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.

B-complex associated immunity to Salmonella enteritidis challenge in congenic chickens.

Six trials were conducted during which a total of 12 congenic lines (University of California-Davis, UCD) homozygous for various B-complex haplotypes, were challenged as neonates by intraperitoneal injection with either of two isolates of Salmonella enteritidis. Because these B haplotypes were expressed on a common genetic background (highly inbred Line UCD 003), and mortality differences among lines were statistically significant in three of the six trials, and morbidity (body weight) differences were significant in another trial; it is suggested that B-complex alleles affect the degree of immunity to these isolates. When all lines and trials were compared, line 342 (BC/BC) emerged as particularly resistant, whereas lines 253 (B18/B18) and 254 (B15/B15) were more susceptible. The remainder of the lines were of neutral (intermediate) susceptibility. Sex did not appear to influence the results of the challenge, but more resistance was observed with an increase in the age at inoculation. Although the mechanism that determined this resistance is unknown it was present as early as 3 d of age, and it is suggested that complement proteins, which have a known role in protection from bacterial infections, and are encoded by genes located within the B-complex, or acute phase proteins, may account for these observations. The results provide additional evidence for the importance of the B-complex in determining immunity to Salmonella.

Resistance, susceptibility, and immunity to Eimeria tenella in major histocompatibility (B) complex congenic lines.

The major histocompatibility (B) complex influence on resistance, susceptibility, and immunity to Eimeria tenella was examined in UCD B complex congenic chicken lines. In Experiment 1, 6-wk-old chicks from 12 UCD congenic lines were weighed and assigned to either challenge or control groups. The challenge group received a dose of 10,000 E. tenella oocysts. Response to challenge was evaluated by body weight gain and cecal lesion scores. Cecal lesion scores in B3B3 chickens were significantly lower than those of all other genotypes. Genotype B2B2 had the highest lesion scores, which were significantly different from the lesion scores calculated for B3B3, B18B18, and B21B21 chickens but were not significantly different from B14B14, B15B15, B17B17, B19B19, B24B24, BCBC, BJBJ, and BQBQ genotypes. The B21B21 chickens had significantly lower lesion scores than B2B2, B14B14, and BCBC chickens. No other significant lesion score differences were found among the remaining lines. The highest weight gain found in B19B19 chickens was significantly different from that of B3B3, B14B14, B15B15, B17B17, B18B18, B24B24, and BCBC chickens. The B15B15 chickens had the lowest weight gain, which was significantly different from that of B2B2, B19B19, B21B21, B24B24, BJBJ, and BQBQ chickens. Experiment 2 tested the immune response to E. tenella after low dose oocyst immunization. Chicks from 10 UCD 003 congenic lines were divided into three groups: control, challenge, and immune. At 5 wk of age, the immune group was immunized with 500 E. tenella oocysts for 5 consecutive d. Fourteen days after the last immunization all chicks were weighed, and 10,000 E. tenella oocysts were administered to the challenge and immune groups. Significant lesion score differences existed among all three treatments: control (0), immune (2.14 +/- 0.1); challenge (3.13 +/- 0.1). Among immune birds, B3B3 and BQBQ chickens had significantly lower lesion scores than B19B19, B24B24, B14B14, and B2B2 chickens. Neither B19B19 nor B24B24 chickens were well-protected, as indicated by their higher lesion scores. No significant differences in weight gain were found in immune birds. The B complex affected resistance and susceptibility as well as the immune response to E. tenella. Cecal lesion scores following challenge in naive birds or after immunization were influenced by the B complex, whereas weight gain was affected in naive birds only. These effects may be manifested through differences in immune competence at the time of challenge or immunization, the amount of parasite antigen production, or the threshold doses for effective immunization.

Differential resistance to Staphylococcus aureus challenge in major histocompatibility (B) complex congenic lines.

Ten inbred B-congenic Leghorn lines were challenged with two isolates of Staphylococcus aureus at 3 days and 6 wk of age. Significant differences in mortality were observed among such lines when challenged at 3 days with either S. aureus Isolate P4L (moderately pathogenic) or S. aureus Isolate 3727 (highly pathogenic). Line 331 (B2/B2 genotype) had lower mortality than either Line 004 (B17/B17, chi 2 = 4.13, P < .05) or Line 253 (B18/B18, chi 2 = 4.23, P < .05) challenged with Isolate P4L. The use of a susceptibility index allowed for the detection of additional differences among the various lines challenged by Isolate 3727. Line 336 (BQ/BQ) was more resistant than either Line 335 (B19/B19, P < .01) or Line 330 (B21/B21, P < .01). No significant differences were found among the lines challenged at 6 wk by either isolate. The results provide additional evidence for the importance of the B complex in genetically determined disease resistance, and further demonstrate the usefulness of congenic lines in such investigations.

