Immune effects of chicken non-MHC alloantigens.

Alloantigen systems are a broad group of molecules found on various cell types, including erythrocytes and lymphocytes. These alloantigens, identified via specific polyclonal or monoclonal antibodies or molecular methods, have demonstrated effects on immune responses. Erythrocyte alloantigens include the A, B, C, D, E, H, I, J, K, L, N, P, and R systems. Highly polymorphic alloantigen B has been identified as the chicken major histocompatibility complex (MHC). The other twelve systems have a variable degree of polymorphism as well as impact on immune measurements or responses against pathogens. Selection for immune characters altered allele frequencies for particular alloantigen systems. Three lymphocyte alloantigens, Bu-1, Ly-4 and Th-1 have more limited polymorphism but still influence responses against viral pathogens, Rous sarcoma virus and Marek's disease. Together, these erythrocyte and lymphocyte systems contribute to the overall immunity. Identification of the specific alloantigen proteins remains crucial to understanding their immune contribution.

Research Note: Rous sarcoma growth differs among congenic lines containing major histocompatibility (B) complex recombinants.

New arrangements of chicken major histocompatibility complex (MHC) class I BF and class IV BG genes are created through recombination. Characterizing the immune responses of such recombinants reveals genes or gene regions that contribute to immunity. Inbred Line UCD 003 (B17B17) served as the genetic background for congenic lines, each containing a unique MHC recombinant. After an initial cross to introduce a specific recombinant, 10 backcrosses to the inbred line produced lines with 99.9% genetic uniformity. The current study compared Rous sarcoma virus (RSV) tumor growth in 5 congenic lines homozygous for MHC recombinants (003.R1 = BF24-BG23, 003.R2 = BF2-BG23, 003.R4 = BF2-BG23, 003.R5 = BF21-BG19, and 003.R13 = BF17-BG23). Two experiments used a total of 70 birds from the 5 congenic lines inoculated with 20 pock forming units of RSV subgroup C at 6 wk of age. Tumor size was scored 6 times over 10 wk postinoculation followed by assignment of a tumor profile index (TPI) based on the tumor size scores. Tumor growth over time and rank transformed TPI values were analyzed by least squares ANOVA. Tumor size increased over the experimental period in all genotypes through 4 wk postinoculation. After this time, tumor size increased in Lines 003.R1, plateaued in Lines 003.R2, 003.R4, and 003.R13, and declined in 003.R5. Tumor growth over time was significantly lower in Line 003.R5 compared with all other genotypes. In addition, Line 003.R5 chickens had significantly lower TPI values compared with Lines 003.R2, 003.R4, and 003.R13. The TPI of Line 003.R1 did not differ significantly from any of the other genotypes. The BF21 in Line 003.R5 produced a greater response against subgroup C RSV tumors than did BF24, found in 003.R1; BF2 found in 003.R2 and R4 as well as BF17 found in 003.R13.

Major histocompatibility complex recombinant R13 antibody response against bovine red blood cells.

Recombination within the chicken major histocompatibility complex (MHC) has enabled more precise identification of genes controlling immune responses. Chicken MHC genes include BF, MHC class I; BL, MHC class II; and BG, MHC class IV that are closely linked on chromosome 16. A new recombination occurred during the 10th backcross generation to develop congenic lines on the inbred Line UCD 003 (B17B17) background. Recombinant R13 (BF17-BG23) was found in a single male chick from the Line 003.R1 (BF24-BG23) backcross. An additional backcross of this male to Line UCD 003 females increased the number of R13 individuals. Two trials tested this new recombinant for antibody production against the T cell-dependent antigen, bovine red blood cells. Fifty-one progeny segregating for R13R13 (n = 10), R13B17 (n = 26), and B17B17 (n = 15) genotypes were produced by a single R13B17 male mated to 5 R13B17 dams. One milliliter of 2.5% bovine red blood cell was injected intravenously into all genotypes at 4 and 11 wk of age to stimulate primary and secondary immune responses, respectively. Blood samples were collected 7 d after injection. Serum total and mercaptoethanol-resistant antibodies against bovine red blood cell were measured by microtiter methods. The least squares ANOVA used to evaluate all antibody titers included trial and B genotype as main effects. Significant means were separated by Fisher's protected least significant difference at P < 0.05. R13R13 chickens had significantly lower primary total and mercaptoethanol-resistant antibodies than did the R13B17 and B17B17 genotypes. Secondary total and mercaptoethanol-resistant antibodies were significantly lower in R13R13 chickens than in R13B17 but not B17B17 chickens. Gene differences generated through recombination impacted the antibody response of R13 compared with B17. Secondary antibody titers were not substantially higher than the primary titers suggesting that the memory response had waned in the 7-wk interval between injections. Overall, the results suggest that the lower antibody response in R13R13 homozygotes may be caused by recombination affecting a region that contributes to higher antibody response.

