Identification of BoLA DRB3.2 Alleles Present in White Fulani and Muturu Cattle Breeds

Cattle production is an important aspect of animal agriculture. Disease predisposition in cattle can lead to lowered productivity and poor animal welfare. To select and breed for the fittest cattle population, it is expedient that we understand the genetic basis of disease tolerance/resistance. Major histocompatibility complex (MHC) is a vital component of the immune system in vertebrates. Its genes are crucial determinants for immune response and resistance to infectious diseases. The bovine MHC is referred to as Bovine Lymphocyte antigen (BoLA) with its most functional and highly variable region located in the exon 2 (BoLA-DRB3.2). Over 100 alleles of BoLA-DRB3.2 have been identified in cattle and many studies have associated polymorphism in this region with disease resistance/susceptibility. In this study, we investigated the polymorphic nature of BoLA-DRB3.2 in the White Fulani and Muturu cattle breeds using a single PCR-sequence based typing. We identified 26 and 25 alleles in White Fulani and Muturu breeds, respectively, with only six alleles being mutual in the two breeds. Some of the alleles identified in this study have been noted as markers for disease status in cattle. BoLA-DRB3*014:01:01, BoLA-DRB3*011:01, and BoLA-DRB3*008:01 alleles have been associated with Bovine leukemia virus (BLV) resistance in cattle. BoLA-DRB3*014:01:01, BoLA-DRB3*001:01, and BoLA-DRB3*011:01 were linked with mastitis resistance in Japanese Holstein cows. While no inference can be drawn in terms of association with disease status, this study confirms the highly polymorphic and diverse nature of BoLA-DRB3 in White Fulani and Muturu cattle breeds.

人外周血淋巴细胞增殖试验的优化及其应用

目的:优化人外周血淋巴细胞增殖试验条件;考察动物源材料的Gal抗原含量与淋巴细胞增殖效应的相关性;并比较人外周血淋巴细胞与小鼠脾脏淋巴细胞增殖试验对动物源材料的敏感性差异。方法:从细胞接种量,阳性对照(刀豆球蛋白A,Con A)的工作浓度与细胞培养时间,优化人外周血淋巴细胞增殖试验的最佳条件;利用优化的试验方法,通过人外周血淋巴细胞与动物源性材料的(牛跟腱或由牛跟腱纯化的胶原蛋白海绵)匀浆液或浸提液共培养,考察材料的不同前处理方式对人外周血淋巴细胞增殖的影响;参考标准YY/T 1561-2017,检测材料匀浆液或浸提液的Gal抗原含量,分析其抗原含量与淋巴细胞增殖效应的相关性;同时参考标准YY/T 1465.1-2016,考察材料匀浆液与浸提液对小鼠脾脏淋巴细胞增殖效应的影响,并对比分析对动物源材料的敏感性差异。结果:人外周血淋巴细胞增殖试验最佳反应条件经优化确定为:细胞接种量为1×10^5,阳性对照(Con A)的终浓度选择5~10μg·mL^-1,培养时间为4 d。人外周血淋巴细胞与胶原蛋白海绵(匀浆组与浸提液组)共培养后,样品组与正常细胞对照组相比不存在明显差异;与牛跟腱匀浆液共培养后,与正常细胞对照组相比不存在显著性差异。胶原蛋白海绵(匀浆组与浸提液组)对小鼠脾脏淋巴细胞均无明显增殖效应。牛跟腱匀浆液在5倍稀释与50倍稀释后,对小鼠脾脏淋巴细胞均有明显增殖效应(增殖率为143.20%和128.71%)。牛跟腱匀浆液的Gal抗原含量为(3.98±1.06)×10^13个·mg^-1、胶原蛋白海绵匀浆液为(2.24±0.60)×10^13个·mg^-1、胶原蛋白海绵浸提液为(1.89±0.64)×10^13个·mg^-1。结论:淋巴细胞增殖效应与动物源性生物材料中残留Gal抗原含量的正向相关性不强。与小鼠脾脏淋巴细胞增殖试验相比,采用人外周血淋巴细胞增殖试验评价含Gal抗原的动物源性生物材料细胞免疫的敏感性未见优势。淋巴细胞增殖试验是评价动物源生物材料细胞免疫的可用方法之一。