Effects of Escherichia coli Dairy Cow Mastitis on the Expressions of Milk-producing Trait Related Genes

doi:10.13560/j.cnki.biotech.bull.1985.2022-0826

Abstract

Abstract:

This work aims to explore the reasons of milk yield and milk quality declining from dairy cows with Escherichia coli mastitis at the genetic level. This experiment established a cell inflammation model by lipopolysaccharide(LPS)-induced bovine mammary epithelial cells（bMECs）. The expressions of milk yield and milk protein-related genes（CSN1S1, CSN2 and LALBA）, milk fat regulation-related genes（FABP3 and LPL）and inflammation and milk yield-related gene MFGE8 were detected by real-time quantitative PCR（qPCR）. The results showed that the expressions of the above 6 genes were significantly down-regulated in the mammary tissues of dairy cows with E.coli mastitis（P < 0.01）compared with the control group（0 h）. In addition, the expressions of the six genes were also significantly down-regulated in LPS-induced bMECs at 3, 6, 12 and 24 h（P < 0.01）. This indicated that the expressions of the six genes were inhibited after E. coli mastitis or bMECs inflammation occurred, which explained the reasons for E.coli mastitis reducing milk yield, milk protein percentage and milk fat percentage.

Key words: Escherichia coli, mastitis, milk-producing trait, mammary epithelial cells, gene expression

LI Yan-xia, WANG Jin-peng, FENG Fen, BAO Bin-wu, DONG Yi-wen, WANG Xing-ping, LUORENG Zhuo-ma. Effects of Escherichia coli Dairy Cow Mastitis on the Expressions of Milk-producing Trait Related Genes[J]. Biotechnology Bulletin, 2023, 39(2): 274-282.

Figures/Tables 8

Table1 qPCR primer information

基因名称Gene name	GenBank登录号GenBank ID	引物序列Primer sequence（5'-3'）	片段大小Product length/bp
IL-6^[17]	NM_173923.2	F：CTGGGTTCAATCAGGCGAT R：CAGCAGGTCAGTGTTTGTGG	205
IL-8^[18]	JN559767.1	F：ACACATTCCACACCTTTCCAC R：ACCTTCTGCACCCACTTTTC	149
TNF-α	NM_173966.3	F：TCTGGGCAGGTCTACTTTG R：CCTGAGCCCATAATTCCCT	139
CSN1S1	NM_181029	F：ATGTGCCCTCTGAGCGTTAC R：AGGCACCAGATGGATAGGC	237
CSN2	XM_015471671.2	F：CTATGGCTCCTAAGCACAAAGA R： GCAGAGGCAGAGGAAGGTG	117
LPL	NM_001075120	F：GTTTATGAACTGGATGGCGG R：GAGAAAGGCGACTTGGAGC	188
FABP3	NM_174313	F：TACTTACGAGAAACAGGCATGA R：CCTCAGAGCACCCTTTGG	232
LALBA	NM_174378	F：AAAGACGACCAGAACCCTCA R：GAAAGAGGACAGAAGCAGCAA	223
MFGE8	NM_176610	F：CCCGCCCTCCTGATTGTA R：CCCACCGTGCCTAAGAAAA	141
RPS18^[18]	NM_001033614.2	F：GTGGTGTTGAGGAAAGCAGACA R：TGATCACACGTTCCACCTCATC	79
GAPDH^[18]	NM_001034034.2	F：GGCATCGTGGAGGGACTTATG R：GCCAGTGAGCTTCCCGTTGAG	186

Fig. 1 Expressions of CSN1S1, CSN2 and LALBA gene in the cow mammary tissues of dairy cows with E.coli type mastitis **** refers to P < 0.000 1，E.coli means mammary tissue of cow with E.coli type mastitis; control means mammary tissue of healthy cow. The same below

Fig. 2 Expressions of FABP3 and LPL gene in the mam-mary tissues of dairy cows with E.coli mastitis

Fig. 3 Expression of MFGE8 gene in mammary tissue of dairy cows with E.coli mastitis

