Characterization of two distinct disulfide-linked B-G molecules in the chicken

Alloantisera specific for B-G antigens recognized a complex of molecules of apparent molecular weights of 90 and 98 Kd under nonreducing conditions and molecules of 40, 44, and 48 Kd under reducing conditions on both embryo- and adult-derived peripheral red blood cells (RBC). The chicken B-G molecules produced a unique two-dimensional "diagonal" pattern. Two antisera permitted the characterization of the complex B-G molecular profile as a homodimer composed of 48-Kd subunits and as a heterodimer composed of 40- and 44-Kd subunits. A rabbit antiserum produced against B-G molecules preferentially recognized the 48-Kd reduced molecules, suggesting that the 90-Kd molecule was a homodimer composed of two 48-Kd molecules. One B-G reagent was capable of recognizing only the 98-Kd nonreduced B-G molecule that gave rise to 40- and 44-Kd molecules under reducing conditions, suggesting that the 98-Kd molecule was a heterodimer composed of 44- and 40-Kd subunits. Adult chicken B-G2 molecules produced a variety of two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) patterns depending on the characteristics of the reagent employed in the immunoprecipitation. B-G molecules were immunoprecipitated from primitive and definitive chicken RBCs but not from any nonerythroid cells tested. B-G molecules were not expressed by avian erythroblastosis virus (AEV)-transformed erythroleukemia cells, nor were they induced to appear with butyric acid-induced erythroid differentiation.     

Biochemical identification of B-F and B-G allelic variants of the chicken major histocompatibility complex

Biochemical methods were used to analyse B-F and B-G antigens of the chicken major histocompatibility complex (MHC). In a panel of 12 inbred or partially inbred chicken lines the MHC haplotypes, originally defined by serological and histogenetical methods, were compared. Using monoclonal 18-6G2, allele-specific B-G patterns were obtained by immunoblotting. Comparison of B-G12 and B-G2 revealed a shared banding pattern, but additional products were detected for B-G12. The B-F products of B2 and B12 had identical IEF patterns. The identical B-F products and partially shared B-G products might explain the serological cross-reaction between these haplotypes. In addition, the IEF pattern of B-F21 appeared similar to B-F2 and B-F12, but the partial proteolysis map showed a clear difference. Although two B-F bands could be detected per haplotype, no evidence for the expression of more than one B-F locus was found. The biochemical methods enabled a precise definition of expressed MHC products and can be a useful tool for the identification of B-alleles in other chicken lines or outbred chickens for their MHC antigens.     

Genotypic variability at the major histocompatibility complex (B and Rfp-Y) in Camperos broiler chickens

Evidence for the importance of major histocompatibility complex (MHC) genotype in immunological fitness of chickens continues to accumulate. The MHC B haplotypes contribute resistance to Marek's and other diseases of economic importance. The Rfp-Y, a second cluster of MHC genes in the chicken, may also contribute to disease resistance. Nevertheless, the MHC B and Rfp-Y haplotypes segregating in broiler chickens are poorly documented. The Camperos, free-range broiler chickens developed in Argentina, provide an opportunity to evaluate MHC diversity in a genetically diverse broiler stock. Camperos are derived by cross-breeding parental stocks maintained essentially without selection since their founding. We analysed 51 DNA samples from the Camperos and their parental lines for MHC B and Rfp-Y variability by restriction fragment pattern (rfp) and SSCP typing methods for B-G, B-F (class Ia), B-Lbeta (class II) and Y-F (class Ib) diversity. We found evidence for 38 B-G genotypes. The Camperos B-G patterns were not shared with White Leghorn controls, nor were any of a limited number of Camperos B-G gene sequences identical to published B-G sequences. The SSCP assays provided evidence for the presence of at least 28 B-F and 29 B-Lbeta genotypes. When considered together B-F, B-L, and B-G patterns provide evidence for 40 Camperos B genotypes. We found even greater Rfp-Y diversity. The Rfp-Y class I-specific probe, 163/164f, revealed 44 different rfps among the 51 samples. We conclude that substantial MHC B and Rfp-Y diversity exists within broiler chickens that might be drawn upon in selecting for desirable immunological traits.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.     