Marek's disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines.

Lines of chickens selected from a common ancestral population for either resistance or susceptibility to Marek's disease developed contrasting frequencies of particular B alloalleles. Comparison of inoculated sibs in backcross-families revealed that the B alloalleles characterizing the two lines accounted for an eightfold difference in tumor incidence. This genetic difference in tumorigenesis associated with the alloalleles of the major histocompatibility complex is probably expressed through the cell-mediated immune system.

Susceptibility to an avian leukosis-sarcoma virus: close association with an erythrocyte isoantigen.

A dominant gene for susceptibility to early steps of cellular infection by subgroup B avian leukosis-sarcoma viruses is associated with the presence of an erythrocyte isoantigen. This gene may control both an isoantigen and a cell membrane receptor for an oncogenic virus.

Resistance to a malignant lymphoma in chickens is mapped to subregion of major histocompatibility (B) complex.

A genetic recombinant within the major histocompatibility (B) complex of the chicken has revealed the chromosomal subregion effecting resistance to Marek's disease--a malignant lymphoma induced by a herpesvirus. The recombinant, BF21-G19, occurred spontaneously among the progeny of a male heterozygous for resistant BF21-G21 and susceptible BF19-G19 haplotypes. Exposure to Marek's disease of families segregating for the recombinant showed that this new F-G arrangement conferred a level of resistance equivalent to that of the resistant parental haplotype. Thus, a gene, or genes, within or closely linked to the B-F region of the B complex appears to be responsible for the observed resistance to Marek's disease.

Rous sarcoma growth in lines congenic for major histocompatibility (B) complex recombinants.

Rous sarcoma virus (RSV)-induced tumor growth was examined in congenic lines of chickens with different major histocompatibility (B) complex recombinant haplotypes on the highly inbred line UCD 003 (B17B17) genetic background. Males bearing an individual B complex recombinant were mated to UCD 003 females followed by 10 backcross generations. Matings among heterozygotes for each recombinant produced homozygous chickens estimated to contain 99.9% of the line UCD 003 background genome. The 5 lines having distinct serologically identified MHC recombinant haplotypes, which arose from separate recombinational events, were as follows: 003.R1, 003.R2, 003.R4, 003. R5, and 003.R6. Chicks from each of the recombinant lines were challenged with 10 pfu subgroup A RSV at 6 wk of age. Tumors were scored for size 6 times over 10 wk postinoculation. Each bird was assigned a tumor profile index (TPI) based on the 6 tumor size scores. Hatch and B genotype were main effects in the statistical analysis. Least squares ANOVA was used to evaluate rank-transformed TPI values and mean tumor sizes through a repeated measures design. Tumor growth and TPI values were greater for 003.R1 and 003.R4 chickens than for the other 3 congenic lines. Among serologically similar recombinants 003.R2 and 003.R4, higher tumor growth and TPI in 003.R4 indicate unique genetic variation affecting RSV tumors compared with 003.R2. The similar tumor growth of 003.R5 and 003.R6 chickens, which have BF/BL21 but different BG regions, demonstrated no BG effect on RSV tumors.

Developmental expression of alloantigen systems in the chicken.

