基于高通量测序技术分析四川两个奶牛场乳汁的菌群差异
作者:
基金项目:

四川省科技厅科技计划(2019YJ0650);四川农业大学学科建设双支计划(03571537)


Analysis of the differences in the microflora of milk from two dairy farms in Sichuan province based on high-throughput sequencing technology
Author:
  • 摘要
  • | |
  • 访问统计
  • |
  • 参考文献 [33]
  • |
  • 相似文献 [20]
  • |
  • 引证文献
  • | |
  • 文章评论
    摘要:

    [目的] 本试验测定了两个奶牛场健康乳汁和乳房炎乳汁中微生物菌群的变化,以揭示不同奶牛场之乳汁菌群的异同,评估其对乳汁代谢的影响是否相同。[方法] 采用16S rRNA高通量测序技术,分别测定两个奶牛场6头健康奶牛和6头乳房炎奶牛乳汁中微生物16S rRNA V4区序列,并对菌群群落结构和多样性进行比较,分析场内及场间的乳汁菌群差异。[结果] 四组乳汁样本共获得4013234条原始序列,经过滤后获得2887024条优化序列。Alpha多样性Chao指数、Ace指数、Shannon指数、Simpson指数差异均不显著(P>0.05);Beta多样性四组样本均分别聚类;在场1和场2中,引起奶牛乳房炎的优势菌属分别是克雷伯氏菌属和埃希氏菌属;在2个奶牛场的健康乳汁中,场2的埃希氏菌属、葡萄球菌属的丰度显著高于场1;在2个奶牛场的乳房炎乳汁中,场2的埃希氏菌属、乳球菌属的丰度显著高于场1;2个奶牛场健康乳汁中的嗜冷菌总丰度分别为31.87%和38.72%;关联分析及功能预测分析表明,2个奶牛场健康乳汁与乳房炎乳汁优势物种之间的关系差异较大;场1无论是Level 1还是Level 2水平,均发现显著性差异的代谢通路,而场2均未发现显著性差异的代谢通路。[结论] 本试验研究了两个奶牛场健康乳汁和乳房炎乳汁微生物菌群之间的异同,为两个奶牛场在乳房炎的预防工作以及原料奶在冷链运输过程中质量控制提供理论依据。

    Abstract:

    [Objective] This experiment studied the microbial flora in healthy milk and mastitis milk from two dairy farms to explore the similarities and differences of milk flora in different dairy farms, and to assess whether their effects on milk metabolism are the same.[Methods] The 16S rRNA high-throughput sequencing technology was used to determine the 16S rRNA V4 region sequence of microorganisms in the milk of 6 healthy cows and 6 mastitis cows in two dairy farms. The structure and diversity of bacterial community were compared, and the differences in milk flora within and between the two farms were analyzed.[Results] A total of 4013234 Raw Reads were obtained from the four groups of milk samples, and 2887024 Clean Reads were obtained after filtering. No significant differences were found in Chao index, Ace index, Shannon index and Simpson index of Alpha diversity (P>0.05); Four groups of samples with Beta diversity are clustered respectively; In farm 1 and farm 2, the dominant bacteria causing cow mastitis were Klebsiella and Escherichia, respectively; the abundances of Escherichia and Staphylococcus in the healthy milk in farm 2 were significantly higher than those in farm 1; the abundance of Escherichia and Lactococcus in the mastitis milk in farm 2 were significantly higher than those in farm 1; the abundance of Psychrophilic bacteria in the healthy milk of the two dairy farms were 31.87% and 38.72% respectively; correlation analysis and functional prediction analysis showed that there was a significant difference in the relationship between the healthy milk and the dominant species of mastitis in the two dairy farms; significant differences in metabolic pathways were found in both Level 1 and Level 2 in farm 1, while no significant differences in metabolic pathways were found in farm 2.[Conclusion]] This experiment studied the similarities and differences of microflora between healthy milk and mastitis milk in two dairy farms, and provided a theoretical basis for the prevention of mastitis in the two dairy farms and the quality control of raw milk during cold chain transportation.

