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1 China Dairy Conference 2015 University of Minnesota 2015 中国奶牛大会 - 明尼苏达大学 Determining the cause of mastitis 判定乳腺炎病因 Phil Durst 菲尔德斯特 Michigan State University Extension 美国密歇根州立大学 durstp@anr.msu.edu

2 If mastitis pathogens are the enemy... 如果乳房炎病原体是敌人 It is said that if you know your enemies and know yourself, you will not be imperiled in a hundred battles; Sun Tzu 孫子, The Art of War 知己知彼, 百战不殆孙子孙子谋攻

3 Know the enemy 了解敌人 Is it caused by a contagious organism? 它是由传染性微生物引起的吗? Is there a change in mastitis caused by environmental organisms? 由环境微生物引起的乳腺炎有什么变化吗? Is it an organism that was not previously identified in the herd? 它是一个以前未在牛群确定的微生物吗? Will it likely respond to treatment? 治疗有效吗? How should you refocus prevention in light of what is causing mastitis? 鉴于是什么原因造成乳腺炎, 你应该如何重新关注预防?

Is it an organism that was not previously identified in the herd? 它是一个以前未在牛群确定的微生物吗?

4 The problems with wrong assumptions 错误假设带来的问题 Misplaced blame 错位的指责 Misdirected effort 工作上的误导 Misspent monies 浪费金钱 Frustration 沮丧

5 Information is only valuable when it is used 信息只有被使用时才有价值 Determining the type of organisms causing mastitis enables us to: 确定引起乳腺炎的微生物类型使我们能够 : Improve the potential that treatment will be effective 提高有效治疗的潜力 Reduces the amount of antibiotics used by not treating where the likelihood of response is low 对于治疗效果差的治疗, 降低抗生素使用量 Directs our prevention efforts to what is most important 指导我们什么是预防工作最重要的

effective 提高有效治疗的潜力 Reduces the amount of antibiotics used by not treating where the likelihood of response

6 How we determine the organism 我们怎样确定微生物 There are various methods, including: 有很多方式, 包括 : Milk culture 牛奶培养 PCR PCR 技术 And they can be done various places: 可以在不同地方操作 Commercial laboratory 商业实验室 Community 社区 On-farm 农场

培养 PCR PCR 技术 And they can be done various places: 可以在不

7 What cows to sample for culture 从哪头牛身上采集来培养 Mild and moderate cases of clinical mastitis (using culture results for treatment decisions will delay treatment for 24 hrs. That should not be a problem) 轻, 中度的临床乳腺炎情况下 ( 根据培养结果决定治疗方案会拖延 24 小时, 这不应该是个问题 ) Fresh cows that had mastitis at dry-off 在干奶时有乳腺炎的新奶牛 New herd additions 新增的牧群 Sick cows - may need immediate care with supportive therapy (NSAID and fluids) 病奶牛 - 可能需要立即治疗与支持疗法 (NSAID 和液体 )

That should not be a problem) 轻, 中度的临床乳腺炎情况下 ( 根据培养结果决定治疗方案会拖延 24 小时, 这不应该是个问题 ) Fresh cows

8 Sampling technique 采样技术 Sample individual quarters (not composites) 单个乳房采样 ( 而不混合 ) Aseptic sampling - to capture only milk, uncontaminated by any other bacteria, in a sterile vial. 无菌采样 - 仅采取未受到任何其他细菌的污染的牛奶, 在无菌瓶中 Have a designated person(s) do all the sampling. 指定专人完成所有的采样 Either plate or refrigerate/freeze sample immediately 立即铺盘或冷藏 / 冷冻样品

无菌采样 - 仅采取未受到任何其他细菌的污染的牛奶, 在无菌瓶中 Have a designated person(s) do all the

9 While diagnostic lab culture may be the gold standard, much can be learned from simple procedures that can be carried out on the farm. 虽然诊断实验室的培养可能是黄金标准, 农场进行的简单方法也可以学到很多知识

10 Setting up the farm lab 建立农场的实验室 Dedicated area 专用区域 Not a high traffic area 不是一个高流动区域 Work area that can be kept clean and free of clutter 保持清洁和无杂乱的工作区 Work surface that can be disinfected 工作台面是可消毒的 All supplies readily available 所有的用品一应俱全 Not subject to temperature swings 不受温度变化影响

