Infection with Eimeria tenella: modulation of lymphocyte blastogenesis by specific antigen, and evidence for immunodepression

The blastogenic effects of specific parasite antigen and of mitogens on the lymphocytes of chickens infected with Eimeria tenella were examined. Lymphocytes from infected chickens were stimulated to divide when cultured with parasite antigen, but their responses to the T-cell mitogen, phytohemagglutinin (PHA), were depressed throughout the period of infection. Responses to the B-cell mitogen, lipopolysaccharide (LPS), were depressed during the first week of infection but enhanced in the second week. The inclusion of plasma samples from infected chickens in the culture medium depressed the responses of normal spleen lymphocytes to PHA, suggesting that soluble suppressor factors are generated during infection.     

Infection with Eimeria tenella: Modulation of Lymphocyte Blastogenesis by Specific Antigen,and Evidence for Immunodepression1

ABSTRACT. The blastogenic effects of specific parasite antigen and of mitogens on the lymphocytes of chickens infected with Eimeria tenella were examined. Lymphocytes from infected chickens were stimulated to divide when cultured with parasite antigen, but their responses to the T-cell mitogen, phytohemagglutinin (PHA), were depressed throughout the period of infection. Responses to the B-cell mitogen, lipopolysaccharide (LPS), were depressed during the first week of infection but enhanced in the second week. The inclusion of plasma samples from infected chickens in the culture medium depressed the responses of normal spleen lymphocytes to PHA, suggesting that soluble suppressor factors are generated during infection.     

Marek’s disease virus prolongs survival of primary chicken B-cells by inducing a senescence-like phenotype

Marek’s disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.     

Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection

Using a novel cDNA microarray prepared from sources of actively responding immune system cells, we have investigated the changes in gene expression in the target tissue during the early stages of infection of neonatal chickens with infectious bursal disease virus. Infections of two lines of chickens previously documented as genetically resistant and sensitive to infection were compared in order to ascertain early differences in the response to infection that might provide clues to the mechanism of differential genetic resistance. In addition to major changes that could be explained by previously described changes in infected tissue, some differences in gene expression on infection, and differences between the two chicken lines, were observed that led to a model for resistance in which a more rapid inflammatory response and more-extensive p53-related induction of apoptosis in the target B cells might limit viral replication and consequent pathology. Ironically, the effect in the asymptomatic neonatal infection is that more-severe B-cell depletion is seen in the more genetically resistant chicken. Changes of expression of many chicken genes of unknown function, indicating possible roles in the response to infection, may aid in the functional annotation of these genes.     

Activation of the c-myb locus is insufficient for the rapid induction of disseminated avian B-cell lymphoma.

We have previously reported that infection of 9- to 13-day-old chicken embryos with RAV-1 results in rapid development of a novel B-cell lymphoma in which proviral insertion has activated expression of the c-myb gene (E. Pizer and E. H. Humphries, J. Virol. 63:1630-1640, 1989). The biological properties of these B-cell lymphomas are distinct from those associated with the B-cell lymphomas that develop following avian leukosis virus proviral insertion within the c-myc locus. In an extension of this study, more than 200 chickens, infected as 10- to 11-day-old embryos, were examined for development of lymphomas that possess disrupted c-myb loci. Fourteen percent developed disseminated B-cell lymphoma. In the majority of these tumors, the RAV-1 provirus had inserted between the first and second exons that code for p75c-myb. However, insertions between the second and third exons and between the third and fourth exons were also detected. In situ analysis of myb protein expression in tumor tissue revealed morphological features suggesting that the tumor originates in the bursa. Within the bursa, the lymphoma appeared to spread from follicle to follicle without compromising the structural integrity of the organ. Tumor masses in liver demonstrated heterogeneous levels of myb protein suggestive of biologically distinct subpopulations. In contrast to the morbidity data, immunohistological analysis of bursae from 4- to 6-week-old chickens at risk of developing lymphomas bearing altered c-myb loci revealed lesions expressing elevated levels of myb in 16 of 19 birds. The activated myb lymphoma displayed very poor capacity to proliferate outside its original host. Only 1 of 33 in vivo transfers of tumor to recipient hosts established a transplantable tumor. None of the primary tumor tissue nor the transplantable tumor exhibited the capacity for in vitro proliferation. Similar experimental manipulation has yielded in vitro lines established from avian B-cell lymphomas expressing elevated levels of c-myc or v-rel. The dependence on embryonic infection for development of activated-myb lymphoma suggests a requirement for a specific target cell in which c-myb is activated by proviral insertion. It is likely, moreover, that continued tumor development requires elevated expression of myb proteins within a specific cell population in a restricted stage of differentiation.     

