Antibody responses to sheep erythrocytes for White Leghorn chickens differing in haplotypes of the major histocompatibility complex (B)

Summary. Lines of White Leghorn chickens were developed by selection for high (HA) or low (LA) antibody response to sheep red blood cells (SRBC) and then backcrossed to provide individuals segregating for haplotypes B ¹³ and B ²¹ of the major histocompatibility complex (MHC) within each selected line. Although antibody response to SRBC was consistently higher in background genome HA than LA, there was a significant interaction between background genome and MHC haplotypes. The interaction resulted from higher antibody response in B¹³/B²¹ individuals of line HA and in B²¹/ B ²¹ individuals of line LA. Thus, response to SRBC was dependent on particular haplotype combinations present at the MHC as well as the background genome in which they were expressed.     

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.     

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.     

Diversity and locus specificity of chicken MHC B class I sequences

The major histocompatibility complex B (MHC B) region in a standard haplotype of Leghorn chickens contains two closely linked class I loci, B-FI and B-FIV. Few sequences of B-FI alleles are available, and therefore alleles of the two loci have not been compared with regard to sequence diversity or locus specificity. Here, we report eight new B-F alpha 1/alpha 2-coding sequences from broiler chicken MHC B haplotypes, and a unique recombinant between the two B-F loci. The new sequences were combined with existing B-F sequences from Leghorn and broiler haplotypes for analysis. On the basis of phylogenetic analysis and conserved sequence motifs, B-F sequences separated into two groups (Groups A and B), corresponding to B-FIV and B-FI locus, respectively. Every broiler haplotype had one B-F sequence in Group A and the second B-F sequence, if it existed, clustered in Group B. Group B (presumptive B-FI locus) sequences identified in broiler haplotypes resembled the human MHC class I HLA-C locus in their distinctive pattern of allelic polymorphism. Compared with B-FIV, B-FI alleles were less polymorphic and possessed a conserved locus-specific motif in the alpha1 helix, but nevertheless demonstrated evidence of diversifying selection. One B-FI alpha 1/alpha 2-coding nucleotide sequence was completely conserved in four different broiler haplotypes, but each allele differed in the exon encoding the alpha 3 domain.     

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.     

Casein haplotypes and their association with milk production traits in the Finnish Ayrshire cattle

Polymorphism of casein genes was studied in half-sib families of artificial insemination bulls of the Finnish Ayrshire dairy breed. Ten grandsires and 300 of their sons were genotyped for the following polymorphisms: α_S1-casein (B, C), β-casein (A¹, A²), the microsatellite within the K-casein gene (ms⁵, ms⁴) and K-casein (A, B, E). Nine different combinations of these alleles, casein haplotypes, were found. Associations between casein haplotypes and milk production traits (milk and protein yield, fat and protein percentage and milking speed) were studied with ordinary least-squares analysis to find a direct effect of the haplotypes or an association within individual grandsire families using the granddaughter design. Estimated breeding values of sons were obtained from cow evaluation by animal model. No direct effect of the casein haplotypes on the traits was found. Within grandsire families, in one out of four families the chromosomal segment characterized by haplotype 3 (B-A²-ms⁴-A) was associated with an increase in milk yield ( P <0.01) and a decrease in fat percentage ( P < 0.01) when contrasted with haplotype 8 (B-A¹-ms⁴-E). The results provide evidence that in the Finnish Ayrshire breed at least one quantitative trait locus affecting the genetic variation in yields traits is segregating linked to either haplotype 3 (B-A²-ms⁴-A) or 8 (B-A¹-ms⁴-E).     

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of β -casein expression in milk. Alleles CSN2 ^A , CSN2 ^{A 1}, CSN2 ^B , CSN2 ^C , CSN2 ^D and CSN2 ^E have been associated with normal content (allele effects of about 5 g of β -casein per litre), whereas the CSN2 ⁰ and CSN2 ⁰¹ alleles have been associated with non-detectable levels of β -casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region ( AJ011018 :g.1311T>C), which is associated with the absence of β -casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.     