和无杂乱的工作区 Work surface that can be disinfected 工作台面是可消毒的 All supplies

11 On-farm culture lab 农场培养实验室 Simple 简单 Clean 干净 Everything handy 一切都方便 Dedicated 专用

13 On-farm milk culture 农场牛奶培养 Results available generally < 24 hrs. <24 小时可得到结果 Low cost 低成本 High probability of identifying the most common mastitiscausing organisms 确定最常见导致乳腺炎微生物概率高 Takes a committed person 需要一个敬业的人 Training in culture technique and interpretation 培养技术和结果解读的培训 Minnesota Easy Culture System or use of various tests 明尼苏达简易培养系统或使用各种测试

organisms 确定最常见导致乳腺炎微生物概率高 Takes a committed person 需要一个敬业的人 Training in culture

14 Minnesota Easy Culture System 明尼苏达简易培养系统 3 types of growth media: 3 种类型的培养基 : Factor media selects for Gram-positive bacteria such as Staphylococci, Streptococci, Bacillus, and Corynebacteria 选择因子培养基用于革兰氏阳性菌如葡萄球菌, 链球菌, 芽孢杆菌和棒状杆菌 MacConkey media selects for Gram-negative organisms, typically Coliforms such as E. coli and Klebsiella 麦氏培养基用于革兰氏阴性菌, 通常大肠菌群如大肠杆菌和克雷伯菌 Modified TKT media selects for Streptococcus and Streptococcuslike bacteria such as Enterococcus and Aerococcus species. 改良 TKT 培养基用于链球菌和链球菌状菌如肠球菌和 Aerococcus 菌种

Gram-negative organisms, typically Coliforms such as E.

15 三板块 3 rd 涂改良 TKT 培养基 2 nd 涂麦氏培养 1 st 涂因子培养基

16 Alternatively: 其他选择 : Simple Gram-positive vs. Gram-negative and maybe... 简单革兰氏阳性 VS 革兰阴性, 也许 Catalase test for colonies that grow on blood agar to distinguish Streps. from Staph. 生长在血琼脂菌群的过氧化氢酶试验来区分金黄色葡萄球菌和链球菌 CAMP test to diagnose Strep. ag. CAMP 测试来诊断链球菌 Coagulase test to diagnose Staph. aureus vs. CNS 凝固酶试验诊断金黄色葡萄球菌和 CNS

17 Culture technique 培养技术 Prepare the area first 首先准备区域 Incubator should be at target temperature 培养箱设目标温度 Handle swabs aseptically 保证无菌棉签 Avoid getting clumps on the plate 避免盘子上有凝块 Re-dip the swab for each section of the plate 对盘子的各部分再次浸湿棉签 Swab plate in order: Factor, MacConkey, MTKT 依次涂盘 : 因子, 麦氏, 和 MTKT 培养基 Turn plate upside down in incubator 将板块倒置在培养箱 Freeze the remaining milk sample 冷冻剩下的牛奶样本

each section of the plate 对盘子的各部分再次浸湿棉签 Swab plate in order: Factor, MacConkey, MTKT 依次涂盘 : 因子, 麦

18 Reading the plates 读盘 First determine if the sample was contaminated 先确定样本是否污染 Note which media has growth 注意哪些培养基有菌生长 Look for low or high number of colonies (CFU) 找出菌落数量 (CFU) 的多少 Look for zone of hemolysis on Factor and MTKT sections 在因子和 MTKT 部分找溶血区

for low or high number of colonies (CFU) 找出菌落数量 (CFU) 的多少 Look

19 Information to make decisions: 用来做决定的信息 : If you can determine whether it grows anything, you can make decisions. 如果你能确定它是否长出了什么, 你就可以做决定 If you can determine whether it is a Coliform vs. Strep., you can make decisions. 如果你能确定它是大肠杆菌还是链球菌, 就可以做出决定 If you can determine whether it is Strep. vs. Staph. you can make decisions. 如果你能确定它是链球菌还是金黄色葡萄球菌你可以做出决定

如果你能确定它是否长出了什么, 你就可以做决定 If you can determine whether it is a Coliform vs. Strep.

