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The chicken MHC has been associated with disease resistance, though the mechanisms are not understood. The functions of macrophages, critical to both innate and acquired immunity, were compared between the more infectious bronchitis virus-resistant B2 and the more infectious bronchitis virus-susceptible B19 lines. In vivo peripheral blood concentrations of monocytes were similar in B2 or B19 homozygous haplotypes. Peripheral blood-derived macrophages were stimulated with poly I:C, simulating an RNA virus, or IFNγ, a cytokine at the interface of innate and adaptive immunity. Not only did B2-derived peripheral monocytes differentiate into macrophages more readily than the B19 monocytes, but as determined by NO production, macrophages from B2 and B2 on B19 genetic background chicks were also significantly more responsive to either stimulant. In conclusion, the correlation with resistance to illness following viral infection may be directly linked to a more vigorous innate immune response.

Clinical respiratory illness was compared in five homozygous chicken lines, originating from homozygous B2, B8, B12 and B19, and heterozygous B2/B12 birds after infection with either of two strains of the infectious bronchitis virus (IBV). All chickens used in these studies originated from White Leghorn and Ancona linages. IBV Gray strain infection of MHC homozygous B12 and B19 haplotype chicks resulted in severe respiratory disease compared to chicks with B2/B2 and B5/B5 haplotypes. Demonstrating a dominant B2 phenotype, B2/B12 birds were also more resistant to IBV. Respiratory clinical illness in B8/B8 chicks was severe early after infection, while illness resolved similar to the B5 and B2 homozygous birds. Following M41 strain infection, birds with B2/B2 and B8/B8 haplotypes were again more resistant to clinical illness than B19/B19 birds. Real time RT-PCR indicated that infection was cleared more efficiently in trachea, lungs and kidneys of B2/B2 and B8/B8 birds compared with B19/B19 birds. Furthermore, M41 infected B2/B2 and B8/B8 chicks performed better in terms of body weight gain than B19/B19 chicks. These studies suggest that genetics of B defined haplotypes might be exploited to produce chicks resistant to respiratory pathogens or with more effective immune responses., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Avian influenza virus (AIV) specific CD8(+) T lymphocyte responses stimulated by intramuscular administration of an adenovirus (Ad) vector expressing either HA or NP were evaluated in chickens following ex vivo stimulation by non-professional antigen presenting cells. The CD8(+) T lymphocyte responses were AIV specific, MHC-I restricted, and cross-reacted with heterologous H7N2 AIV strain. Specific effector responses, at 10 days post-inoculation (p.i.), were undetectable at 2 weeks p.i., and memory responses were detected from 3 to 8 weeks p.i. Effector memory responses, detected 1 week following a booster inoculation, were significantly greater than the primary responses and, within 7 days, declined to undetectable levels. Inoculation of an Ad-vector expressing human NP resulted in significantly greater MHC restricted, activation of CD8(+) T cell responses specific for AIV. Decreases in all responses with time were most dramatic with maximum activation of T cells as observed following effector and effector memory responses., (Copyright 2010 Elsevier Inc. All rights reserved.)

Evaluation of peripheral blood mononuclear cells (PBMC) in avian species by flow cytometry is complicated by the presence of large numbers of nucleated thrombocytes. With light scattering properties similar to those of lymphocytes, variations in the proportion of thrombocytes in PBMC can result in apparent shifts in percentages of lymphocyte subpopulations. We have applied a dual-labeling procedure for flow cytometric analyses to exclude thrombocytes from the analyzed population by labeling with K55 monoclonal antibody (MAb), which differentially labeled leukocyte and thrombocyte populations. Flow cytometric analyses with K55 labeled chicken PBMC differentiated leukocytes into three populations according to their intensity of fluorescence. Using the Kl MAb, the K55-low population was shown to consist of thrombocytes. Dual-labeling with K55 MAb and MAb specific for B lymphocyte, CD4 or CD8 markers indicated that the K55 intermediate population consisted of lymphocytes. Therefore, concentrations of CD4+ and CD8+ T lymphocytes could be determined from the lymphocyte fraction by gating specifically on the K55 intermediate cells. Selecting cross-reactive chicken MAbs that included K55, this protocol was shown to identify CD4+ and CD8+ T lymphocytes in PBMC of another avian species, the endangered Attwater's prairie chicken.

Infectious bronchitis virus (IBV), the first coronavirus described, has been a continuing problem in poultry for more than 70 years. IBV, causing a highly contagious respiratory disease in chickens, resembles the recently described severe acute respiratory syndrome virus in pathogenesis and genome organization. While previous studies demonstrated that effector and memory CD8(+) T lymphocytes are critical in controlling acute IBV infection and disease in chickens, here chicken anti-IBV antibody (IgG) secreting cells (ASC) in both peripheral blood mononuclear cells (PBMC) and spleens collected following IBV Gray infection were evaluated using an ELISPOT assay. The ASC in peripheral blood and spleens can be detected from 3 to 7 days post-infection (p.i.), which is 3-7 days earlier than anti-IBV IgG detected in the serum. The ASC frequency reached a maximum at 7-10 days p.i., and decreased more than 90% in the spleen and 70% in PBMC by 14 days p.i. The ASC levels in the PBMC then decreased gradually to 0.5 ASC/10(6) over the next 8 weeks. The higher concentration of about 20 ASC/10(6) cells in spleens may, at least partially, account for the presence of antibody in the serum although bone marrow ASC were not determined. In vitro stimulation of PBMC and splenocytes with IBV antigen demonstrated that memory B cells can be activated to secrete antibody by 3 weeks p.i. ELISPOT detection of primary B cells could be useful in the early detection of infection following infection with respiratory coronaviruses.