Effects of heterozygosity of major histocompatibility complex haplotypes on performance of Leghorn hens sharing a common inbred background.

Four sets of diallele crosses between partially congenic lines of Leghorns differing in their MHC haplotypes (B blood group) were made in 4 successive yr. Hens heterozygous for B haplotypes resulting from the crosses were then compared with those representing the homozygous congenic parent lines. Heterozygotes superior in performance to the average of their parental homozygotes were found to occur predominantly for survivor egg production, egg weight, and viability to 40 wk of age. Body weight of adult hens showed least heterosis. Surprisingly few crosses produced heterozygous embryos with superior hatchability, although heterotic combinations did exist. Additive differences between parent homozygotes were of greater magnitude than dominance effects measured as average excess of heterozygotes over homozygote averages of parents. A control genotype heterozygous for the B2/BQ combination was included in all tests and proved heterotic in most cases. This same genotype also had been found to confer resistance to Marek's disease under experimental inoculation.

Reproductive performance of inbred congenic Leghorns carrying different haplotypes for the major histocompatibility complex.

Twelve congenic lines of White Leghorn chickens carrying distinct haplotypes of the MHC (B blood group) but sharing a common genetic background from a highly inbred line (UCD 003) were compared. Each of the lines had been bred back to Line UCD 003 for five generations before intercrossing its members to establish its distinct genetically homozygous MHC type. Seven generations of these congenic lines were compared between 1981 and 1987 for fertility, hatchability of fertile eggs, egg production, mortality to 40 wk of age, 40-wk egg weight, and 40-wk body weight. Statistically significant differences between MHC haplotypes were found for all traits except fertility.

Effects of the sex-linked dwarf gene (dw) on the expression of the muscular dystrophy gene (am) in chicken.

The sex-linked dwarf gene (dw) was introduced into companion muscular dystrophic (am) and nondystrophic (Am+) New Hampshire chicken lines to investigate influences of the dwarf gene on breast muscle weights, muscle fiber area, and the histological expression of muscular dystrophy. Dystrophic and nondystrophic chickens within dwarf or nondwarf genotypes were similar in body and carcass weights. Pectoralis and supracoracoideus muscle weights (as a percentage of adjusted carcass weight) were similar in nondystrophic dwarf and nondwarf males and females. In addition, pectoralis weight was similar in dystrophic dwarf males and dystrophic nondwarf males and females. However, pectoralis weight was significantly smaller in dystrophic dwarf females than in dystrophic nondwarf females, whereas supracoracoideus weight was significantly larger in dystrophic dwarf males than in dystrophic nondwarf males. Supracoracoideus weight was similar in dystrophic dwarf males and females and dystrophic nondwarf females. Pectoralis muscle fiber area was influenced by sex and by dwarf and dystrophy genotype. Muscle fiber area was larger in females than in males, smaller in dwarfs than in nondwarfs, and smaller in dystrophic than in nondystrophic muscles. Muscle fiber degeneration and adipose infiltration was more extensive in dystrophic than in nondystrophic females and males, and it was more advanced in dwarfs than in nondwarfs. Excessive acetylcholinesterase staining patterns were characteristic of dystrophic muscle in both dwarf and nondwarf genotypes. Nondystrophic and dystrophic dwarf male and female chickens are comparable substitutes for nondwarfs as biomedical models with respect to pectoralis histology, acetylcholinesterase staining pattern, and pectoralis muscle hypertrophy.

Abnormalities associated with the bursal microenvironment in spontaneous dysgammaglobulinemia of UCD line 140 chickens.