Molecular characterization of major histocompatibility complex (B) haplotypes in broiler chickens

In Leghorn (laying) chickens, susceptibility to a number of infectious diseases is strongly associated with the major histocompatibility ( B ) complex. Nucleotide sequence data have been published for six class I ( B-F ) alleles and for class II ( B-Lβ ) alleles or isotypes from 17 Leghorn haplotypes. It is not known if classical B-L or B-F alleles in broilers are identical, at the sequence level, to any Leghorn alleles. This report describes molecular and immunogenetic characterization of two haplotypes from commercial broiler breeder chickens that were originally identified by serology as a single haplotype, but were differentiated serologically in the present work. The two haplotypes, designated B ^A4 and B ^A4variant, shared identical B-G restriction fragment length polymorphism patterns, but differed in one B-Lβ fragment that cosegregated with the serological B haplotype. Furthermore, the nucleotide sequences of the highly variable exons of an expressed B-LβII family gene and B-F gene from the two haplotypes were markedly different from each other. Both the B-LβII family and B-F gene sequences from the B ^A4 haplotype were identical to the sequences obtained from the reference B ²¹ haplotype in Leghorns; however, in the B ^A4 haplotype the B-Lβ ²¹ and B-F ²¹ alleles were in linkage with B-G alleles that were not G ²¹. The nucleotide sequences from B ^A4variant were unique among the reported chicken B-LβII family and B-F alleles.     

Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus

The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition of S-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-gamma) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition of N(G)-monomethyl L-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-gamma plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC], B(21)B(21)) and MDV-susceptible S(13) (MHC, B(13)B(13)) and P2a (MHC, B(19)B(19)) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-gamma plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-gamma plus LPS reduced the inhibition. MDV infection of chickens treated with S-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.     

Live haploid-diploid and other unusual mosaic chickens (Gallus domesticus)

Sixteen haploid-diploid chickens, four diploid-triploids, and one haploid-diploid-triploid chicken have been found in a layer line of crossbred origin. Live haploid-euploid vertebrates have not been reported previously. Flow cytometric analysis revealed 1-80% haploid red blood cells (RBCs) in the haploid-euploid chickens and 5-30% triploid RBCs in the diploid-triploids. Mosaic cell lines were also found in leukocytes and many tissues. The haploid-diploid chickens were all phenotypically normal and fertile, but the phenotype and fertility of the diploid-triploid chickens depended on their sex-chromosome complements and the ratio of diploid to triploid cells. The haploid cell lines are thought to originate from supernumerary spermatozoa, and the diploid-triploids from binucleated oocytes with meiotic errors. It is likely that genetic factors are partly responsible for the occurrence of these mosaic chickens.     

Antigens similar to major histocompatibility complex B-G are expressed in the intestinal epithelium in the chicken

A monoclonal antibody directed against the erythrocytic B-G antigens of the major histocompatibility complex (MHC) of the chicken, an antiserum raised against purified erythrocytic B-G protein, and a cDNA probe from the BeckmanB-G subregion were used to look for evidence of the expression ofB-G genes in tissues other than blood. Evidence has been found in northern hybridizations, in immunoblots, and in immunolabeled cryosections for the presence of B-G-like antigens in the duodenal and caecal epithelia. Additional B-G-like molecules may be expressed in the liver as well. The BG-like molecules in these tissues appear larger and somewhat more heterogeneous than the B-G antigens expressed on erythrocytes. Further characterization of these newly recognized B-G-like molecules may help to define a function for the enigmatic B-G antigens of the MHC. al. 1977; Miller et al. 1982, 1984; Salomonsen et al. 1987; Kline et al. 1988), and in the multiplicity of B-G restriction fragment patterns found in genomic DNA from different haplotypes (Goto et al. 1988; Miller et al. 1988; Chaussé et al. 1989). The B-G antigens have contributed, together with the B-F (class I) and B-L (class II) antigens, to the definition of over 27 B system haplotypes in experimental flocks (Briles et al. 1982). Yet the function of the B-G antigens remains entirely unknown. No mammalian counterparts have been identified, although the possibility remains that there may be similar antigens among the blood group systems of mammals. In an effort to define a function of the B-G antigens, a recently cloned B-G sequence (Miller et al. 1988; Goto et al. 1988) and antibodies to the B-G polypeptides (Miller et al. 1982, 1984) were used to examine other tissues for evidence of B-G expression.     