Seroprevalence of Toxoplasma gondii infection in chickens in Durango State, Mexico

Little is known concerning the seroprevalence of Toxoplasma gondii infection in chickens (Gallus domesticus) in Mexico. Antibodies to T. gondii were determined in 519 chickens in Durango, Mexico using the modified agglutination test (MAT). Two groups (A, B) of chickens were sampled. Group A chickens (n = 51) were raised in backyards in 7 municipalities in 3 geographical regions in Durango State. Group B chickens were raised in farms in the Mexican States of Sinaloa (n = 289) and Nayarit (n = 179) but slaughtered in 2 abattoirs in Durango City. Overall, antibodies to T. gondii were found in 36 (6.9%) of 519 chickens, with MAT titers of 1∶25 in 22, 1∶50 in 8, 1∶100 in 2, 1∶200 in 3, and 1∶400 in 1. Seroprevalence of T. gondii increased significantly with age and was significantly higher in Group A chickens than in Group B chickens. In Group A chickens, a 25.5% seroprevalence of T. gondii infection was found. Seropositive chickens were found in all 7 municipalities sampled. In Group B chickens, the seroprevalence of T. gondii infection was 4.9%. This is the first report of T. gondii infection in chickens in Durango State, Mexico.     

Cross-protection among lethal H5N2 influenza viruses induced by DNA vaccine to the hemagglutinin.

Inoculation of mice with hemagglutinin (HA)-expressing DNA affords reliable protection against lethal influenza virus infection, while in chickens the same strategy has yielded variable results. Here we show that gene gun delivery of DNA encoding an H5 HA protein confers complete immune protection to chickens challenged with lethal H5 viruses. In tests of the influence of promoter selection on vaccine efficacy, close correlations were obtained between immune responses and the dose of DNA administered, whether a cytomegalovirus (CMV) immediate-early promoter or a chicken beta-actin promoter was used. Perhaps most important, the HA-DNA vaccine conferred 95% cross-protection against challenge with lethal antigenic variants that differed from the primary antigen by 11 to 13% (HA1 amino acid sequence homology). Overall, the high levels of protection seen with gene gun delivery of HA-DNA were as good as, if not better than, those achieved with a conventional whole-virus vaccine, with fewer instances of morbidity and death. The absence of detectable antibody titers after primary immunization, together with the rapid appearance of high titers immediately after challenge, implicates efficient B-cell priming as the principal mechanism of DNA-mediated immune protection. Our results suggest that the efficacy of HA-DNA influenza virus vaccine in mice extends to chickens and probably to other avian species as well. Indeed, the H5 preparation we describe offers an attractive means to protect the domestic poultry industry in the United States from lethal H5N2 viruses, which continue to circulate in Mexico.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Chicken B-cell marker chB6 (Bu-1) is a highly glycosylated protein of unique structure

 The chB6 molecule is expressed on chicken B cells throughout most of their development, as well as on some non-lymphoid cells. It has long been used as an allotypic marker in important studies of B-cell development, though its function is unknown. We isolated a chB6 cDNA by expression cloning and sequenced two further alleles following polymerase chain reaction amplification. The results show that chB6 is a typical type I transmembrane protein, highly glycosylated in the extracellular region and carrying a large intracellular region. It has no recognizable similarity to known mammalian molecules and thus represents a unique B-cell marker. Its presence in chickens may be related to differences in the properties of B-cell development between chickens and mammalian species. The sequences of the different alleles of this gene revealed a higher level of polymorphism than expected. A restriction fragment length polymorphism linked to the CHB6 gene has been used to determine its location on the linkage map of the chicken genome, which will allow the definitive evaluation of reported associations with disease resistance. Received: 21 February 1996 / Revised: 26 March 1996     

Highly pathogenic H5N1 influenza virus causes coagulopathy in chickens

Severe hemorrhage at multiple organs is frequently observed in chickens infected with highly pathogenic avian influenza (HPAI) A viruses. In this study we examined whether HPAI virus infection leads to coagulation disorder in chickens. Pathological examinations showed that the fibrin thrombi were formed in arterioles at the lung, associated with the viral antigens in endothelial cells of chickens infected intravenously with HPAI virus. Hematological analyses of peripheral blood collected from the chickens revealed that coagulopathy was initiated at early stage of infection when viral antigens were detected only in the endothelial cells and monocytes/macrophages. Furthermore, gene expression of the tissue factor, the main initiator of blood coagulation, was upregulated in the spleen, lung, and brain of HPAI virus-infected chickens. These results suggest that dysfunction of endothelial cells and monocytes/macrophages upon HPAI virus infection may induce hemostasis abnormalities represented by the excessive blood coagulation and consumptive coagulopathy in chickens.     