The different allelic forms of nine non-Mhc alloantigen systems of the chicken were examined for developmental expression on erythrocytes isolated from embryos and young chicks. Polyclonal alloantisera raised against the different antigens were used to detect these antigens on the cell surface by hemagglutination as well as by indirect immunofluorescence. The developmental stage of initial expression on erythrocytes for each of the genetic systems investigated (i.e., A, E, C, D, H, I, K, L and P) varied from day 4 to day 14 of incubation. The different antigens of each system appeared simultaneously at a particular stage of development except for those of the I system, where the I8 allelic form appeared earlier than I2.

Graft-versus-host response in sex-linked dwarf, autosomal dwarf and control K strain chickens.

Two independent experiments were conducted to examine the ability of 1) the autosomal-dwarf (ADW) strain and 2) the sex-linked (SLD) strain chicken to make a Graft-versus-Host (GvH) response. In each experiment the GvH response of the dwarf strain was compared to the GvH response of a normal growing control strain, the Cornell K strain chicken. All 3 strains were homozygous B15/B15 at the major histocompatibility complex. GvH responsiveness of peripheral blood lymphocytes (PBL) from females of each strain was assessed at various intervals from 1.5 to 20 months of age using the chorio-allantoic membrane (CAM) assay. The GvH response in female chickens from the ADW strain was significantly higher than in K strain females after the birds had reached sexual maturity (after 5.5 months). The GvH response in females from the SLD strain was, however, significantly lower than in the K strain females throughout the experiment. All strains tended to have a biphasic GvH response. There was a significant increase in GvH responsiveness from 1.5 to 5.5 months of age in chicks from all strains. In the SLD and K strain chickens, this was followed by a drop in the GvH response until 8.5 months of age. In 16- to 20-month-old SLD and K strain hens, the ability to mount a GvH response returned to levels observed in younger (5.5 months) pullets. The GvH responsiveness of the ADW strain remained at a constant level from 5.5 to 12 months of age. However, a second peak in GvH responsiveness was observed in 16-month-old ADW strain hens. Strain differences in GvH responsiveness may be due to the known hormonal abnormalities in the dwarf strains. The suitability of these dwarf strains for the study of endocrine-immune interrelationships is discussed.

Cellular expression of MHC glycoproteins on erythrocytes from normal and aneuploid chickens.

The major histocompatibility complex (MHC) in the chicken (B-complex) encodes glycoproteins homologous in function and distribution to the mammalian MHC. These are the B-F (class I) and B-L (class II) glycoproteins. In addition, a third glycoprotein (B-G) is also encoded by the chicken MHC. We are interested in examining gene regulation and cellular expression of these MHC gene products in the chicken. The trisomic line of chickens is being developed as an animal model for this purpose. Birds from this line contain either 2, 3, or 4 MHC-encoding chromosomes. In this study, we investigated the hypothesis that the quantities of B-complex glycoproteins on the membranes of fully differentiated erythrocytes are proportional to the number of MHC-encoding chromosomes present in particular birds. Hemagglutination final titer and quantitative adsorption assays were carried out using erythrocytes from disomic and aneuploid chickens homozygous for the B15 haplotype. The average hemagglutination final titers were higher for tetrasomic cells as compared to disomic cells. Furthermore, in adsorption assays, employing a B15 cross-reacting alloantiserum, trisomic and tetrasomic erythrocytes displayed increased adsorption capabilities (1.6 and 3.1 fold, respectively) compared to disomic control cells. These results indicate a step-wise increase in the amounts of erythrocyte surface glycoproteins per cell in the trisomics and tetrasomics, respectively. Such findings are consistent with a MHC-dosage-dependent model of gene expression in homeothermic vertebrates.

A non-MHC genetic influence on response to Rous sarcoma virus-induced tumors in chickens.

