Sandra J. Ewald

2004 nomenclature for the chicken major histocompatibility (B and Y) complex

The first standard nomenclature for the chicken (Gallus gallus) major histocompatibility (B) complex published in 1982 describing chicken major histocompatibility complex (MHC) variability is being revised to include subsequent findings. Considerable progress has been made in identifying the genes that define this polymorphic region. Allelic sequences for MHC genes are accumulating at an increasing rate without a standard system of nomenclature in place. The recommendations presented here were derived in workshops held during International Society of Animal Genetics and Avian Immunology Research Group meetings. A nomenclature for B and Y (Rfp-Y) loci and alleles has been developed that can be applied to existing and newly defined haplotypes including recombinants. A list of the current standard B haplotypes is provided with reference stock, allele designations, and GenBank numbers for corresponding MHC class I and class IIβ sequences. An updated list of proposed names for B recombinant haplotypes is included, as well as a list of over 17 Y haplotypes designated to date.

Characterization and differential expression of protein kinase C isoforms in PC12 cells Differentiation parallels an increase in PKC beta11

Nerve growth factor (NGF) treatment of PC12 cells induced a 2.8‐fold increase in protein kinase C activity concomitant with differentiation and acquisition or neurites. PKC protein isoforms were separated by sequential chromatography on DEAE‐Sephacel/hydroxylapatite. A broad peak or PKC activity eluted which corresponded to the alpha PKC isoform. In control cells, message for all six PKC isoforms was detected and expressed as epsilon>zeta=gamma>delta>beta>alpha. Western blot or whole cell lysates revealed a large increase in the beta11, while slight changes were observed for the other five PKC isoforms during treatment (12‐14 days) with NGF (50 ng/ml). In parallel, coordinate changes in the expression of the individual transcripts for the six isoforms occurred during NGF treatment. Induction and accumulation of PKC beta11 may play a role in maintenance or neuronal morphology.

The MHC of a broiler chicken line: serology, B-G genotypes, and B-F/B-LB sequences.

Abstract Although the major histocompatibility complex of chickens (encoded in the B complex) has been studied for a number of years, almost all work has focused on the White Leghorn breed. Broiler (meat-type) chickens were derived from other breeds, including Cornish and Plymouth Rock. It was our hypothesis that new B haplotypes, not previously identified in White Leghorns, might be present in lines of broiler chickens. Furthermore, alloantisera used to identify B serotypes in Leghorn lines reportedly do not work well outside the line in which they were raised, with the result that broiler B haplotypes have not been incorporated into the universal nomenclature system. Our approach was to use a panel of B alloantisera produced to identify B serotypes within a commercial broiler breeder line (designated line A). B homozygotes identified serologically were compared by B-G genotyping using restriction fragment length polymorphism analysis. Furthermore, reverse transcription-polymerase chain reaction was used to amplify variable domains of expressed B-LB and B-F genes of homozygotes of most of the B serotypes in Line A, followed by cloning and nucleotide sequence determination. Comparison of B-LB and B-F sequences with standard Leghorn haplotypes demonstrated the existence of new alleles of B-L and B-F in a broiler breeder line, as well as the presence of alleles previously identified in Leghorns. In some cases, Leghorn-type alleles were in linkage with different B-G alleles in the broiler line than the common haplotypic associations found in Leghorn lines.

Association of Mx1 Asn631 variant alleles with reductions in morbidity, early mortality, viral shedding, and cytokine responses in chickens infected with a highly pathogenic avian influenza virus

Myxovirus-resistance (Mx) proteins are produced by host cells in response to type I interferons, and some members of the Mx gene family in mammals have been shown to limit replication of influenza and other viruses. According to an early report, chicken Mx1 variants encoding Asn at position 631 have antiviral activity, whereas variants with Ser at 631 lack activity in experiments evaluating Mx1 complementary DNA (cDNA) expressed ectopically in a cell line. We evaluated whether the Mx1 631 dimorphism influenced pathogenesis of highly pathogenic avian influenza virus (HPAIV) infection in chickens of two commercial broiler lines, each segregating for Asn631 and Ser631 variants. Following intranasal infection with HPAIV strain A/Chicken/Queretaro/14588-19/1995 H5N2, chickens homozygous for Asn631 allele were significantly more resistant to disease based on early mortality, morbidity, or virus shedding than Ser631 homozygotes. Higher amounts of splenic cytokine transcripts were observed in the Ser631 birds after infection, consistent with higher viral loads seen in this group and perhaps contributing to their higher morbidity. Nucleotide sequence determination of Mx1 cDNAs demonstrated that the Asn631 variants in the two chicken lines differed at several amino acid positions outside 631. In vitro experiments with a different influenza strain (low pathogenicity) failed to demonstrate an effect of Mx1 Asn631 on viral replication suggesting that in vivo responses may differ markedly from in vitro, or that choice of virus strain may be critical in demonstrating effects of chicken Mx1. Overall, these studies provide the first evidence that Mx1 has antiviral effects in chickens infected with influenza virus.