20 Treatment decisions: 治疗决定 Develop treatment protocols ahead of time based on culture results AND history & status: 根据培养结果和历史与现状, 提前制定治疗方案 : Gram positive or negative 革兰氏阳性或阴性 type of organism 微生物类型 severity of sickness 疾病的严重程度 colony count 菌落计数 previous treatment 早先治疗 lactation number, stage of lactation 哺乳次数, 哺乳期

negative 革兰氏阳性或阴性 type of organism 微生物类型 severity of sickness 疾病的严重程度

21 What does no growth mean? 没有菌生长是什么意思? No growth is diagnosed at 48 hours of incubation. Up to 40% of samples, depending on the farm, may be no growth. 经 48 小时培养没有生长高达 40% 的样本可能是没有菌生长, 这取决于农场 Quarter was not infected 乳房未感染 Pathogen was killed in the udder 病原体在乳房里被杀死 Contamination of the sample by disinfectant 消毒剂感染了样品 Conditions for incubation did not allow bacteria to grow 细菌在培养箱条件无法生长

22 No growth = No treatment 无生长 = 无治疗 No intramammary antibiotic, but if cow is systemically sick, provide supportive care and maybe systemic antibiotic. 无乳房内抗生素, 但如果牛是全身病, 提供支持治疗, 也许全身抗生素

23 Contaminated samples or plates 污染的样品或培养皿 Growth of more than one type of bacteria 多于一种类型的细菌生长 Recheck sampling technique 重新检查采样技术 Check sample handling 检查样品处理 Recheck the work area 重新检查工作区 Check supplies (don t re-use no-growth plates) 检查用品 ( 不要重复使用无增长的培养皿 ) Recheck culture technique 重新检查培育技术 Target: < 5% contaminated samples 目标 : 污染的样品 <5%

24 Biosecurity 生物安全 You are growing pathogens; protect person doing cultures, protect the herd, protect others 你在培养病原体 ; 保护操作人员, 保护牛群, 保护他人 Wear gloves 戴上手套 No food or drink in lab room 实验室不允许食物或饮料 Kill the plate 销毁培养皿 Dispose of properly 妥善处置废物

25 Essential Records 基本记录 Label the vial before drawing the sample (Cow ID, Date, DIM, Quarter) 取样前标记样品瓶 ( 牛编号, 日期,DIM, 乳房区 ) Label the bottom of the plate before swabbing (Cow ID, Date, DIM, Quarter) 涂拭前标注盘底部 ( 牛编号, 日期,DIM, 乳房区 ) Enter info into lab record book when you plate the sample 当你在处理样本时, 将信息输入实验室记录本, 当你在处理样本时 Record results by 48 hour reading, and action 48 小时查看记录结果, 采取行动 Enter into computer record system 输入电脑记录系统 Weekly or monthly summary 每周或每月总结

26 Date and time 日期时间 Cow and quarter 牛和乳房 Extent of bacterial growth on media 在培养基上细菌生长的程度 Diagnosis of bacteria type 细菌类型的诊断

27 Use the information 使用信息 Look for trends or changes in patterns 寻找趋势或变化 Monitor treatment effectiveness 监测治疗效果 Measure change in drug use? 在使用药物时测量变化? Note frequency of infection in the same cow and quarter 注意相同奶牛和乳房感染频率 Investigate potential reasons for an outbreak 调查爆发的潜在原因

28 Check yourself 检查自己 More than one person to do the culture (maybe work with someone from another farm) 不止一个人做培养 ( 也许与另一个农场的人合作 ) Routine checks with veterinarian 与兽医例行检查 Send occasional samples to diagnostic lab 间或发送样品到诊断实验室 Resource websites: 资源网站 University of Minnesota Dairy Extension Milk Quality 明尼苏达大学奶牛推广 - 牛奶质量 National Mastitis Council 国家乳腺炎理事会 Quality Milk Alliance 优质牛奶联盟 University of Wisconsin Milk Quality 威斯康星大学 - 牛奶质量

29 Know your enemy, and use that knowledge to control mastitis 了解你的敌人用这些知识来控制乳腺炎 The value of knowing the cause of mastitis on your farm is worth the time, expense and delay 花时间和费用, 甚至延迟工作来了解乳腺炎的在你的农场发生的原因是值得的 It enables you to be proactive 它让您变得主动 It enables you to target treatment 它让你进行针对性治疗 It enables you to improve. 它使你能进行改善

30 Thank you! 谢谢! Phil Durst Michigan State University Extension

China Dairy Conference 2015 University of Minnesota 2015 中国奶牛大会 - 明尼苏达大学 Determining the cause of mastitis 判定乳腺炎病因 Phil Durst 菲尔德斯特 Michigan State Un

China Dairy Conference 2015 University of Minnesota 2015 中国奶牛大会 - 明尼苏达大学 Determining the cause of mastitis 判定乳腺炎病因 Phil Durst 菲尔德斯特 Michigan State University Extension 美国密歇根州立大学 durstp@anr.msu.edu If