Avian-leukosis-free females from Regional Poultry Research Laboratory (RPRL) inbred lines 6(1) and 7(2) were inseminated with pooled semen from RPRL line 15I5 males. The progeny resulting from the two crosses 15I5 X 6(1) and 15I5 X 7(2) were of the B2/B15 major histocompatibility complex (MHC) genotype, the B2 haplotypes from lines 6(1) and 7(2) being indistinguishable. When challenged with Rous sarcoma virus (RSV) at 6 weeks of age, 30 out of 31 progeny of cross 15I5 X 6(1) developed tumors that rapidly regressed, whereas 26 of 29 progeny of cross 15I5 X 7(2) died with progressive tumor growth. On the other hand, 15 of 18 B2/B15 segregants from the three-way cross (15I5 X 6(3]F1 X 7(2), identical in source to the MHC of the (15I5 X 7(2]F1 progressors, were characterized by tumor regression. Thus influences associated with non-MHC background genes from lines 6(1), 6(3), and 7(2) appeared to be critical to host response to RSV-induced sarcomas. Similar findings suggesting a strong non-MHC influence on anti-sarcoma response were obtained using 107 F2 and 77 backcross progeny of RPRL line 100 and noninbred University of New Hampshire (UNH) line 105. The B2 haplotype of line 100 was associated with tumor regression when combined with the line 105 background and with tumor progression when expressed on the line 100 background. Suppression of the anti-tumor response associated with line 100 appeared to be fairly generalized, since a poor response to tumor was observed regardless of which of three subgroups of RSV was used to induce tumor.

The effect of serologically defined major histocompatibility complex haplotypes on Marek's disease resistance in commercially bred White Leghorn chickens.

In commercial pure white leghorn lines, A, B, and C, the effects on resistance against a virulent strain of Marek's disease virus were assessed for B19 and B21 haplotypes of the chicken major histocompatibility complex. B haplotypes were identified by direct hemagglutination using alloantisera raised against erythrocyte antigens. In homozygous B21 female chicks from lines A and B, mortality upon challenge with virus was 16% and 9%, respectively; in B19 chicks, mortality was 42% and 60%, respectively. Intermediate mortality was observed in heterozygous B19/B21 birds. When line A and B hens were crossed with B15/B15 or B5/B19 cocks from line C, differences between B19 and B21 were significant only in the progeny from B5/B19 sires. Therefore, it was concluded that selection for major histocompatibility complex-associated disease resistance markers may be useful only when B haplotypes complement each other in commercial line crosses and when interactions with genetic background do not severely obscure the differential haplotype effects, as are observed within pure lines.

Non-major histocompatibility complex alloantigen genes affecting immunity.

An alloantigen is a genetically determined cell-surface molecule detected by specific antisera. An identifying letter has been assigned to each genetic locus responsible for the 12 distinct families of alloantigens: A, B, C, D, E, H, I, J, K, L, P, and R. The genes of each system segregate independently of the other systems, except that the A and E are very closely linked (0.5 centimorgans). Selection experiments over numerous generations have revealed distinct changes in gene frequency of the A-E alloantigens, suggesting immune responses associated with susceptibility to coccidiosis, response to immunizations with SRBC, and selection for size of the bursa of Fabricius. Immune response effects of the C system of alloantigen genes are indicated by distinct gene frequency changes following selection for response to SRBC, selection for size of bursa of Fabricius, and macrophage nitrite production after lipopolysaccharide (LPS) stimulation. Immune response effects of the D system of antigens are indicated by data from genetic selection for response to immunization with SRBC, selection for bursa size, and macrophage nitrite and cytokine interleukin (IL)-6 production following LPS stimulation. Immune response effects of the I system genes are indicated by distinct gene frequency changes in lines selected for bursa size and within family comparisons for macrophage nitrite and cytokine IL-6 production following LPS stimulation. Effects of the L system, consisting of only 2 alleles, are indicated by the gene frequency changes following selection for bursa size, direct comparison of genotypes within families for monocyte phagocytosis, susceptibility to coccidiosis, outcome of Rous sarcomas, and immune responses to SRBC and Brucella abortus. Genotypes of the P alloantigen system were directly compared within families of fully pedigreed chicks with significant differences for monocyte phagocytosis. An experimental procedure for simultaneously testing for immune responses of genotypes of 9 of the alloantigen systems (A, B, C, D, E, H, I, L, and P) has been established by producing test progeny from a single cross of parent lines segregating for genes of each of the systems.