High-resolution typing for chicken BF2 (MHC class I) alleles by automated sequencing

Sequence-based typing (SBT) was developed for major histocompatibility complex (MHC) class I and class II alleles in humans. We report here the development and application of a SBT method for alleles of the chicken BF2 locus (the more polymorphic of the two MHC class I loci in chickens). Exon 2 of the BF2 gene was selectively amplified from genomic DNA using a BF2 locus-specific PCR primer. Exon 2 sequences were sufficient to identify the 21 distinct BF2 alleles described in standard B haplotypes of Leghorns and in commercial broiler-breeder lines. Sixty-six samples from MHC typed, pedigreed chickens were tested, including 50 different heterozygous combinations. BF2 sequences from all B homozygotes were successfully amplified, and all combinations of BF2 alleles in heterozygotes were co-amplified equally. The two different BF2 alleles in heterozygotes could be identified unambiguously by distinct sequence motif patterns. In tests of samples of unknown B genotype in commercial broiler-breeder flocks, we identified expected BF2 alleles as well as an allele not previously encountered in one of the lines.     

Phage types and pulsed-field gel electrophoresis patterns of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis isolated from humans and chickens

We analyzed 66 Salmonella Enteritidis isolates in 2002. Thirty isolates were obtained from human patients with diarrhea, and 36 were obtained from chickens. A total of ten phage types (PT) were identified in the human and chicken isolates. PT1 and PT21 were the predominant PTs in both the human (20% and 13%) and chicken (17% and 47%) isolates. Twelve pulsotypes were generated by PFGE and divided into two major groups. Most of the PFGE types were categorized into cluster group 1. Eighteen chicken isolates in cluster group 1 showed high-level genetic association (>95%) with 22 other human isolates. Additionally, six chicken isolates from cluster group 2 showed fairly high-level genetic association (>95%) with the other seven human isolates. The highest levels of genetic association in humans and chickens were seen with A5-PT21 (11 isolates), A2-PT1 (7 isolates), and B1-PT4 (6 isolates). The Pulsed-Field Gel Electrophoresis (PFGE) and phage typing provided conclusive evidence that human Salmonella infections are attributable to the consumption of contaminated chicken.     

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes.

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.     

A PCR method for typing B-LβII family (class II MHC) alleles in broiler chickens

Certain haplotypes of the major histocompatibility (B) complex are strongly associated with resistance or susceptibility to several infectious diseases in Leghorn chickens. Identification of chicken haplotypes based on the nucleotide sequence of B complex loci could provide more precise identification of haplotypes than traditional serological methods. We report the development and application of polymerase chain reaction with sequence specific primers (PCR-SSP) to type broiler chicken B haplotypes based on the DNA sequence of B-L beta II family genes. Five well-defined standard B haplotypes from White Leghorns and 12 recently characterized B haplotypes from a broiler breeder line were used to develop the test system. The B-L beta II family loci were amplified from genomic DNA by B-L beta II family specific primers and then characterized by PCR-SSP. In total, ten pairs of primers, derived from the sequences of expressed B-L beta II family alleles, were used in the PCR typing test to discriminate the chicken B haplotypes identified previously by serological means. The PCR-SSP showed that each haplotype had a different amplification pattern, except those haplotypes known or suspected to have the same B-L beta alleles. Cloning and sequencing of the family specific PCR products indicated that two loci in the B-L beta II family, presumably B-L beta I and B-L beta II, were amplified. Finally, B-L beta PCR-SSP typing was used in combination with B-G RFLP analyses to characterize unusual (variant) B serotypes; the results indicate that some of these are natural recombinants within the B complex.     

Chemical and immunological characterization of developmentally expressed chicken erythroid surface membrane antigens

The expression of two hematopoietic-lymphoid membrane antigens, referred to as chicken fetal antigen (CFA) and chicken adult antigen (CAA) were investigated on primitive and definitive peripheral red blood cells (RBC) from different-aged chickens using chemical and immunological techniques. Differential adsorptions of antisera specific for adult RBC membrane antigens permitted the serological dissection of CAA into eight antigenic determinants. CFA and CAA were assayed by hemagglutination, hemolysis, and immune precipitation of radioiodinated surface antigens of RBC from different-aged chickens. Primitive RBC express CFA, while definitive RBC express three distinct phenotypes: CFA, both CFA and CAA, or CAA, depending on the developmental age of the chicken from which the RBC were obtained. When solubilized membrane extracts of radioiodinated peripheral RBC from chickens at 5 and 18 days embryonic development (E5 and E18, respectively), 13 days posthatch development (H13), and adult chickens were immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the major antigen detected by anti-CFA sera was associated with proteins having apparent molecular weights (M_r) of 50,000 daltons (50 kd). The antigens detected by anti-CAA sera were associated with proteins having apparent M_r of 102, 81, 48, and 43 kd.     