Fig. 4 Expression of main inflammatory factor genes in LPS-induced bMECs at different times A: bMECs cultured in vitor（200×）. B: The relative expression level of TNF-α. C: The relative expression of IL-6. D: The relative expression level of IL-8.*P < 0.5; **P < 0.01; and ****P < 0.000 1. The same below

Fig. 5 Expressions of milk protein-related genes in LPS-induced bMECs at different times A: Relative expression of CSN1S1. B: The relative expression of CSN2. C: The relative expression of LALBA

Fig. 6 Expressions of milk fat regulation related genes in LPS-induced bMECs at different times A: Relative expression level of FABP3; B: The relative expression level of LPL

Fig. 7 Expressions of MFGE8 gene in LPS-induced bMECs at different times

References 37

[1]	le Maréchal C, Thiéry R, Vautor E, et al. Mastitis impact on technological properties of milk and quality of milk products—a review[J]. Dairy Sci Technol, 2011, 91(3): 247-282. doi: 10.1007/s13594-011-0009-6 URL
[2]	卢世杰, 吴红翔, 习学勇, 等. 奶牛乳房炎对主要牛奶成分的影响[J]. 中国奶牛, 2019(5): 27-32.
	Lu SJ, Wu HX, Xi XY, et al. Effect of cow mastitis on major milk components[J]. China Dairy Cattle, 2019(5): 27-32.
[3]	Ju ZH, Jiang Q, Wang JP, et al. Genome-wide methylation and transcriptome of blood neutrophils reveal the roles of DNA methylation in affecting transcription of protein-coding genes and miRNAs in E. coli-infected mastitis cows[J]. BMC Genomics, 2020, 21(1): 102. doi: 10.1186/s12864-020-6526-z URL
[4]	Blum SE, Heller ED, Leitner G. Long term effects of Escherichia coli mastitis[J]. Vet J, 2014, 201(1): 72-77. doi: 10.1016/j.tvjl.2014.04.008 URL
[5]	Liu HY, Kai LX, Du HH, et al. LPS inhibits fatty acid absorption in enterocytes through TNF-α secreted by macrophages[J]. Cells, 2019, 8(12): 1626. doi: 10.3390/cells8121626 URL
[6]	He XX, Liu WJ, Shi MY, et al. Docosahexaenoic acid attenuates LPS-stimulated inflammatory response by regulating the PPARγ/NF-κB pathways in primary bovine mammary epithelial cells[J]. Res Vet Sci, 2017, 112: 7-12. doi: S0034-5288(16)30149-7 pmid: 28095338
[7]	Luoreng ZM, Wang XP, Mei CG, et al. Expression profiling of peripheral blood miRNA using RNAseq technology in dairy cows with Escherichia coli-induced mastitis[J]. Sci Rep, 2018, 8(1): 12693. doi: 10.1038/s41598-018-30518-2 URL
[8]	Luoreng ZM, Wang XP, Mei CG, et al. Comparison of microRNA profiles between bovine mammary glands infected with Staphylococcus aureus and Escherichia coli[J]. Int J Biol Sci, 2018, 14(1): 87-99. doi: 10.7150/ijbs.22498 URL
[9]	Bionaz M, Loor JJ. ACSL1, AGPAT6, FABP3, LPIN1, and SLC27A6 are the most abundant isoforms in bovine mammary tissue and their expression is affected by stage of lactation[J]. J Nutr, 2008, 138(6): 1019-1024. doi: 10.1093/jn/138.6.1019 URL
[10]	Liu XL, Shen JL, Zong JX, et al. Beta-sitosterol promotes milk protein and fat syntheses-related genes in bovine mammary epithelial cells[J]. Animals(Basel), 2021, 11(11): 3238.
[11]	Song N, Chen YT, Luo J, et al. Negative regulation of αS1-casein(CSN1S1)improves β-casein content and reduces allergy potential in goat milk[J]. J Dairy Sci, 2020, 103(10): 9561-9572. doi: 10.3168/jds.2020-18595 URL
