A semi‐nested polymerase chain reaction (snPCR) assay was developed for the rapid detection of resistant/susceptible BF haplotypes to Marek’s disease (MD) using the cDNA samples from peripheral blood leucocytes, liver, spleen and heart from Xiayan homozygous chickens: A11, C23, D8 and D12 (resistant to MD), A5 and B21 (susceptible to MD). The snPCR was utilized to span alternative splicing‐out of the sequence encoding the second segment of the cytoplasmic part of the mature BF molecules (exon 7). This alternative exon 7 splice variant was detected in BF*A5 and BF*B21 (susceptible to MD), but not in the MD‐resistant BF*A11, BF*C23, BF*D8 and BF*D12 haplotypes, suggesting a potential role of exon 7 for the detection of resistant/susceptible BF haplotypes to MD.

1. The aim was to analyse the variability of the BLB2/BF2 genes of Xiayan chickens to identify homozygous birds with resistance or susceptibility to Marek's disease (MD). 2. The experiment used two lines: birds from a common line were divided into Group A (unvaccinated) and Group B (vaccinated with herpesvirus of turkeys (HVT)); and birds from an MD-resistant line were divided into Group C (unvaccinated) and Group D (vaccinated with HVT). They were challenged intra-abdominally with Marek's disease virus (MDV) and genomic DNA extracted from peripheral blood lymphocytes so that polymorphism of the BLB2/BF2 genes could be analysed using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and nucleotide sequence analysis. 3. A 374-bp fragment of the BLB2 gene was amplified from the samples and, after digesting with restriction enzymes Alu I, Cai I, Cfr I, Hin1 I, Hinf I and Rsa I for RFLP analysis, the 6 electrophoretic patterns were analysed. Seven homozygous genotypes were found and used tentatively to identify alleles of the BLB2 gene. 4. A 765-bp fragment of the BF2 gene was amplified from the 7 samples for cloning and sequencing. 5. Six homozygous birds were confirmed from the sequenced BLB2/BF2 gene. Four birds were resistant to MD. Three birds had identical nucleotide sequences and were highly homologous with MHC haplotype B⁶, which is MD resistant. One bird had high homology with the highly MD-resistant B²¹ haplotype, and two birds were susceptible and highly homologous to the B¹⁹ haplotype, which is highly MD susceptible.

The study was to analyse the relationship between the alternative exon 7 splice variant of the BF gene and MHC-related Marek's disease (MD) resistance in chickens. The experiment first determined whether or not the cocks of Xiayan chickens have alternative splicing-out of the exon 7 of the BF gene from peripheral blood leucocytes (PBLs). Then, selected two groups: Group K included the offspring of the selected cocks which have no alternative splicingout of the exon 7 of the BF gene; Group Y included the offspring of the selected cocks which have alternative splicing-out of the exon 7 of the BF gene. All hens used in the cross-breeding were non-selected. The experimental chickens were challenged with a very virulent strain of Marek's disease virus (MDV) at 4 days old and were raised for 12 weeks. At this time, all the surviving chickens were killed and necropsy was also performed during the experiment whenever chickens died from the infection. Tumour incidence and mortality were calculated using SPSS, and the tissues were collected to detect MDV by PCR. The results showed that the mortalities of Group K and Y were 52.75% and 70.65%, respectively; and that the tumour incidences of non-alternative splicing-out of the exon 7 of the BF for Groups K and Y were 15.38% and 38.89%, respectively; the tumour incidences for the alternative splicing-out of the exon 7 were 46.15% and 56.76%, respectively. The results demonstrated the tumour incidence was highly related with the alternative exon 7 splice variant of the BF gene (P < 0.05).

The three gldCDE genes from Lactobacillus diolivorans, that encode the three subunits of the glycerol dehydratase, were cloned and the proteins were co-expressed in soluble form in Escherichia coli with added sorbitol and betaine hydrochloride. The purified enzyme exists as a heterohexamer (α2β2γ2) structure with a native molecular mass of 210 kDa. It requires coenzyme B12 for catalytic activity and is subject to suicide inactivation by glycerol during catalysis. The enzyme had maximum activity at pH 8.6 and 37 °C. The apparent K m values for coenzyme B12, 1,2-ethanediol, 1,2-propanediol, and glycerol were 1.5 μM, 10.5 mM, 1.3 mM, and 5.8 mM, respectively. Together, these results indicated that the three genes gldCDE encoding the proteins make up a coenzyme B12-dependent diol dehydratase and not a glycerol dehydratase.