Molecular characterization of major and minor MHC class I and II genes in B21-like haplotypes in chickens

The major histocompatibility complex (MHC) sequences of three B21-like haplotypes deriving from very different origins including the Red Jungle Fowl Gallus Gallus gallus were compared with the MHC sequences of the standard B21 haplotype from Scandinavian White Leghorn Gallus domesticus. The present analysis reveals two cDNA sequences for B-F and two cDNA sequences for B-LB for every B21-like haplotype, including B21 itself. Contrary to expectation, no sequence polymorphism in the antigen-binding domains of the MHC genes, between the investigated haplotypes, was found. The relative level of MHC class I molecules on the surface of leukocytes measured by flow cytometry was also analysed and found to be low in Marek's Disease (MD)-resistant B haplotypes (B21 and B21-like) and high in MD-susceptible B haplotypes (B15 and B19). However, in heterozygous (resistant/susceptible) animals, the relative level was almost as high as in susceptible haplotypes.     

Genotyping bovine milk proteins using allele discrimination by primer length and automated DNA sizing technology

A method for genotyping K-casein ( A, B, E ), β-casein ( A ¹, A ², A ³, A⁵, B ) and β-lactoglobulin ( A, B ) simultaneously by the use of allele discrimination by primer length combined with automated detection of fragments with a sequencing instrument is described. Seven different mutations within the milk protein genes were analysed in order to distinguish between the alleles examined. The samples were amplified in two separate multiplex polymerase chain reactions (PCRs), which were then pooled and separated according to size in a single lane on the gel. By using stringent PCR conditions, we have been able to achieve allele-specific amplifications and minimize amplification of mismatched primer for all seven mutations.     

Characterization of amplified esterase Estβ12 associated with organophosphate resistance in a multi-resistant population of the mosquitoCulex quinquefasciatus from Cuba

Esterase amplification is the major organophosphorus (OP) insecticide resistance mechanism in Culex mosquitoes. The amplified Estα2 ^1\ Estβ2 ¹ esterases are found in > 90% of resistant populations worldwide, whereas amplified DNAs (amplicons) containing Estβ1s are much rarer. Individuals with the Estβ1 amplicons appear to be at a selective disadvantage in competition with those carrying the Estα2 ¹\ Estβ2 ¹ amplicons. To test the hypothesis that this is because Estβ1 is less able to bind insecticide than the common amplified esterases, Estβ1² was purified from the multi-resistant Habana strain of Culex quinquefasciatus , from Cuba. In its native form Estβ1 is a monomeric enzyme of 66 kDa, with a pI of 4.8. The bimolecular rate constants for interaction of Estβ1² with several OP insecticides were similar to those for the commonly elevated esterases Estα2¹ and Estβ2¹, and much higher than for the electrophoretically identical non-elevated Estβ1³ and Estα3. Hence the apparent selective advantage of the Estα2 ¹\ Estβ2 ¹ amplicon is not due to its greater efficiency of insecticide binding, as OP insecticides are significantly better inhibitors of all the amplified esterases than of their non-amplified counterparts and therefore should be equally effective at conferring resistance.     

Interaction between superantigen and T-cell receptor Vβ element determines levels of superantigen-dependent cell-mediated cytotoxicity of CD8+ T cells in induction and effector phases

Specific superantigens activate different T-cell fractions with distinct TCR Vβ elements in association with MHC class II molecules and also induce SDCC against MHC class II⁺ target cells. In the present study, to determine whether the responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the TCR Vβ, we compared the levels of proliferation and SDCC in Vβ3⁺ and Vβ11⁺ T cells upon stimulation with SEA. Upon stimulation with SEA_wt, the levels of proliferation were higher in Vβ3⁺ T cells than in Vβ11⁺ T cells. The levels of SDCC were also higher for the combination of Vβ3⁺ T cells and SEA_wt than for the combination of Vβ11⁺ T cells and SEA_wt during both the induction phase and the effector phase. In addition, upon stimulation with SEA_m, the levels of proliferation were higher in Vβ11⁺ T cells than in Vβ3⁺ T cells. And then, the levels of SDCC were also higher for the combination of Vβ11⁺ T cells and SEA_m than for the combination of Vβ3⁺ T cells and SEA_m during both the induction phase and the effector phase. These results suggest that the SAG-responsiveness of each CD8⁺ T-cell fraction expressing a different TCR Vβ element is primarily determined by the interaction between the TCR Vβ element and the SAG.     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