[12]	Atabai K, Fernandez R, Huang XZ, et al. Mfge8 is critical for mammary gland remodeling during involution[J]. Mol Biol Cell, 2005, 16(12): 5528-5537. pmid: 16195353
[13]	Sabha BH, Alzahrani F, Almehdar HA, et al. Disorder in milk proteins: lactadherin multifunctionality and structure[J]. Curr Protein Pept Sci, 2018, 19(10): 983-997. doi: 10.2174/1389203719666180608091849 URL
[14]	罗仍卓么, 王晋鹏, 焦鹏, 等. 奶牛乳腺炎模型的建立及炎症相关因子基因mRNA转录水平的分析[J]. 畜牧兽医学报, 2022, 53(8): 2763-2772.
	Luo R, Wang JP, Jiao P, et al. Construction of dairy cow mastitis model and analysis of mRNA transcription level of inflammation related cytokine genes[J]. Acta Vet Zootechnica Sin, 2022, 53(8): 2763-2772.
[15]	Yang J, Hu QC, Wang JP, et al. RNA-seq reveals the role of miR-29c in regulating inflammation and oxidative stress of bovine mammary epithelial cells[J]. Front Vet Sci, 2022, 9: 865415. doi: 10.3389/fvets.2022.865415 URL
[16]	Wang XP, Luoreng ZM, Zan LS, et al. Bovine miR-146a regulates inflammatory cytokines of bovine mammary epithelial cells via targeting the TRAF6 gene[J]. J Dairy Sci, 2017, 100(9): 7648-7658. doi: 10.3168/jds.2017-12630 URL
[17]	焦鹏, 王兴平, 汪书哲, 等. 奶牛乳腺炎差异表达lncRNA BCL2对炎症及凋亡相关mRNA表达的影响[J]. 畜牧兽医学报, 2022, 53(7): 2160-2171.
	Jiao P, Wang XP, Wang SZ, et al. Effect of differentially expressed lncRNA BCL2 in dairy cow with mastitis on the expression of inflammation and apoptosis related mRNA[J]. Acta Vet Zootechnica Sin, 2022, 53(7): 2160-2171.
[18]	李宇航, 王兴平, 杨箭, 等. miR-665在奶牛乳腺上皮细胞炎症中的表达及功能分析[J]. 生物技术通报, 2022, 38(5): 159-168.
	Li YH, Wang XP, Yang J, et al. Expression and functional analysis of miR-665 in bovine mammary epithelial cell inflammation[J]. Biotechnol Bull, 2022, 38(5): 159-168.
[19]	Liu MJ, Zhang C, Xu XL, et al. Ferulic acid inhibits LPS-induced apoptosis in bovine mammary epithelial cells by regulating the NF-κB and Nrf2 signalling pathways to restore mitochondrial dynamics and ROS generation[J]. Vet Res, 2021, 52(1): 104. doi: 10.1186/s13567-021-00973-3 pmid: 34256834
[20]	Sharun K, Dhama K, Tiwari R, et al. Advances in therapeutic and managemental approaches of bovine mastitis: a comprehensive review[J]. Vet Q, 2021, 41(1): 107-136. doi: 10.1080/01652176.2021.1882713 pmid: 33509059
[21]	李莲, 唐娟, 吴洁, 等. 基于iTRAQ技术的脂多糖刺激下奶牛乳腺上皮细胞的蛋白组学分析[J]. 畜牧兽医学报, 2016, 47(9): 1853-1860.
	Li L, Tang J, Wu J, et al. Analysis of expression protein profiles of bovine mammary epithelial cell based on isobaric tag for relative and absolute quantification proteomics and bioinformatics technique[J]. Chin J Animal Vet Sci, 2016, 47(9): 1853-1860.
[22]	Chen JB, Wu YJ, Sun YW, et al. Bacterial endotoxin decreased histone H3 acetylation of bovine mammary epithelial cells and the adverse effect was suppressed by sodium butyrate[J]. BMC Vet Res, 2019, 15(1): 267. doi: 10.1186/s12917-019-2007-5 pmid: 31357995
[23]	Visker MHPW, Dibbits BW, Kinders SM, et al. Association of bovine β-casein protein variant I with milk production and milk protein composition[J]. Anim Genet, 2011, 42(2): 212-218. doi: 10.1111/j.1365-2052.2010.02106.x pmid: 24725229
