ABSTRACT. For genotyping of feline major histocompatibility complex (FLA) class II DRB, the polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method using group-specific primers was tried. Sixty-six DRB genes were classified into 8 groups according to differences in the first 5' amino acid sequences. The group-specific primers were designed as forward ones, which were specific for 5' base sequences of genes in each group. Three to 7 appropriate restricted enzymes were selected by computer analysis for RFLP typing of the genes divided into each group. In 6 out of 9 cats, the results of DRB typed by direct sequence method agreed with results of the PCR-RFLP method using group-specific primers. In the other 3 cats, the number of genes amplified by group-specific primers was 1 or 2 more than those detected by direct sequence method. The direct sequence method in 9 cats identified 5 new FLA-DRB genes. The PCR-RFLP method using group-specific primers could divide 66 genes into 37 genes and 10 subgroups from the RFLP pattern. One to 6 genes in each cat, and a total of 203 genes and subgroups were detected in 68 domestic cats. The genes detected might be biased to the subgroup G1-1a (28.8%), DRB*0501 (10.3%), G1-2a (9.4%) and G6b (7.4%). The PCR-RFLP method using group-specific primers may be useful in typing FLA class II DRB. KEY WORDS: feline, FLA class II DRB genotyping, group-specific primer, PCR-RFLP method.J. Vet. Med. Sci. 62(12): 1283-1289, 2000 Major histocompatibility complex (MHC) class II molecules are heterodimeric glycoproteins involved in the regulation of the immune responses [21]. Human leukocyte antigen (HLA) class II genes consist of various components, DR, DP, DQ and etc. [11]. However, the structure of feline MHC (FLA) class II has not been well established, and only DR genes have been detected at the present time [29]. The DRB gene which encodes the β chain of DR molecule is the most polymorphic in class II genes in humans [13], pigs [8] and dogs [3]. Polymorphism of DRB gene accumulates at the second exon, which encodes the first extracellular domain, β 1 domain of the DR β chain [10]. The type of DRB gene is associated with the result of mixed lymphocyte reaction (MLR) [1], percent of 1-year renal graft survival [22], as well as susceptibility to autoimmune diseases [21,25] in humans. Polymerase chain reaction (PCR)-sequence specific oligonucleotide probe (SSOP) method or PCR-restriction fragment length polymorphism (RFLP) method has been used commonly to distinguish the genotypes of MHC class II [9,11]. It has also been reported that digestions of PCR products by some restriction endonucleases after the PCR for grouping of alleles by group-specific primers (modified PCR-RFLP method combined with group-specific primers) were used for genotyping of human DRB1 [16].Chronic renal failure (renal sclerosis) is one of the frequent and important diseases in feline medicine. The basic therapies for this failure are fluid therapy, and administrations of antibiotics, steroids,...

ABSTRACT. In order to confirm the effects of matching of expressed feline major histocompatibility complex (FLA) class II DRB genotype on transplantation immunity in cats, skin-allogeneic transplantation was carried out between cats, in which DRB genes expressed were genotyped by the RT-PCR-RFLP method using group-specific primers. Duration until grafts were rejected was 14.63 ± 1.69 days (mean ± standard deviation) in the pairs that had the same type of subgroups, 7.25 ± 0.71 days in the pairs that had one different type of subgroup and 6.88 ± 0.35 days in the pairs that had two different types of subgroups. The duration of graft survival in the pairs with the same type of subgroups was significantly longer (P<0.01) than those in the pairs with different types. Although FLA components involved in transplantation immunity should not only be DRB genes, it was suggested that the expressed FLA-DRB genotype might associate with feline transplantation immunity, and that typing and matching of expressed FLA-DRB genes might be one of the important factors in the control of feline transplantation immunity.

ABSTRACT. The optimal condition for methods of lymphocytotoxic crossmatch test for feline renal transplantation was investigated. On separation of viable lymphocytes from whole blood, the best results were obtained when Ficoll-diatrizoate with 1.078 of a specific gravity at 20°C was centrifuged with 800 × g for 30 min at 4°C. A nylon wool column was used to separate T and B cells from lymphocyte fraction. The ratio of T cells in nylon wool effluent cells was 95%, while the ratio of B cells in adherent cells was 41%. Lymphocytotoxic crossmatch tests were performed by using the effluent cells as T cells and the adherent cells as B cells, at 37°C (warm) and 4°C (cold). The ratio of B cells in adherent cells was low, however, the result was utilized as a matching test before transplantation by combining with the T cell result. The trypan blue stain method made it easier than the eosin stain method to distinguish living and dead cells. The lymphocytotoxic crossmatch tests were performed on 15 pairs of healthy cats, and only one pair showed doubtful positive against anti-B cell cold antibodies. During acute rejection after renal transplantation in two pairs which were negative on any anti-lymphocyte antibodies before the transplantation, the anti-T cell warm antibodies became positive in both pairs, and the anti-T cell cold antibodies became positive on one of the two pairs.-KEY WORDS: anti-lymphocyte antibody, feline, lymphocytotoxic crossmatch test, renal transplantation, trypan blue stain.