Infectious bronchitis has remained one of the most difficult to control diseases in poultry since it was first described in 1931. Previous studies demonstrated that primary CD8(+) T lymphocytes collected at 10 days post-infection (p.i.) are important in controlling acute infection. To further investigate the role of memory T cells in protection, T lymphocytes collected from B19/B19 chicken spleens at 2, 3, 4, and 6 weeks p.i. were transferred to six-day-old syngeneic chicks one day prior to challenging with 10(6) EID(50) of the IBV Gray strain. Memory immune T cells collected at 3 to 6 weeks p.i. provided dose responsive protection from clinical illness. The greatest protection was observed after the transfer of 10(7) T cells collected at 6 weeks p.i., whereas T cells collected at 2 weeks p.i. did not protect. Annexin-V staining of the spleen cells demonstrated that the cells collected at 2 weeks p.i. were undergoing significantly more apoptosis than cells collected at 10 days p.i. Specific antibody production in sera collected at 7 days p.i. did not correlate with protection. T cell subtype depletion demonstrated that CD8(+), not CD4(+), T cells were critical. Memory T cells can be detected in peripheral blood mononuclear cells up to at least 10 weeks p.i. These results demonstrated that IBV specific CD8(+) memory T cells generated at 3 to 6 weeks p.i. can protect syngeneic chicks from acute IBV infection.

Infectious bronchitis virus (IBV) infection and associated illness may be dramatically modified by passive transfer of immune T lymphocytes. Lymphocytes collected 10 days postinfection were transferred to naive chicks before challenge with virus. As determined by respiratory illness and viral load, transfer of syngeneic immune T lymphocytes protected chicks from challenge infection, whereas no protection was observed in the chicks receiving the MHC compatible lymphocytes from uninfected chicks. Protection following administration of T lymphocytes could be observed in chicks with three distinct MHC haplotypes: B(8)/B(8), B(12)/B(12), and B(19)/B(19). Nearly complete elimination of viral infection and illness was observed in chicks receiving cells enriched in alphabeta lymphocytes. In contrast, removal of gammadelta T lymphocytes had only a small effect on their potential to protect chicks. The adoptive transfer of enriched CD8(+) or CD4(+) T lymphocytes indicated that protection was also a function primarily of CD8-bearing cells. These results indicated that alphabeta T lymphocytes bearing CD8(+) antigens are critical in protecting chicks from IBV infection., (Copyright 2000 Academic Press.)

A recombinant DNA probe with specificity for the 3' end of genomic RNA from the Ark 99 strain of infectious bronchitis virus (IBV) was found to hybridize with extracted RNA of three strains with the Ark serotype, as well as the Mass41, Holl52, Gray, JMK, Conn, Fla and SE17 strains of IBV. Viral infection was detected in the cytoplasm of chicken embryo kidney cells inoculated with Mass41, Ark99, SE17 or two recent field isolates of IBV using in situ cytohybridization and a biotinylated probe. In vivo infections were detected in individual cells of tracheas and lungs 2,4, and 6 days after inoculation of chicks with Mass41 and Ark99. In situ hybridization of Ark99 infected tissue sections using 32P-dATP labelled probe indicated that more viral replication was present in the trachea on day 4 than either days 2 or 6; whereas more viral RNA was found in the lungs on day 6 than days 2 or 4 after inoculation.

Avian reoviruses have been associated with several pathologic conditions, but correlative relationships between genotypes and specific diseases have not been demonstrated. Six avian reoviruses (883, 176, 81-5, S1133, FC, and TX) were selected for this study, and a comparative study of the pathogenic properties of the viruses in chickens, following peroral and footpad inoculation, was carried out, along with a comparison of the electrophoretic mobility of viral genomic segments and viral proteins encoded by the gene segments. The pathogenic properties of the viruses were shown to be diverse, with three distinct pathotypes being defined: Pathotype I (883) caused only a syndrome that we have termed "transient digestive system disorder" (TDSD); Pathotype II (FC, TX, and S1133) caused only "viral arthritis syndrome" (VAS), whereas Pathotype III (176 and 81-5) caused both TDSD and VAS. Likewise, the genomes of the viruses were shown to be extremely polymorphic, with a maximum of five segments co-migrating between any two strains. Considerable variation in the electrophoretic mobility of the encoded proteins also was demonstrated with pronounced variation in the molecular size of the sigma 4 protein, the purported viral attachment protein, being evident. These results show that the genomes of avian reoviruses were extremely polymorphic, preventing correlation between genotypes and pathotypes. But these studies have provided us with the genetic elements needed to characterize the gene functions involved in viral pathogenesis.