A chicken embryo model for the maintenance and amplification of Cryptosporidium parvum and Cryptosporidium baileyi oocysts

Cryptosporidium is a genus of apicomplexan parasites that inhabit the respiratory and gastrointestinal tracts of vertebrates. Research of these parasites is limited by a lack of model hosts. This study aimed to determine the extent to which infection at the embryo stage can enhance the propagation of Cryptosporidium oocysts in chickens. Nine-day-old chicken embryos and one-day-old chickens were experimentally infected with different doses of Cryptosporidium baileyi and Cryptosporidium parvum oocysts. Post hatching, all chickens had demonstrable infections, and the infection dose had no effect on the course of infection. Chickens infected as embryos shed oocysts immediately after hatching and shed significantly more oocysts over the course of the infection than chickens infected as one-day-olds. In chickens infected as embryos, C. baileyi was found in all organs except the brain whereas, C. parvum was only found in the gastrointestinal tract and trachea. In chickens infected as one-day-olds, C. baileyi was only found in the gastrointestinal tract and trachea. Chickens infected as embryos with C. baileyi died within 16 days of hatching. All other chickens cleared the infection. Infection of chickens as embryos could be used as an effective and simple model for the propagation of C. baileyi and C. parvum.     

禽白血病病毒J亚群（ALV—J）的血清学调查及PCR诊断

禽骨髓细胞性白血病 (ｍｙｅｌｏｉｄｌｅｕｃｏｓｉｓ) (或称禽骨髓细胞瘤 ,ｍｙｅｌｃｙｔｏｍａｔｏｍａｔｏｓｉｓ) (ＭＬ)是由禽白血病病毒 (Ａｖｉａｎｌｅｕｋｏｓｉｓｖｉｒｕｓ)Ｊ亚群 (ＡＬＶ Ｊ)引起的禽的一种肿瘤性传染病[1] ,ＡＬＶ -Ｊ是英国的Ｐａｙｎｅ于 199     

Seroimmunological and parasitological surveys of leucocytozoon caulleryi infection in chickens in several asian countries

Epizootiological surveys of Leucocytozoon caulleryi infection in chickens in Japan, Taiwan, Philippines, Singapore, Malaysia and Thailand were undertaken by means of the immunodiffusion test. The rate of infection of L. caulleryi confirmed by the examination of parasites in the peripheral blood of chickens coincided with that of positive antibody response in the immunodiffusion test. Antibodies against L. caulleryi were found in chickens in all the countries surveyed in the present investigation. The prevalence of L. caulleryi infection in chickens was confirmed by the immunodiffusion test. Several chickens in each country showed the presence of serum antigens of L. caulleryi at the times of serum sample collection. These results seemed to indicate that the immunodiffusion test is a method efficient enough to be applicable to the epizootiological surveys and diagnosis of L. caulleryi infection in chickens in the field. As a result, the antibodies or soluble antigens in the sera of chickens infected with L. caulleryi present, respectively, in each country may have the same immunological characters.     

Characterization of activation of a partially defective B cell subpopulation with immunocompetence restricted to IgM and IgA

A B-cell subpopulation (BM-A cell) responding to an antigen with the production of IgM and IgA plaque-forming cells but not of IgG plaque-forming cells was isolated from neonatally bursectomized chickens and was examined for the mode of activation by B-cell mitogens. The BM-A cells did not elevate both glucose consumption and protein synthesis with the B-cell mitogens, in striking contrast to normal B cells. The BM-A cells were also not activated by an activator of protein kinase C, phorbol myristate acetate. Both anti-Ig and a calcium ionophore, A23187, however, primed the BM-A cells to increase intracellular free calcium ion as well as normal B cells. From these results it is conceived that the lack of protein kinase C activation may be responsible for the failure of activation of BM-A cells.     

Cytokine signaling in splenic leukocytes from vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enteritidis

In order to design a new Salmonella enterica vaccine, one needs to understand how naive and immune chickens interact differently when exposed to S. enterica. In this study we therefore determined the immune response of vaccinated and non-vaccinated chickens after intravenous infection with Salmonella enterica serovar Enteritidis (S. Enteritidis). Using flow cytometry we showed that 4 days post infection (DPI), counts of CD4 and B-lymphocytes did not change, CD8 and γδ T-lymphocytes decreased and macrophages and heterophils increased in the spleen. When vaccinated and non-vaccinated chickens were compared, only macrophages and heterophils were found in significantly higher counts in the spleens of the non-vaccinated chickens. The non-vaccinated chickens also expressed higher anti-LPS antibodies than the vaccinated chickens. The expression of interleukin (IL)1β, IL6, IL8, IL18, LITAF, IFNγ and iNOS did not exhibit any clear pattern in the cells sorted from the spleens of vaccinated or non-vaccinated chickens. Only IL17 and IL22 showed a differential expression in the CD4 T-lymphocytes of the vaccinated and non-vaccinated chickens at 4 DPI, both being expressed at a higher level in the non-vaccinated chickens. Due to a similar IFNγ expression in the CD4 T-lymphocytes in both the vaccinated and non-vaccinated chickens, and a variable IL17 expression oscillating around IFNγ expression levels, the IL17∶IFNγ ratio in CD4 T-lymphocytes was found to be central for the outcome of the immune response. When IL17 was expressed at higher levels than IFNγ in the non-vaccinated chickens, the Th17 immune response with a higher macrophage and heterophil infiltration in the spleen dominated. However, when the expression of IL17 was lower than that of IFNγ as in the vaccinated chickens, the Th1 response with a higher resistance to S. Enteritidis infection dominated.     