Genetics and classification of major histocompatibility complex antigens of the chicken.

The chicken MHC consists of a gene complex divisable into three chromosomal regions, each producing a series of genetically antithetical molecules with regionally specific functions. Most of the genes (alleles) of each region are present in unique haplotype combinations with genes (alleles) from the other two regions; relatively few haplotypes appear to share identical regional genes. Even with the high degree of polymorphism existing within and between regions, general typing of erythrocytes for MHC haplotypes can still be performed as economically as ever with alloimmune reagents of known specificity.

Immunogenetics of the A-E alloantigen complex.

The close linkage (0.5%) between the A and E erythrocyte alloantigen loci present a special challenge in the production of locus-specific typing antisera. The objective of the investigation was to determine immunogenetically the A-E haplotypes (genetically linked combinations of A and E antigens) existing in the locally maintained individuals of the New Hampshire (NH) and White Plymouth Rock (WR) breeds. The A and E alloantigens in these populations were identified using reference antisera previously produced in White Leghorns. A total of four A-E haplotypes were identified within each of the two breeds; A2E1, A6E2, A6E4, and A8E2 in WR and A2E1, A3E7, A7E4, and A8E2 in NH. Individuals of these two brown-egg breeds were backcrossed over several generations to a line of Ancona chickens homozygous at the A and E loci. Genetic segregation occurring over four generations resulted in nonrecombinant and recombinant progeny that were immunized reciprocally with the blood of siblings to raise antibodies reactive with the individual A and E antigens of the NH and WR stocks. The antisera resulting from the within-family alloimmunizations confirmed the haplotypes deduced in the WR and NH lines from the initial tests with the A and E reference antisera.

Resistance, susceptibility, and immunity to cecal coccidiosis: effects of B complex and alloantigen system L.

This study examined alloantigen system L effects on resistance to initial infection and acquired immunity to Eimeria tenella infection in three B complex genotypes. Experimental progeny segregating for B and L genotypes were produced from pedigree matings of B2B5 L1L2 sires and dams. Chicks were weighed and inoculated with 30,000 E. tenella oocysts at 6 wk of age to evaluate resistance in four trials (n = 262). Immunity was studied in four additional trials (n = 244) by immunizing progeny with 500 E. tenella oocysts per day for 5 d beginning at 5 wk of age. Two weeks after the last immunization dose, the birds were weighed and challenged with 30,000 E. tenella oocysts. All birds were weighed again and scored for cecal lesion 6 d after the 30,000 oocyst dose challenge. Weight gain and cecal lesion scores were evaluated by ANOVA. Major histocompatibility (B) complex genotypes B2B2 and B5B5 did not affect resistance to initial challenge with E. tenella based on lesion score and weight gain. However, after immunization, the B5B5 and B2B5 genotypes had significantly lower cecal scores than the B2B2 genotype when the birds were rechallenged. Weight gain was not affected among immunized birds. No significant L system effects with or without immunization were detected. These results are consistent with previous research demonstrating B complex effects on immunity to cecal coccidiosis.

Resistance to Marek's disease in chickens with recombinant haplotypes to the major histocompatibility (B) complex.

Genetic resistance to Marek's disease (MD) is associated with the B-F region of the MHC. The resistance of chickens possessing either of two MHC haplotypes to challenge with different strains of MDV was examined. Chickens with serologically similar MHC recombinants BR2 and BR4 (both BF2-G23) were backcrossed for four generations to the highly inbred UCD-003 line (B17B17). Heterozygotes (B17BF2-G23) were mated to produce BR2BR2 and BR4BR4 homozygotes with 93% background gene uniformity. Both genotypes were highly resistant to GA-5 MDV, having an incidence of 0 and 8% MD for BR2BR2 and BR4BR4, respectively, whereas the incidence of MD in the UCD-003 birds was above 80%. Challenge with the very virulent RB-1B strain caused 10% and 31% MD in the BR2BR2 and BR4BR4 chickens, respectively, compared with 100% and 52% in the B17B17 (UCD-003) and B23B23 (New Hampshire 105) lines, respectively. Viremia levels at 5 and 6 d postinfection were significantly lower in BR2BR2 and B23B23 than in B17B17 genotypes.