Inheritance of adenosine deaminase variants in chickens and turkeys

Blood cell lysates of chickens and turkeys were subjected to starch gel electrophoresis and the gels were stained for adenosine deaminase. Two zones were observed singly or together in the electrophoretic patterns of each lysate. Zones of chicken lysates were analogous in electrophoretic mobility to those of turkeys. An extra zone which appeared in patterns of a sample stored over one month was not detected in patterns of a second aliquot of stored sample treated with a reducing agent prior to electrophoresis.
Family data involving 110 chicken progeny and 221 turkey progeny supported the hypothesis that these zones were controlled by two codominant alleles designated ADA.⁴ and ADA^B. In the two Leghorn strains studied ADA^B was much more frequent than ADA.⁴, but the frequency distribution was reversed in the Small White turkey strain examined.     

In Human Fetal Astrocytes Exposure to Interleukin-1β Stimulates Acquisition of the GD3+ Phenotype and Inhibits Cell Division

Abstract: In human astrocyte cultures established from second-trimester fetal brain tissue, ∼5–10% of total astrocyte population in unstimulated cultures were GD3⁺/glial fibrillary acidic protein (GFAP)⁺. The GD3⁺ cells were always GFAP⁺ and grew as flat, highly spread cells but changed to process-bearing cells after interleukin-1β (IL-1β) stimulation. It is interesting that IL-1β, a known mitogen for rat astrocytes, suppressed human fetal astrocyte proliferation as determined by [³H]thymidine incorporation, bromodeoxyuridine (BrdU) labeling, and cell counting. The GD3⁺ population, however, consistently increased in absolute number after IL-1β stimulation, in a dose- and time-dependent manner. The IL-1β-mediated increase in number of GD3⁺ astrocytes was independent of initial cell density or serum concentration. By flow cytometry, IL-1β enhanced both the mean fluorescence intensity and the percentage of GD3⁺ cells. To investigate whether the increase in GD3⁺ astrocyte cell number was due to proliferation of preexisting GD3⁺ astrocytes or due to conversion of GD3⁻ to GD3⁺ cells, we performed BrdU/GD3 double immunocytochemistry. BrdU/GD3 double-positive cells were extremely rare in both control and IL-1β-stimulated cultures. Moreover, an increase in number of GD3⁺ astrocytes was still observed in control and IL-1β-stimulated cultures where GD3⁺ cells had been initially eliminated by cell sorting. These results indicate that GD3⁺ astrocytes in human fetal culture may represent a postmitotic, differentiated, distinct phenotype.     

Determination of pig apolipoprotein B genotype by gene amplification and restriction fragment length polymorphism analysis

Summary
Sequence differences within the pig apoB gene can be used to identify rapidly four of eight known pig apoB alleles, designated LPB ¹- LPB ⁸. We describe the use of gene amplification, followed by endonuclease digestion and agarose gel electrophoresis, to discern size and restriction site differences. LPB ⁵, a common allele associated with reduced low density lipoprotein clearance and hypercholesterolaemia in pigs, is identified by a 283-bp insertion in intron 28. LPB ³ and LPB ⁷ are distinguished by a unique Hind III site; LPB ⁸ shares a unique Hinc II site with LPB ⁵. This method facilitates identification of the apoB genotype of pigs used in lipoprotein research and allows for further investigation into the association of particular apoB alleles with lipoprotein metabolism abnormalities.     

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.     

The MHC haplotypes of the chicken

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as standard B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The standard haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.