    参考文献
    [1] Bhattarai D, Worku T, Dad R, Rehman ZU, Gong XL, Zhang SJ. Mechanism of pattern recognition receptors (PRRs) and host pathogen interplay in bovine mastitis. Microbial Pathogenesis, 2018, 120:64-70.
    [2] Miles AM, Huson HJ. Graduate Student Literature Review:Understanding the genetic mechanisms underlying mastitis. Journal of Dairy Science, 2021, 104(1):1183-1191.
    [3] El-Ashker M, Gwida M, Monecke S, Ehricht R, Elsayed M, El-Gohary F, Reißig A, Müller E, Paul A, Igbinosa EO, Beshiru A, Maurischat S. Microarray-based detection of resistance genes in coagulase-negative staphylococci isolated from cattle and buffalo with mastitis in Egypt. Tropical Animal Health and Production, 2020, 52(6):3855-3862.
    [4] Keller D, Sundrum A. Comparative effectiveness of individualised homeopathy and antibiotics in the treatment of bovine clinical mastitis:randomised controlled trial. The Veterinary Record, 2018, 182(14):407.
    [5] Azooz MF, El-Wakeel SA, Yousef HM. Financial and economic analyses of the impact of cattle mastitis on the profitability of Egyptian dairy farms. Veterinary World, 2020, 13(9):1750-1759.
    [6] Taponen S, Salmikivi L, Simojoki H, Koskinen MT, Pyörälä S. Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing. Journal of Dairy Science, 2009, 92(6):2610-2617.
    [7] Oikonomou G, Machado VS, Santisteban C, Schukken YH, Bicalho RC. Microbial diversity of bovine mastitic milk as described by pyrosequencing of metagenomic 16s rDNA. PLoS One, 2012, 7(10):e47671.
    [8] Kuehn JS, Gorden PJ, Munro D, Rong RC, Dong QF, Plummer PJ, Wang C, Phillips GJ. Bacterial community profiling of milk samples as a means to understand culture-negative bovine clinical mastitis. PLoS ONE, 2013, 8(4):e61959.
    [9] Marchand S, Heylen K, Messens W, Coudijzer K, De Vos P, Dewettinck K, Herman L, De Block J, Heyndrickx M. Seasonal influence on heat-resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples. Environmental Microbiology, 2009, 11(2):467-482.
    [10] Katholm J, Bennedsgaard TW, Koskinen MT, Rattenborg E. Quality of bulk tank milk samples from Danish dairy herds based on real-time polymerase chain reaction identification of mastitis pathogens. Journal of Dairy Science, 2012, 95(10):5702-5708.
    [11] Shome BR, Das Mitra S, Bhuvana M, Krithiga N, Velu D, Shome R, Isloor S, Barbuddhe SB, Rahman H. Multiplex PCR assay for species identification of bovine mastitis pathogens. Journal of Applied Microbiology, 2011, 111(6):1349-1356.
    [12] Kuang Y, Tani KR, Synnott AJ, Ohshima K, Higuchi H, Nagahata H, Tanji Y. Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR-DGGE method. Biochemical Engineering Journal, 2009, 45(1):76-81.
    [13] Kennedy R, Lappin DF, Dixon PM, Buijs MJ, Zaura E, Crielaard W, O'Donnell L, Bennett D, Brandt BW, Riggio MP. The microbiome associated with equine periodontitis and oral health. Veterinary Research, 2016, 47:49.
    [14] Oultram JWH, Ganda EK, Boulding SC, Bicalho RC, Oikonomou G. A metataxonomic approach could be considered for cattle clinical mastitis diagnostics. Frontiers in Veterinary Science, 2017, 4:36.
    [15] Cremonesi P, Ceccarani C, Curone G, Severgnini M, Pollera C, Bronzo V, Riva F, Addis MF, Filipe J, Amadori M, Trevisi E, Vigo D, Moroni P, Castiglioni B. Milk microbiome diversity and bacterial group prevalence in a comparison between healthy Holstein Friesian and Rendena cows. PLoS ONE, 2018, 13(10):e0205054.
    [16] Zeng XQ, Liu CJ, Yang X, Li XR. Microbial community structure and diversity of mastitis cows by 16S rRNA high-throughput sequencing. Acta Agriculturae Zhejiangensis, 2019, 31(9):1437-1445. (in Chinese) 曾学琴, 柳陈坚, 杨雪, 李晓然. 高通量测序法检测奶牛乳房炎关联微生物群落结构及多样性. 浙江农业学报, 2019, 31(9):1437-1445.
    [17] Levison LJ, Miller-Cushon EK, Tucker AL, Bergeron R, Leslie KE, Barkema HW, DeVries TJ. Incidence rate of pathogen-specific clinical mastitis on conventional and organic Canadian dairy farms. Journal of Dairy Science, 2016, 99(2):1341-1350.