[24]	Dixit SP, Singh S, Ganguly I, et al. Genome-wide runs of homozygosity revealed selection signatures in Bos Indicus[J]. Front Genet, 2020, 11: 92. doi: 10.3389/fgene.2020.00092 pmid: 32153647
[25]	Wu YJ, Chen JB, Sun YW, et al. PGN and LTA from Staphylococcus aureus induced inflammation and decreased lactation through regulating DNA methylation and histone H3 acetylation in bovine mammary epithelial cells[J]. Toxins, 2020, 12(4): 238. doi: 10.3390/toxins12040238 URL
[26]	赵笑, 白沙沙, 孔凡华, 等. 乳蛋白功能特性及其分析检测技术研究进展[J]. 中国乳品工业, 2022, 50(1): 37-42.
	Zhao X, Bai SS, Kong FH, et al. Research progress on the functional properties of milk protein and its analytical detection technology[J]. China Dairy Ind, 2022, 50(1): 37-42.
[27]	Neville MC. Introduction: alpha-lactalbumin, a multifunctional protein that specifies lactose synthesis in the Golgi[J]. J Mammary Gland Biol Neoplasia, 2009, 14(3): 211-212. doi: 10.1007/s10911-009-9149-1 URL
[28]	单曦, 吉丽, 张永云, 等. 槟榔江水牛LALBA基因克隆及序列特征分析[J]. 云南农业大学学报: 自然科学, 2017, 32(5): 830-837.
	Shan X, Ji L, Zhang YY, et al. Cloning, sequence characterization of Binglangjiang buffalo gene LALBA[J]. J Yunnan Agric Univ Nat Sci, 2017, 32(5): 830-837.
[29]	Spitzer AJ, Tian Q, Choudhary RK, et al. Bacterial endotoxin induces oxidative stress and reduces milk protein expression and hypoxia in the mouse mammary gland[J]. Oxid Med Cell Longev, 2020, 2020: 3894309.
[30]	Li AN, Wu LJ, Wang XY, et al. Tissue expression analysis, cloning and characterization of the 5'-regulatory region of the bovine FABP3 gene[J]. Mol Biol Rep, 2016, 43(9): 991-998. doi: 10.1007/s11033-016-4026-7 URL
[31]	Zhao WS, Hu SL, Yu K, et al. Lipoprotein lipase, tissue expression and effects on genes related to fatty acid synthesis in goat mammary epithelial cells[J]. Int J Mol Sci, 2014, 15(12): 22757-22771. doi: 10.3390/ijms151222757 URL
[32]	Liang MY, Hou XM, Qu B, et al. Functional analysis of FABP3 in the milk fat synthesis signaling pathway of dairy cow mammary epithelial cells[J]. In Vitro Cell Dev Biol Anim, 2014, 50(9): 865-873. doi: 10.1007/s11626-014-9780-z URL
[33]	Yang YW, Pan QQ, Sun BX, et al. miR-29b targets LPL and TDG genes and regulates apoptosis and triglyceride production in MECs[J]. DNA Cell Biol, 2016, 35(12): 758-765. doi: 10.1089/dna.2016.3443 URL
[34]	Zhong WQ, Shen JL, Liao XD, et al. Camellia(Camellia oleifera Abel.)seed oil promotes milk fat and protein synthesis-related gene expression in bovine mammary epithelial cells[J]. Food Sci Nutr, 2019, 8(1): 419-427. doi: 10.1002/fsn3.1326 URL
[35]	Fitz-Gerald CH, Deeth HC, Kitchen BJ. The relationship between the levels of free fatty acids, lipoprotein lipase, carboxylesterase, N-acetyl-beta-D-glucosaminidase, somatic cell count and other mastitis indices in bovine milk[J]. J Dairy Res, 1981, 48(2): 253-265. pmid: 7298962
[36]	Hanayama R, Nagata S. Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8[J]. Proc Natl Acad Sci USA, 2005, 102(46): 16886-16891. pmid: 16275924
[37]	Nakatani H, Aoki N, Nakagawa Y, et al. Weaning-induced expression of a milk-fat globule protein, MFG-E8, in mouse mammary glands, as demonstrated by the analyses of its mRNA, protein and phosphatidylserine-binding activity[J]. Biochem J, 2006, 395(1): 21-30. pmid: 16401186