UCD line 140 chickens have been previously reported to develop a syndrome of spontaneous 7s immunoglobulin deficiency and the presence of autoantibodies. Earlier studies demonstrated that these inbred birds have normal peripheral blood T and B cell numbers; they also respond normally to allogeneic stimulation. Although the 7s immunodeficiency does not manifest itself until several months of age, line 140 birds have a premature degeneration of bursa. Because of the recent development of monoclonal reagents specific for bursal elements, including surface epithelium, basement membrane associated epithelium, follicle associated epithelium, and lymphoid subpopulations, we have examined line 140 and control birds for the expression of bursal epithelial cell antigens. Line 140 birds, in contrast to control chickens, have a dramatic early alteration in the expression of an epithelial cell marker in the bursa, thymus, and intestine. Moreover, to further address this issue, we transplanted bursa from 10-day embryos onto the chorioallantoic membrane, a privileged site. Bursae from control birds became abnormal when transplanted onto line 140 CAM; they remained normal when transplanted among several control chicken lines. In contrast, line 140 bursa remained abnormal independent of the transplant procedure. Due to the marked bursal abnormality observed specific to the dysgammaglobulinemia chicken line, we propose that the microenvironmental features of line 140 bursa may predispose these birds to the development of humoral immunodeficiency and autoantibodies.

Ontogeny of T cell development in avian scleroderma.

UCD line 200 chickens develop an inherited fibrotic disease associated with the production of antinuclear antibodies, antibodies to type II collagen, and early skin lesions characterized by intense T lymphocyte infiltrates. In the present study we have investigated the hypothesis that developmental abnormalities in T lymphocyte differentiation predispose the line 200 chickens to autoimmune disease. The status of the thymic microenvironment in these birds during ontogeny was studied with an extensive panel of monoclonal antibodies (mAbs) reactive with distinct stromal cell subsets including MHC determinants. In addition, their T-cell graft-versus-host reactivity and responses to mitogenic stimulation and interleukin (IL)-2 were also analyzed. Line 200 chickens have profound defects in thymic structure with a virtual complete absence of antigens specific for type I epithelium which lines the thymic subcapsular and perivascular regions. There are excessive levels of MHC class II positive cells, particularly in the cortex, and B cells/subset macrophages identified by mAb MUI 36. These defects are found from the late embryonic period, long before clinical disease is manifest. Furthermore, by FACS analysis, line 200 thymocytes have a major increase in IL-2 receptor density. In addition, line 200 chicken peripheral blood lymphocytes (PBL) respond poorly to mitogenic agents and have a reduced response to IL-2. Finally, it is important to note that line 200 PBL produce a normal graft-versus-host reaction. We propose that these abnormalities in T-cell differentiation are selective, not global, and may be reflective of a defect in thymic education resulting in an inappropriate response to self-antigens.

Euploid chicken embryos from eggs containing one, two or several yolks.

A high incidence of haploid/diploid chimerism in chick embryos from strains of chickens selected for large size was postulated to be caused by the propensity of such hens to ovulate erratically. To test the hypothesis karyological analysis was made of embryos in eggs containing 1 or greater than 1 yolk. The eggs were from a line selected for multiple ovulation for 20 generations. Double and multiple-yolk eggs are a manifestation of an irregular ovulatory pattern. Ova in multiple yolk eggs were significantly less fertile and significantly fewer embryos survived to 18 h of incubation than single ovulated ova. In the sample of 342 embryos analysed, only 2 forms of heteroploidy occurred in frequencies of greater than 1.2%; 2n/4n mosaicism (5.8%) and 3n (5.0%). Only triploidy occurred significantly more frequently in eggs containing greater than 1 yolk (7.0%) than in single yolk eggs (none). The overwhelming majority of 3n embryos had a digynic origin (i.e from ova with 2 maternal pronuclei), as inferred from the sex chromosome complement. Erratic ovulation therefore resulted in suppression of second polar body extrusion leading to digynic triploidy. Multiple yolks had no effect on dispermy, the primary cause of 1n/2n chimaeric embryos, in single-yolked chicken eggs.

Genetic control of avian scleroderma.

The inheritance of avian scleroderma, a fibrotic autoimmune disease of chickens resembling human scleroderma, was investigated. Comb inflammations and lesions were used to determine the state of disease of 4-week-old chickens. All line 200 males and 60% of female line 200 chicks showed abnormalities. Crosses (F1) between line 200 and eight partially inbred lines of chickens maintained at the University of California at Davis were all normal. Backcrosses of F1 cocks to line 200 hens showed a higher incidence of scleroderma in males than in females for all lines. The incidence of affected birds varied between backcrosses from a low of 42% for backcross line 217 males derived from a New Hampshire line, to 88% for males of backcross line 213 derived from a partially inbred Leghorn line, demonstrating the presence of genes modifying the penetrance of presumed major genes causing the disease. Backcross genotypes segregating for haplotypes of the major histocompatibility complex (MHC) derived from inbred lines showed consistently lower penetrance of scleroderma than homozygotes carrying the line 200 haplotype. Thus B3Bs (lines 211 and 215), B14Bs (line 217), and B15Bs (lines 212, 213, 216, and 218) all had fewer affected individuals than BsBs homozygotes from the same families.

