Bu-1a and Bu-1b in the respective lines.

A flow cytometric method for identifying Marek's disease virus pp38 expression in lymphocyte subpopulations.

Identification of Marek's disease virus-infected lymphocytes is essential for a full understanding of the pathogenesis of Marek's disease. This paper describes the development of a simple, quantitative and reproducible flow cytometric technique for the identification of the phenotype of Marek's disease virus-infected lymphocytes. The method is based upon the detection of the Marek's disease virus-specific phosphoprotein pp38, in saponin-permeabilized lymphocytes, using the monoclonal antibody BDI, and the identification of the phenotype of pp38+ lymphocytes using monoclonal antibodies against lymphocyte surface markers. pp38 expression in the spleen was demonstrated in the cytolytic phase of infection. The mean proportion of pp38+ lymphocytes at 4 days post-infection was 0.43%. Of these, 95% were B lymphocytes, while only 4% were T lymphocytes (both CD4+ and CD8+). The potential of the technique for the investigation of the cytolytic phase of Marek's disease is discussed.