The chicken BF1 (classical MHC class I) gene shows evidence of selection for diversity in expression and in promoter and signal peptide regions

The chicken MHC B contains two classical class I genes, BF1 and BF2, with the exception of two related haplotypes lacking BF1 due to insertion/rearrangement. In light of functional specialization of BF1 and BF2 molecules, we were interested in evaluating their relative expression at the mRNA level. We evaluated several MHC haplotypes for class I gene expression by RT-quantitative PCR. BF1 transcript levels were approximately two- to fivefold lower than BF2, with the exception of one haplotype in which BF1 expression was very low. To investigate molecular explanations for differences in BF locus or allele expression, we determined nucleotide sequences of Enhancer A and proximal promoter elements of nine different BF1 alleles, as well as their signal peptide sequences. Results showed that all BF1 alleles exhibit conservation of most of the identified promoter elements, but divergence from the Enhancer A sequence identified in the more highly expressed BF2 locus. Nonetheless, extensive BF1 allelic polymorphism was found in the promoter region and in the signal peptide, with two strongly separated allelic lineages identified for both. Patterns of promoter lineages, signal peptide lineages, and exon 2 mature protein coding sequences in individual BF1 alleles suggest that recombination among these elements has contributed to diversification of BF1 alleles. Finally, identification of a novel inactivating mutation in one BF1 allele suggests past selective pressure to eliminate BF1 function.

Serological evidence of chicken infectious anemia virus in the United States at least since 1959.

Abstract A retrospective, serological survey was performed to determine an approximate time frame for when chickens were first exposed to chicken anemia virus (CAV) in the southeastern United States. A serum collection covering most of the period between 1959 and 2005 was available for the present study. These sera were obtained from adult chicken flocks that were maintained in experimental chicken farms at Auburn Universitys Department of Poultry Science. Sera were tested for the presence of CAV-specific antibodies using a commercially available competitive enzyme-linked immunosorbent assay (ELISA) kit. Values <0.6 were considered positive. Fresh sera obtained from hens in 2005 showed 45.5% negative and 54.5% positive for CAV antibodies. The assessment of serum samples covering the time period of 1959 through 1979 resulted in most sera being positive for CAV antibodies. The percentage of positive samples between years varied from 43% to 100%. These serological results support assumptions based on circumstantial evidence that CAV must have been present in the United States long before its first isolation in 1989.

B-haplotype control of CD4/CD8 subsets and TCR Vβ usage in chicken T lymphocytes

The major histocompatibility (B) complex of the chicken contains genes similar to Class I (B-F) and Class II (B-L beta) genes in mammals, as well as a highly-polymorphic gene family (B-G) whose exact function is not known. Specific B-haplotypes are strongly associated with resistance to a number of infectious diseases, and with immune responses to soluble and cellular antigens. In mammals, Class I and Class II molecules control development of the T cell repertoire, including selection of CD4+ and CD8+ T cells. One study of chickens reported that low CD4:CD8 ratio was associated with the B4 haplotype, which shares expressed B-F/B-L genes with the B13 haplotype. In studies reported here, chickens of two haplotypes carried in the Auburn R line, B302 and B305 (which is B13-related), were evaluated for percentages of T cells expressing the CD4, CD8, CD3, TCR1, TCR2 and TCR3 antigens in peripheral blood lymphocytes (PBL), thymus, and spleen. These two haplotypes were chosen for comparison because they differ in resistance to Mareks disease (MD) and are closely-related in B-F and B-L genes by restriction fragment length polymorphism analyses. Homozygous birds of each B haplotype were produced from crosses of (B302 x B305)F1 sires and dams. PBL, thymocytes, and splenocytes from B302 homozygotes had higher CD4:CD8 ratios than B305 homozygotes. However, CD4:CD8 ratio differences could not be attributed to haplotype-controlled differences in V beta usage within CD4/CD8 subsets, as has been described for certain V beta families in mice and humans. These results indicate that thymic selection events involving CD4 and CD8 subsets and TCR V beta usage are controlled by a gene or genes closely-linked to the B-complex, which may or may not be Class I or Class II genes.

Thymic alterations in feline GM1 gangliosidosis.