Identification of T cells in early dermal lymphocytic infiltrates in avian scleroderma.

University of California, Davis (UCD) line 200 chickens spontaneously develop a progressive fibrotic syndrome with features similar to those observed in human autoimmune connective tissue diseases, including fibrosis, vascular occlusion, and lymphocytic infiltration of the comb, skin, digits, and viscera. Beginning at 2 weeks post hatch, line 200 chickens develop intense lymphocytic infiltration of the comb and dorsal neck skin. To further characterize the nature of these cellular infiltrates, weekly serial skin biopsy specimens from line 200 and control birds were examined using hematoxylin and eosin staining and indirect immunofluorescence with a library of mouse anti-chicken monoclonal antibodies specific for lymphocyte markers. In situ staining performed on serial skin sections revealed the presence of large groups of T cells beginning at 2 weeks of age. Further characterization of these infiltrates demonstrated the presence of both T helper and T cytotoxic/suppressor cells with a mean +/- SD T4:T8 ratio of 1.44 +/- 0.29 by 4 weeks of age. As the lesions progressed, the infiltrates also contained distinct groups of B cells as characterized by MUI 36. In addition, the lesions were strongly positive for B-L (Ia) antigen, which was noted on B cells, monocytes/macrophages, activated T cells, and fibroblasts. The skin sections were negative for 2 different macrophage monoclonal antibodies at all time-points. Upon extraction from affected skin, 42.0 +/- 13.06% (mean +/- SD) of these cells were positive for B-L, 35.10 +/- 6.51% were T cells, and 31.25 +/- 3.14% were recognized by MUI 36. Although positive staining for IgG was not found in these extracted cells, 7% of the isolated cells were positive for surface IgM.

Immune response versus susceptibility to Marek's disease.

It was hypothesized that the generation of activated T cells through an efficient and rapid immune response during the early pathogenesis of Marek's disease virus (MDV) infection provides a large pool of target cells for transformation. Therefore, the correlation between genetic susceptibility to Marek's disease (MD) and in vitro mitogenic responses of lymphocytes as a measure of cell-mediated immune competence and efficiency was tested. In one series of trials, spleen cells from strains of chickens with differing levels of susceptibility to MD tumors were stimulated with graded doses of Concanavalin A (Con A) or phytohemagglutin (PHA). In a second series of trials, peripheral blood lymphocytes from individual chickens within genetic strains were tested at the same time chickens were challenged with MDV to determine susceptibility. Responsiveness was determined using one-way mixed lymphocyte reaction (MLR) tests as well as mitogen stimulation. Data from the tests comparing chicken strains supported the hypothesis in some but not all cases. The S13 chickens, which are more susceptible than P2a chickens to MD, were significantly more responsive, and highly resistant N2a chickens were significantly less responsive to Con A. In contrast, five other resistant strains were either more responsive (UCD-058, OS13) or equally responsive (UCD-140, OS5, C) to Con A when compared with P2a chickens. The PHA responses were even less predictive of MD susceptibility. No general correlation was observed between responsiveness to either mitogen or MLR tests and subsequent tumor development in trials comparing individuals within strains.

Selection with restriction in a poultry breeding scheme with different selection procedures in both sexes: direct responses.

1. A selection experiment with two lines of White Leghorns originating from a common base population was carried out over 5 generations with the aims of maintaining an unchanged egg weight, reducing age at first egg and reducing adult body weight. Each line consisted of 14 male and 42 female breeders. 2. Males were mass selected for low body weight at 20 weeks of age. To compensate for the expected correlated loss in egg weight, hens were selected according to an index which counteracted this undesirable change while also reducing age at first egg and reducing body weight. 3. An index value was calculated for each individual hen from average egg weight, age at first egg and body weight at 20 weeks. Index weights had to be calculated for each generation and line in accordance with the expected change in egg weight due to male selection on body weight. 4. For control matings hens with an index near the population average were mated either to males with body weight near the population average (control C1) or to the selected males within lines (control C2). 5. Expected and observed total responses agreed well for all traits in line 1 and for body weight in line 2. 6. Phenotypic variances and covariances showed little change during the experiment. However, genetic variances and covariances estimated at the end of the experiment showed some differences, both between lines and compared to the parameters used for index construction.