Allelic complementation between MHC haplotypes B(Q) and B17 increases regression of Rous sarcomas.

Major histocompatibility (B) complex haplotypes B(Q) and B17 were examined for their effect on Rous sarcoma outcome. Pedigree matings of B(Q)B17 chickens from the second backcross generation (BC2) of Line UCD 001 (B(Q)B(Q)) mated to Line UCD 003 (B17B17) produced progeny with genotypes B(Q)B(Q), B(Q)B17, and B17B17. Six-week-old chickens were injected with subgroup A Rous sarcoma virus (RSV). The tumors were scored for size at 2, 3, 4, 6, 8, and 10 weeks postinoculation. A tumor profile index (TPI) was assigned to each bird based on the six tumor scores. Two experiments with two trials each were conducted. In Experiment 1, chickens (n = 84) were inoculated with 30 pock-forming units (pfu) RSV. There was no significant B genotype effect on tumor growth over time or TPI among the 70 chickens that developed tumors. Chickens (n = 141) were injected with 15 PFU RSV in Experiment 2. The B genotype significantly affected tumor growth pattern over time in the 79 chickens with sarcomas. The B(Q)B17 chickens had the lowest TPI, which was significantly different from B17B17 but not B(Q)B(Q). The data indicate complementation because more tumor regression occurs in the B(Q)B17 heterozygote than in either B(Q)B(Q) or B17B17 genotypes at a 15 pfu RSV dose and significantly so compared to B17B17. By contrast, the 30 pfu RSV dose utilized in the first experiment overwhelmed all genotypic combinations of the B(Q) and B17 haplotypes, suggesting that certain MHC genotypes affect the immune response under modest levels of viral challenge.

Further tests for genetic linkages of three morphological traits, three blood groups, and break points of two chromosome translocations on chromosome one in the chicken.

Two matings were conducted to further test the locations of the pea comb (P*), blue egg shell color (O*), and tardy feathering (T*) loci. In each mating a different chromosome rearrangement break point (R(B)) was tested against the three loci. Independent segregation was noted between the traits and the R(B) when the R(B) was on the long arm of chromosome 1. Significant linkage was noted when an R(B) on the short arm was tested against the three markers, indicating that the loci for P*, O*, and T* are on the short arm. Three blood group loci, EAD*, EAI*, and EAP*, were simultaneously tested against the short arm R(B). Independent segregation was noted in each instance, indicating that these blood group loci are not on the short arm of chromosome 1.

Nonmajor histocompatibility complex alloantigen effects on the fate of Rous sarcomas.

Rous sarcoma virus-induced tumor outcome is controlled by the MHC (B). Additional data, using controlled segregation in families, has indicated non-MHC effects as well, but few studies have focused on blood groups other than the B complex. Segregating combinations of genes encoding erythrocyte (Ea) alloantigen systems A, C, D, E, H, I, P, and L in B2B5 and B5B5 MHC (B) backgrounds were examined for their effects on Rous sarcomas. Six-week-old chickens were inoculated in the wing-web with 30 pfu of Rous sarcoma virus (RSV). Tumors were scored six times over a 10-wk period. A tumor profile index (TPI) was assigned to each chicken based on the six tumor size scores. Response was evaluated using tumor size at each measurement period, TPI, and mortality. The genotypes of Ea systems A, C, D, E, H, I, and P had no significant effect on any parameter in either B complex population. The Ea-L system had an effect on Rous sarcomas in the B2B5 intermediate responders and B5B5 progressors. Tumor size, TPI, and mortality were all significantly lower in B2B5 L1L1 chickens than in B2B5 L1L2 chickens. Mortality was lower in the B5B5 L1L1 birds than in B5B5 L1L2 chickens. It appears that the Ea-L system, or one closely linked, is acting in a manner independent of the B complex in response to RSV challenge.