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.

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.

Association of the chicken MHC B haplotypes with resistance to avian coronavirus.

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.

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.

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.

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.

Single-strand conformation polymorphism (SSCP) assays for major histocompatibility complex B genotyping in chickens.

We have developed a DNA-based method for defining MHC B system genotypes in chickens. Genotyping by this method requires neither prior determination of allele-specific differences in nucleotide sequence nor the preparation of haplotype-specific alloantisera. Allelic differences at chicken B-F (class I) and B-L (class II) loci are detected in PCR single-strand conformation polymorphism (SSCP) assays. PCR primer pairs were designed to hybridize specifically with conserved sequences surrounding hypervariable regions within the two class I and two class I loci of the B-complex and used to generate DNA fragments that are heat- and formamide-denatured and then analyzed on nondenaturing polyacrylamide gels. PCR primer pairs were tested for the capacity to produce SSCP patterns allowing the seven B haplotypes in the MHC B congenic lines, and seven B haplotypes known to be segregating in two commercial broiler breeder lines to be distinguished. Primer pairs were further evaluated for their capacity to reveal the segregation of B haplotypes in a fully pedigreed family and in a closed population. Concordance was found between SSCP patterns and previously assigned MHC types. B-F and B-L SSCP patterns segregated in linkage as expected for these closely linked loci. We conclude that this method is valuable for defining MHC B haplotypes and for detecting potential recombinant haplotypes especially when used in combination with B-G (class IV) typing by restriction fragment pattern.

Alloantigen systems L and P influence phagocytic function independent of the major histocompatability complex (B) in chickens.

Synthetic parent stocks were designed to produce progeny among which alleles were simultaneously segregating for nine alloantigen systems, including the MHC (B). Chicks from Ancona-derived B19B19 females crossed with White leghorn B19B21 males were blood typed, resulting in genotypic categories for the A-E, C, D, H, I, L, and P loci with the objective of determining which, if any, of the eight non-MHC alloantigen systems influence or interact with the B system genotypes for blood monocyte phagocytic activity. Leukocytes obtained from whole blood at 2 and 4 wk were separated on a Fico/Lite LymphoH, density gradient and were allowed to adhere to glass coverslips. The resulting adherent monocyte monolayers were incubated with viable Escherichia coli for 1 h and stained with Leukostat, and the phagocytic monocytes and numbers of internalized bacteria per phagocytic monocyte were scored microscopically. The combined results from two separate trials demonstrated that the genotypes of the A-E, C, D, H, and I systems did not differ in the percentage of monocytes exhibiting phagocytosis, whereas significant differences were noted relative to the B system genotype at 2 wk of age (B19B21 > B19B19; P = 0.049), L at 4 wk (L1L1 > L1L2; P = 0.009), and P at 4 wk (P4P4 > P1P1; P = 0.047). The data were further analyzed to determine any interactions of P and L alloantigen genotypes with the B system genotypes; no such interaction was observed. These studies suggest that the L and P non-MHC alloantigen systems have the potential to influence immune responses by modulating phagocytic function in chickens. Furthermore, this modulation seems to be independent of the B (MHC) system.

Adoptive transfer of infectious bronchitis virus primed alphabeta T cells bearing CD8 antigen protects chicks from acute 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.

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.

A consensus linkage map of the chicken genome.

A consensus linkage map has been developed in the chicken that combines all of the genotyping data from the three available chicken mapping populations. Genotyping data were contributed by the laboratories that have been using the East Lansing and Compton reference populations and from the Animal Breeding and Genetics Group of the Wageningen University using the Wageningen/Euribrid population. The resulting linkage map of the chicken genome contains 1889 loci. A framework map is presented that contains 480 loci ordered on 50 linkage groups. Framework loci are defined as loci whose order relative to one another is supported by odds greater then 3. The possible positions of the remaining 1409 loci are indicated relative to these framework loci. The total map spans 3800 cM, which is considerably larger than previous estimates for the chicken genome. Furthermore, although the physical size of the chicken genome is threefold smaller then that of mammals, its genetic map is comparable in size to that of most mammals. The map contains 350 markers within expressed sequences, 235 of which represent identified genes or sequences that have significant sequence identity to known genes. This improves the contribution of the chicken linkage map to comparative gene mapping considerably and clearly shows the conservation of large syntenic regions between the human and chicken genomes. The compact physical size of the chicken genome, combined with the large size of its genetic map and the observed degree of conserved synteny, makes the chicken a valuable model organism in the genomics as well as the postgenomics era. The linkage maps, the two-point lod scores, and additional information about the loci are available at web sites in Wageningen (http://www.zod.wau.nl/vf/ research/chicken/frame_chicken.html) and East Lansing (http://poultry.mph.msu.edu/).

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.

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.

Identification, inheritance, and linkage of B-G-like and MHC class I genes in cranes.

We identified B-G-like genes in the whooping and Florida sandhill cranes and linked them to the major histocompatibility complex (MHC). We evaluated the inheritance of B-G-like genes in families of whooping and Florida sandhill cranes using restriction fragment patterns (RFPs). Two B-G-like genes, designated wcbg1 and wcbg2, were located within 8 kb of one another. The fully sequenced wcbg2 gene encodes a B-G IgV-like domain, an additional Ig-like domain, a transmembrane domain, and a single heptad domain typical of alpha-helical coiled coils. Patterns of restriction fragments in DNA from the whooping crane and from a number of other species indicate that the B-G-like gene families of cranes are large with diverse sequences. Segregation of RFPs in families of Florida sandhill cranes provide evidence for genetic polymorphism in the B-G-like genes. The restriction fragments generally segregated in concert with MHC haplotypes assigned by serological typing and by single stranded conformational polymorphism (SSCP) assays based in the second exon of the crane MHC class I genes. This study supports the concept of a long-term association of polymorphic B-G-like genes with the MHC. It also establishes SSCP as a means for evaluating MHC genetic variability in cranes.

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.

Immunoresponsiveness in chickens: association of antibody production and the B system of the major histocompatibility complex.

Lines of White Leghorn chickens were selected for high or low antibody response to sheep erythrocytes for five generations. The base population from which the experiment started was composed of individuals all of which were heterozygous at the MHC haplotypes B13 and B21. Body weights, egg production traits, and genotypes at the B system were monitored for all individuals in each generation. By Generations 4 and 5 there was separation of the two replicate lines selected for high titer from the two replicate lines selected for low titer. Over the course of the experiment, higher antibody titers and lower BW were associated with B21 and lower antibody titers and higher BW were associated with B13, although these relationships did not occur in every instance. Conclusions were that the B system was associated with antibody response, but that the chickens did not depend entirely upon that association for protection against foreign proteins. Also, the importance of having replicate lines in a selection experiment was shown.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.