A genome-wide survey of copy number variation regions in various chicken breeds by array comparative genomic hybridization method

The discovery of copy number variation (CNV) in the genome has provided new insight into genomic polymorphism. Studies with chickens have identified a number of large CNV segments using a 385k comparative genomic hybridization (CGH) chip (mean length >140 kb). We present a detailed CNV map for local Chinese chicken breeds and commercial chicken lines using an Agilent 400k array CGH platform with custom-designed probes. We identified a total of 130 copy number variation regions (CNVRs; mean length = 25.70 kb). Of these, 104 (80.0%) were novel segments reported for the first time in chickens. Among the 104 novel CNVRs, 56 (53.8%) of the segments were non-coding sequences, 65 (62.5%) showed the gain of DNA and 40 (38.5%) showed the loss of DNA (one locus showed both loss and gain). Overlapping with the formal selective sweep data and the quantitative trait loci data, we identified four loci that might be considered to be high-confidence selective segments that arose during the domestication of chickens. Compared with the CNVRs reported previously, genes for the positive regulation of phospholipase A2 activity were discovered to be significantly over-represented in the novel CNVRs reported here by gene ontology analysis. Availability of our results should facilitate further research in the study of the genetic variability in chicken breeds.     

Prediction and identification of T cell epitopes in the H5N1 influenza virus nucleoprotein in chicken

T cell epitopes can be used for the accurate monitoring of avian influenza virus (AIV) immune responses and the rational design of vaccines. No T cell epitopes have been previously identified in the H5N1 AIV virus nucleoprotein (NP) in chickens. For the first time, this study used homology modelling techniques to construct three-dimensional structures of the peptide-binding domains of chicken MHC class Ι molecules for four commonly encountered unique haplotypes, i.e., B4, B12, B15, and B19. H5N1 AIV NP was computationally parsed into octapeptides or nonapeptides according to the peptide-binding motifs of MHC class I molecules of the B4, B12, B15 and B19 haplotypes. Seventy-five peptide sequences were modelled and their MHC class I molecule-binding abilities were analysed by molecular docking. Twenty-five peptides (Ten for B4, six for B12, two for B15, and seven for B19) were predicted to be potential T cell epitopes in chicken. Nine of these peptides and one unrelated peptide were manually synthesized and their T cell responses were tested in vitro. Spleen lymphocytes were collected from SPF chickens that had been immunised with a NP-expression plasmid, pCAGGS-NP, and they were stimulated using the synthesized peptides. The secretion of chicken IFN-γ and the proliferation of CD8(+) T cells were tested using an ELISA kit and flow cytometry, respectively. The significant secretion of chicken IFN-γ and proliferation of CD8(+) T lymphocytes increased by 13.7% and 11.9% were monitored in cells stimulated with peptides NP(89-97) and NP(198-206), respectively. The results indicate that peptides NP(89-97) (PKKTGGPIY) and NP(198-206) (KRGINDRNF) are NP T cell epitopes in chicken of certain haplotypes. The method used in this investigation is applicable to predicting T cell epitopes for other antigens in chicken, while this study also extends our understanding of the mechanisms of the immune response to AIV in chicken.     

Structural and genetic studies on chicken 7S immunoglobulin allotypes. IV. The presence of an unexpected chicken immunoglobulin heavy chain allotype: subclass or pseudoallele?

Low concentrations of allotypic specificity CS-1.1 were detected in the sera of two inbred chicken lines [University of California, Davis (UCD) 7 and Regional Poultry Research Laboratory 15I4] previously reported to lack this specificity. The CS-1.1 alloantigen in 15I4 chickens has the same specificity as the major allotype in a line of chickens (UCD 2) in which it was initially defined. In 15I4 chickens, CS-1.1 allotype is present on a population of molecules distinct from those which carry the major allotype; thus a second 7S Ig H chain locus, CS-2, is proposed. The concentration of CS-1.1-bearing molecules determined by two different methods was 7 microgram/ml and 230 microgram/ml in 15I4, whereas UCD 2 chickens had 4 mg/ml of CS-1.1 molecules. The levels of CS-1.1 inhibitory activity in 15I4 birds remained relatively constant over a 30-day period. The presence of two 7S Ig populations in 15I4 chickens may be interpreted as evidence either for 7S Ig subclasses with shared allotypes or for a pseudoallelic organization of genes controlling expression of 7S Ig H chains. The results were consistent with the presence of redundant C region genes, differing in allotypes, whose expression is under the control of an as yet undefined regulatory mechanism.