Observations on the Taiwanese Strain of Leucocytozoon caulleryi (Haemosporina) in Chickens

ABSTRACT. The Taiwanese strain of Leucocytozoon caulleryi was isolated from an infected chicken in Taipei, Taiwan, and established in chickens and biting midges Culicoides arakawae from Japan. Sporogony of the strain in C. arakawae was completed on day 3 after the infective blood meals at 25°C. Sporozoites isolated from the salivary glands of C. arakawae on days 3 or 4 after feeding caused infection in all the chickens inoculated. The strain showed high pathogenicity for chickens. Mortality of chickens rose with an increase in the number of sporozoites inoculated. The prepatent period for chickens inoculated with sporozoites was 14 days. Parasites appeared in the peripheral blood of chickens on day 15 and disappeared on day 26 after sporozoite inoculation. Soluble antigens were found in the sera of chickens infected with the strain between 10 and 17 days after inoculation, and homologous antibodies appeared after 17 days. Antigens prepared from sera, schizonts, merozoites, and gametocytes of the Taiwanese strain reacted with the sera of chickens infected witt the same strain or the strain isolated in Japan. The chickens that recovered from a primary infection with the Taiwanese strain demonstrated complete resistance to reinfection with the same strain or the strain isolated in Japan.     

Eimeria acervulina: evaluation of the cellular and antibody responses to the recombinant coccidial antigens in B-congenic chickens

The roles of major histocompatibility complex (MHC) and non-MHC-linked genes in the genetic control of disease susceptibility and the development of protective immunity to Eimeria acervulina infection were investigated in six 15I5-B congenic and four different strains of chickens characterized for the MHC. When oocyst production was assessed, wide variations were noted following initial and challenge infections among the strains of chickens tested. In general, 15.N-21, 15.P-13, B21, B19, SC, and FP chickens were protected following challenge infection whereas 15I5, 15.P-19, 15.7-2, and 15.6-2 chickens were not. Strains of chickens sharing a same B haplotype on different genetic backgrounds did not show comparable levels of protection. These results lead to the view that non-MHC-linked genes have a profound influence on the outcome of the host response to E. acervulina infection. Chickens infected twice at 1-month intervals by an oral inoculation with E. acervulina developed both coccidial-specific antibody and T-cell responses. E. acervulina infected chickens showed T-cell-mediated immune responses to the intact sporozoites as well as to recombinant proteins, p130 of sporozoites and p150 of merozoites. Both p130 and p150 antigens have been identified and characterized previously. Sera obtained from all infected chickens recognized the p150 merozoite protein, but not the p130 sporozoite protein in immunoblots. In general, the cellular response, but not the antibody response to the p150 recombinant surface merozoite antigen correlated with the degree of protection following the challenge infection. These results suggest that the strains of chickens having improved protection against challenge infection demonstrate higher T-cell responses to the recombinant surface merozoite protein, p150.     

Observations on the Taiwanese strain of Leucocytozoon caulleryi (Haemosporina) in chickens

The Taiwanese strain of Leucocytozoon caulleryi was isolated from an infected chicken in Taipei, Taiwan, and established in chickens and biting midges Culicoides arakawae from Japan. Sporogony of the strain in C. arakawae was completed on day 3 after the infective blood meals at 25 degrees C. Sporozoites isolated from the salivary glands of C. arakawae on days 3 or 4 after feeding caused infection in all the chickens inoculated. The strain showed high pathogenicity for chickens. Mortality of chickens rose with an increase in the number of sporozoites inoculated. The prepatent period for chickens inoculated with sporozoites was 14 days. Parasites appeared in the peripheral blood of chickens on day 15 and disappeared on day 26 after sporozoite inoculation. Soluble antigens were found in the sera of chickens infected with the strain between 10 and 17 days after inoculation, and homologous antibodies appeared after 17 days. Antigens prepared from sera, schizonts, merozoites, and gametocytes of the Taiwanese strain reacted with the sera of chickens infected with the same strain or the strain isolated in Japan. The chickens that recovered from a primary infection with the Taiwanese strain demonstrated complete resistance to reinfection with the same strain or the the strain isolated in Japan.