Abstract：
Bovine mastitis is a complex infectious disease that develops in the mammary gland, predominantly caused by a bacterial infection of mammary tissue. Genetic variability of mastitis is well established and depends upon different quantitative trait loci (QTL) related to mastitis resistance or susceptibility. The susceptibility is often attributed to single-nucleotide polymorphisms (SNPs) in the variable cow breed genomes. Several global investigative attempts have resulted in studies mapping mastitis to the variations in the relevant genes. Reports have been attributed to dramatic genetic expression changes in Toll-Like Receptor 4 (TLR4) genes in mastitis-positive cows. However, the mechanism behind this variable genetic expression of TLR4 genes has been studied poorly. The present study aims to investigate SCM through various screening tests like somatic cell count (SCC), electric conductivity (EC), pH, and California mastitis test (CMT) in milk samples. This study also aims to investigate possible mechanisms behind this variable expression of TLR4 by comparative SNP evaluation and transcriptional factor profile mining. So that the important genetic mutations and effects thereof can be exploited in selecting specific breeds with higher mastitis resistance and milk yield. Seventy Holstein Frisian (HF) crossbred dairy cows were selected in the present study. The animals were screened based on various diagnostic tests (SCC, pH, EC, and CMT). Blood samples (5 mL) were collected for extraction of DNA followed by amplification of PPR1 and PPR2 of the promoter region and 5\'UTR of the bovine TLR4 gene using specific primers. Sanger\'s enzymatic DNA sequencing technique sequenced the amplified PCR products. Further, the identification of SNPs was done through various bioinformatic tools used in this study. The findings of the present study revealed that CMT, EC, pH, and SCC could be used for the early detection of subclinical mastitis. In the present study, a significant increase in the EC, pH, and SCC in milk samples of animals affected with SCM was found in comparison to the healthy animals. The present study also revealed 16 SNPs falling in TLR4 promoter and 5\' untranslated region (5\'UTR) sequences in mastitis-positive genotypes compared to reference genomes. The study also investigates the potential transcriptional factor program deployed in response to variable mastitis development resistance. In the present study, the allelic and genotype frequencies of all SNP variants in the three regions viz., PPR1, PPR2, and 5\'UTR, were the same indicating the absence of heterozygous condition at the respective loci. The present study has wide applicability for researchers developing mastitis-resistant breeding programs and the data generated may aid in the selection of better genetic breeds. The transcription factor binding profiles can serve as concrete leads about the studies on bovine mastitis at the molecular level and may also aid global research groups working on transcription factor (TF)-based molecular pathology of mastitis.
摘要：
牛乳腺炎是一种在乳腺中发展的复杂传染病，主要由乳腺组织的细菌感染引起。乳腺炎的遗传变异性已经很好地确定，并且取决于与乳腺炎抗性或易感性相关的不同数量性状基因座（QTL）。易感性通常归因于可变奶牛品种基因组中的单核苷酸多态性（SNP）。一些全球调查尝试已经导致研究将乳腺炎映射到相关基因的变异。报道已归因于乳腺炎阳性母牛中Toll样受体4(TLR4)基因的显著遗传表达变化。然而,TLR4基因这种可变遗传表达背后的机制研究甚少。本研究旨在通过各种筛选测试，如体细胞计数（SCC），电导率(EC)，pH值,和牛奶样本中的加州乳腺炎测试（CMT）。本研究还旨在通过比较SNP评估和转录因子谱挖掘来研究TLR4这种可变表达背后的可能机制。因此,可以利用重要的遗传突变及其效应来选择具有更高乳腺炎抗性和产奶量的特定品种。在本研究中选择了70头荷斯坦弗里斯（HF）杂交奶牛。根据各种诊断测试（SCC，pH值,EC,和CMT）。收集血液样品(5mL)用于提取DNA,随后使用特异性引物扩增牛TLR4基因的启动子区的PPR1和PPR2和5'UTR。Sanger酶促DNA测序技术对扩增的PCR产物进行了测序。Further,SNP的鉴定是通过本研究中使用的各种生物信息学工具完成的。本研究的结果表明，CMT，EC,pH值,SCC可用于亚临床乳腺炎的早期检测。在本研究中,EC的显著增加，pH值,与健康动物相比，发现受SCM影响的动物的牛奶样品中的SCC。本研究还揭示了与参考基因组相比，乳腺炎阳性基因型中TLR4启动子和5'非翻译区（5'UTR）序列中的16个SNP。该研究还调查了针对可变的乳腺炎发展抗性而部署的潜在转录因子程序。在本研究中,三个区域中所有SNP变体的等位基因和基因型频率。,PPR1、PPR2和5'UTR，是相同的，表明在各自的基因座上不存在杂合条件。本研究对开发抗乳腺炎育种计划的研究人员具有广泛的适用性，所产生的数据可能有助于选择更好的遗传品种。转录因子结合谱可以作为在分子水平上研究牛乳腺炎的具体线索，也可以帮助全球研究小组研究基于转录因子（TF）的乳腺炎分子病理学。