Inbred crosses and inherited muscular dystrophy of the chicken.

Inbred normal and genetically dystrophic chickens of New Hampshire and White Leghorn backgrounds, respectively, have been crossed to yield hybrids of normal and dystrophic genotypes in order to provide genetically homogeneous but heterozygous experimental animals. This study examined carcass and pectoral muscle weights, pectoral muscle fiber diameters, serum creatine kinase (CK) levels, muscle acetylcholinesterase (AChE), lactic dehydrogenase (LDH), and creatine kinase, and response to daily injections of corticosterone-21-acetate (C21A) of these hybrid chickens and their inbred parental lines. With the exception of pectoral muscle weight, dystrophic hybrids exhibited symptoms of dystrophy: high serum CK and high muscle AChE and low LDH levels. The results support the hypothesis that neither early muscle fiber hypertrophy nor atrophy is invariably associated with expression of the dystrophic gene; both are the result of secondary gene interactions. One experiment showed that muscle AChE levels decreased and LDH levels rose after C21A treatments.

Genotyping chickens for the B-G subregion of the major histocompatibility complex using restriction fragment length polymorphisms.

Chicken B-G-subregion cDNA probes were used to analyze restriction fragment length polymorphisms (RFLP) of the B-G subregion of the chicken major histocompatibility complex. Genomic DNA from chickens representing 17 of the 27 standard B haplotypes were digested with restriction endonucleases and analyzed in Southern hybridizations with two cDNA clones from the B-G subregion. Each B-G genotype was found to produce a unique pattern of restriction fragments in these Southern hybridizations. With 15 of the 17 genotypes examined, the different genotypes could be readily distinguished in hybridizations produced with DNA digested with a single restriction enzyme, PVU II. The two additional genotypes produced nearly identical patterns in PVU II preparations and with three additional enzymes as well, but were readily distinguishable in Eco RI digestions. For many of the haplotypes, samples from several individuals in different flocks were examined. In every instance, genotyping by RFLP pattern was found to confirm the B-G allele assigned serologically.

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex.

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a lambda gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, lambda bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations lambda bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the lambda bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of lambda bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.

Inherited dysgammaglobulinemia of chickens: reduced incidence of disease in parabiotic chimeras.

University of California, Davis (UCD) line 140 chickens develop a dysgammaglobulinemia characterized as selective 7S immunoglobulin (Ig) deficiency with elevated serum IgM levels. To study the role of bursal development on the expression of dysgammaglobulinemia in these birds, we examined the effect of bursacyte transfer to line 140 birds and parabiosis between UCD 140 and a control line of chickens on changes in serum IgM and 7S Ig levels. Bursacyte transfer was performed by injecting 18-day UCD 140 embryos (which had been cyclophosphamide treated on Day 15) with bursacytes from major histocompatibility complex B-matched control line (11 X 58) F1 birds. This transfer produced little change in the incidence of dysgammaglobulinemia in UCD 140 transfer birds (56%) compared to unmanipulated line 140 birds (60%). These data reflect a failure of line 140, rather than technique, because successful reconstitution was seen using line 11 X 58 birds injected with 11 X 58 bursacytes. In contrast, the generation of UCD 140/line 11 X 58 chimeras significantly reduced the incidence of dysgammaglobulinemia in line UCD birds. Indeed, fusion of the chorioallantoic vascular system (parabiosis) of UCD 140 and 11 X 58 embryos on Day 15 decreased the frequency of dysgammaglobulinemia of UCD 140 parabionts to 14% compared to 66% in unmanipulated line 140 controls. The success of parabiosis was 83% as determined by demonstrating chimerism with allogeneic blood groups. Moreover, the frequency of dysgammaglobulinemia in the 17% of parabionts that did not reveal chimerism was similar to unmanipulated UCD 140 chickens.

Genetic correlations of abdominal fat with production traits of Leghorn hens based on inbred line averages.

Data from 16 inbred lines derived by three generations of full sib mating from a common outbred base population were used to estimate genetic correlations between body weight, abdominal fat content, follicle number, liver weight, egg weight and egg number using between-line variances and covariances. High genetic correlations were found between body weight, fat content and egg weight. Low genetic correlations were found between follicle number and all traits other than egg weights. Theoretical expectations and low correlations within lines led to the conclusion that environmental correlations among the traits considered were very low.