GM1 gangliosidosis is an inherited metabolic disease characterized by progressive neurological deterioration with premature death seen in children and numerous animals, including cats. We have observed that thymuses from affected cats greater than seven months of age (GM1 mutant cats) show marked thymic reduction compared to age-matched normal cats. The studies reported here were done to describe alterations in the thymus prior to (less then 90 days of age) and during the development of mild (90 to 210 days of age) to severe (greater than 210 days of age) progressive neurologic disease and to explore the pathogenesis of the thymic abnormality. Although histologic examination of the thymus from GM1 affected cats less than 210 days of age showed no significant differences from age-matched control cats, thymuses from GM1 mutant cats greater than 210 days of age were significantly reduced in size (approximately 3-fold). Histologic sections of lymph nodes, adrenal glands, and spleens from GM1 gangliosidosis-affected cats showed no significant differences. Flow cytometric analyses showed a marked decrease in the percentage of immature CD4+CD8+ thymocytes (p < 0.001) and significantly increased CD4-CD8+ cells (p < 0.01) in GM1 mutant cats greater than 210 days of age when compared to normal age matched cats. Co-labelling with CD4, CD8, and CD5 indicated an increase in the percentage of GM1 mutant cat thymocytes at this age which were CD5high, suggesting the presence of more mature cells. Cytometric analyses of subpopulations of peripheral lymphocytes indicated an increase in CD4-CD8+ cells (p < 0.05) with concurrent decreases in CD4+CD8- and CD4-CD8- cells (which were not significant). Similar analyses of thymocyte and lymphocyte subpopulations from cats < 210 days of age showed no significant differences between GM1 mutant and normal cells. GM1 mutant cats at all ages had increased surface binding of Cholera toxin B on thymocytes, indicating increased surface GM1 ganglioside expression. Increases were highly significant in GM1 mutant cats greater than 210 days of age. In situ labelling for apoptosis was increased in GM1 mutant cats between 90 to 200 days of age when thymic masses were within normal limits. In GM1 mutant cats over 200 days of age, decreased labelling was observed when thymic mass was reduced and the CD4+CD8+ subpopulation, known to be very susceptible to apoptosis, was significantly decreased. These data describe premature thymic involution in feline GM1 gangliosidosis and suggest that increased surface GM1 gangliosides alters thymocyte development in these cats.

Interferon α–Induced Inhibition of Infectious Bursal Disease Virus in Chicken Embryo Fibroblast Cultures Differing in Mx Genotype

Abstract Interferon (IFN)-induced antiviral activity in cells forms an important early line of defense against viral pathogens. IFN-induced mediators are well documented in mammals, with one of the best-characterized antiviral proteins being Mx. In chickens, many alleles of Mx have been described, but functionally only the polymorphism at a site encoding residue 631 in the protein determines differential antiviral activity against vesicular stomatitis virus and influenza virus in transfection experiments. The role of chicken Mx has not been assessed with regard to infectious bursal disease virus (IBDV), an important pathogen of chickens. To examine the role of chicken IFNα and the Mx631 single-nucleotide polymorphism against IBDV, chicken embryo fibroblast cultures (CEF) that differed in Mx genotype (antivirally positive Mx Asn631 or antivirally negative Mx Ser631) were treated with IFNα and viral yield was assessed following infection with D78 vaccine-strain IBDV. IFNα was shown to have strong antiviral activity in this system in terms of reduced virus yield. Furthermore, the reduction in viral yield did not differ significantly among Mx genotypes, indicating that Mx Asn631 is not a pivotal determinant of resistance to this strain of IBDV in CEF.

Pathogenesis of Infectious Bronchitis Virus in Vaccinated Chickens of Two Different Major Histocompatibility B Complex Genotypes

Abstract Infectious bronchitis (IB) disease progression in vaccinated chickens after challenge was evaluated in a single commercial line of layer chickens presenting two different major histocompatibility complex (MHC) B complex genotypes. MHC B genotypes were determined by DNA sequence-based typing of BF2 alleles. In total, 33 B2/B15 and 47 B2/B21 chickens were vaccinated with an Ark-type IB virus (IBV) attenuated vaccine and challenged with Ark-type IBV field isolate AL/4614/98 14 days later. Additional chickens of both genotypes served as unvaccinated/challenged and unvaccinated/nonchallenged controls. Clinical signs, histopathologic analysis, detection of IBV genomes in tears, and IBV-specific immunoglobulin A (IgA) in tears were used to evaluate disease progression and immune response. The incidence of IBV respiratory signs was significantly higher in B2/21 than in B2/B15 MHC genotype birds. However, neither the severity and duration of respiratory signs nor the severity and incidence of histologic lesions differed significantly with MHC genotype. The levels of IBV-specific IgA in tears of vaccinated and challenged chickens did not differ significantly between MHC genotypes. IBV genomes were present in the tears of vaccinated and challenged birds, and the incidence of detectable IBV genomes did not vary significantly with MHC B genotype. From an applied perspective, these results indicate that vaccinated commercial outbred chickens with these MHC genotypes are equally resistant to IBV.