    [18] Vakkamäki J, Taponen S, Heikkilä AM, Pyörälä S. Bacteriological etiology and treatment of mastitis in Finnish dairy herds. Acta Veterinaria Scandinavica, 2017, 59(1):33.
    [19] Massé J, Dufour S, Archambault M. Characterization of Klebsiella isolates obtained from clinical mastitis cases in dairy cattle. Journal of Dairy Science, 2020, 103(4):3392-3400.
    [20] Kerro Dego O, Oliver SP, Almeida RA. Host-pathogen gene expression profiles during infection of primary bovine mammary epithelial cells with Escherichia coli strains associated with acute or persistent bovine mastitis. Veterinary Microbiology, 2012, 155(2/3/4):291-297.
    [21] Zhang J, Jiang Y, Xia X, Wu J, Almeida R, Eda S, Qi HC. An on-site, highly specific immunosensor for Escherichia coli detection in field milk samples from mastitis-affected dairy cattle. Biosensors and Bioelectronics, 2020, 165:112366.
    [22] Vasquez AK, Ganda EK, Capel MB, Eicker S, Virkler PD, Bicalho RC, Nydam DV. The microbiome of Escherichia coli and culture-negative nonsevere clinical mastitis:Characterization and associations with linear score and milk production. Journal of Dairy Science, 2019, 102(1):578-594.
    [23] 韩天赐. 西藏曲才热泉的细菌多样性及栖热菌属比较基因组学. 云南大学硕士学位论文, 2018.
    [24] Li CF, Li Y, Wu XY, Cao YB, Wang WD, Bao MT. Study on the activation Geobacillus species in Zhan 3 blocks of Shengli Oilfield. Journal of China University of Petroleum: Edition of Natural Science, 2016, 40(1):163-167. (in Chinese) 李彩风, 李阳, 吴昕宇, 曹嫣镔, 汪卫东, 包木太. 胜利油田沾3区块油藏中Geobacillus菌的激活研究. 中国石油大学学报:自然科学版, 2016, 40(1):163-167.
    [25] Hoekstra J, Zomer AL, Rutten VPMG, Benedictus L, Stegeman A, Spaninks MP, Bennedsgaard TW, Bigg慳猠慁欬椠?奥???灩楥摧敨浥楲漠汓漬朠楍捡慴汥?猠瑄畈搬礠?瑵潢?楲渭癓散獨瑬楥杮慳瑴敥?瑴栠敒?椠湋捡楴摨敯湬捭攠?愬渠摋?灶爦攣瘲愲氵攻湣捳攠?漬映?捲氦楯湵業捬愻汭?浥慲猠瑖椬琠楌獥??灥敵牸愠捇甬琠敍?浲慯獮瑩椠瑐椬猠??浮敨瑯愠扌漬氠楓捭?摬楳獫潩爠摓攬爠獓?慰湲搦?瀲攳爳椻瀠態爬琠畓海?摮楫獥潬牳搠敊牍猬??潯湬?慥?搠慍楁爬礠?晡慭爠浔?楇湍?愠?瑯敯浰瀠敇爮愠瑇敥?穯潭湩散?楡湮??慹灳慩湳???椠?????噥敡瑮攠牢楯湶慩牮祥?刼敩猾敓慴牡捰桨??楯????ふ??????????????rom clinical versus subclinical mastitis. Scientific Reports, 2020, 10(1):18172.
    [26] Pérez VKC, Custódio DAC, Silva EMM, Oliveira J, Guimarães AS, Brito MAVP, Souza-Filho AF, Heinemann MB, Lage AP, Dorneles EMS. Virulence factors and antimicrobial resistance in Staphylococcus aureus isolated from bovine mastitis in Brazil. Brazilian Journal of Microbiology, 2020, 51(4):2111-2122.
    [27] Rodrigues MX, Lima SF, Higgins CH, Canniatti-Brazaca SG, Bicalho RC. The Lactococcus genus as a potential emerging mastitis pathogen group:a report on an outbreak investigation. Journal of Dairy Science, 2016, 99(12):9864-9874.
    [28] de Oliveira GB, Favarin L, Luchese RH, McIntosh D. Psychrotrophic bacteria in milk:How much do we really know? Brazilian Journal of Microbiology, 2015, 46(2):313-321.
    [29] Vithanage NR, Dissanayake M, Bolge G, Palombo EA, Yeager TR, Datta N. Biodiversity of culturable psychrotrophic microbiota in raw milk attributable to refrigeration conditions, seasonality and their spoilage potential. International Dairy Journal, 2016, 57:80-90.
    [30] Vithanage NR, Dissanayake M, Bolge G, Palombo EA, Yeager TR, Datta N. Microbiological quality of raw milk attributable to prolonged refrigeration conditions. The Journal of Dairy Research, 2017, 84(1):92-101.
    [31] 席晓敏. 乳房炎牛乳中微生物多样性及代谢组学研究. 呼和浩特:内蒙古农业大学硕士学位论文, 2016.
    [32] Santman-Berends IMGA, Swinkels JM, Lam TJGM, Keurentjes J, van Schaik G. Evaluation of udder health parameters and risk factors for clinical mastitis in Dutch dairy herds in the context of a restricted antimicrobial usage policy. Journal of Dairy Science, 2016, 99(4):2930-2939.
    [33] Fukushima Y, Kino E, Furutani A, Minamino T, Mikurino Y, Horii Y, Honkawa K, S
    引证文献
引用本文

吕倩,马莉,骆巧,罗雪,陈久兵,罗正中,雍康,姚学萍,余树民,沈留红,曹随忠. 基于高通量测序技术分析四川两个奶牛场乳汁的菌群差异[J]. 微生物学报, 2021, 61(10): 3087-3102

复制
分享
文章指标
  • 点击次数:374
  • 下载次数: 1258
  • HTML阅读次数: 1044
  • 引用次数: 0
历史
  • 收稿日期:2020-11-25
  • 最后修改日期:2021-03-11
  • 在线发布日期: 2021-09-29
文章二维码