Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎支原体的诊断方法 Albert Rovira, DVM, MS, PhD 阿尔伯特罗维拉博士 University of Minnesota 明尼苏达大学

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1 Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎支原体的诊断方法 Albert Rovira, DVM, MS, PhD 阿尔伯特罗维拉博士 University of Minnesota 明尼苏达大学

2 Acknowledgements 致谢 Dr. Maria Pieters 玛丽亚彼得斯博士 Dr. Eduardo Fano 爱德华多范诺博士

3 Presentation outline 演讲大纲 Background information on M. hyopneumoniae 猪肺炎支原体的背景资料 : Etiology, Clinical Signs, Lesions, Epidemiology, Diagnostics, Control 病因, 临床症状, 病变, 流行病学, 诊断, 控制 Diagnostics 诊断 : Disease Investigation 疾病调查 Monitoring M. hyopneumoniae in positive farms 阳性猪场监测猪肺炎支原体 Surveillance of M. hyopneumoniae in negative farms 阴性猪场监控猪肺炎支原体 M. hyopneumoniae diagnostics 猪肺炎支原体诊断 Early detection study 早期检测研究

4 Mycoplasmas 支原体 Scientific classification 科学分类 Kingdom 界 : Division 门 : Class 纲 : Order 目 : Family 科 : Genus 属 : Bacteria 菌 Firmicutes 厚壁菌门 Mollicutes 柔膜菌纲 Mycoplasmatales 支原体目 Mycoplasmataceae 支原体科 Mycoplasma 支原体 Lack of cell wall 缺乏细胞壁 Minimum genome 最小基因组 Smallest free-living organisms 最小自由生物体 Pathogens of animals, plants, insects 动物, 植物, 昆虫的病原体

5 Swine Mycoplasmas 猪支原体 Species 种类 Condition(s) 症状 M. hyopneumoniae 猪肺炎支原体 Enzootic pneumonia 地方性肺炎 M. hyorhinis 猪鼻支原体 Polyserositis, arthritis, otitis, among others 多浆膜炎, 关节炎, 耳炎, 等等 M. hyosynoviae 猪关节液支原体 Arthritis 关节炎 M. Suis 猪付红细胞体 Anemia 贫血 M. Flocculare 絮状支原体 M. Sualvi 猪肠枝原体 M. Hyopharyngis 舌咽枝原体 Non pathogenic 非致病性 M. hyoarthrinosa?????? M. hyogenitalium 猪生殖道支原体

6 Mycoplasma hyopneumoniae 猪肺炎支原体 Disease causing agent 致病菌 Species specific 物种特异性 Difficult to culture 难培养 Fastidious growth 难增长 : - Very slow 非常缓慢 - Requires an enriched media 需要丰富培养基

7 Clinical presentation 临床表现 Dry cough 干咳 : starts 14 dpi and stops 70 dpi 开始于 14 dpi, 停止于 70 dpi Decreased weight gain 增重下降 High morbidity 高发病率 Low mortality 低死亡率 Can affect pigs of all ages but more common in finishing pigs 可影响所有年龄段, 但更常见于育肥猪

8 Gross Lesions 肉眼病变 : cranioventral lung consolidation 靠腹部的肺实变

9 Microscopic Lesions 显微病变 Perobronchiolar lymphoid hyperplasia 支气管周围淋巴组织增生 Alveoli filled with macrophages 充满巨噬细胞的肺泡 Alveoli filled with edema fluid 充满水肿液的肺泡

10 Microscopic Lesions 显微病变

12 Ultrastructural lesions超微结构病变

13 Pathogenesis of M. hyopneumoniae 猪肺炎支原体的发病机制 M. hyopneumoniae attaches to the tips of cilia 猪肺炎支原体附纤毛尖端 M. hyopneumoniae destroys the cilia 猪肺炎支原体破坏纤毛 Facilitates lung invasion of secondary pathogens 促进肺二次病原体入侵 M. hyopneumoniae stimulates an exaggerated, ineffective immune response 猪肺炎支原体激活一次扩大的, 无效的免疫反应 M. hyopneumoniae evades the effective immune responses 猪肺炎支原体逃避有效的免疫反应

14 Epidemiology of M. hyopneumoniae 猪肺炎支原体流行病学 Piglets are born free of M. hyopneumoniae 仔猪出生时猪肺炎支原体阴性 Sow to piglet colonization is important 母猪仔猪定殖很重要 Colonized weaned pigs spread the microorganism 定殖断奶猪传播微生物 Prevalence at weaning is a predictor of disease severity 断奶期流行程度预测疾病严重性 Long persistence in infected pigs 在感染猪体内长期存在 Airborne transmission 空气传播 Fomite or vector transmission? 污染物或媒介传播?

15 Transmission chain of Mycoplasma hyopneumoniae 猪肺炎支原体的传播链 Lateral spread 横向传播 (transmission between farms 农场之间传输 ) Level 1 第 1 级 Replacement animals 后备 Level 2 第 2 级 Sow herd 母猪群 Level 3 第 3 级 Growing and finishing pigs 生长和肥育猪 Horizontal transmission 水平传播 Vertical transmission 垂直传播 Horizontal transmission 水平传播

16 Diagnostics for M. hyopneumoniae\ 猪肺炎支原体诊断 Clinical signs 临床症状 Non-specific 非特异性 Gross Lesions 肉眼病变 Non-specific 非特异性 Histopathology 组织病理学 Characteristic lesions 特征病变 Bacterial isolation 细菌分离 - very difficult! 非常困难! PCR: nasal swab, bronchial swab, oral fluids, BALF 支气管拭子, 鼻咽拭子, 口腔液体, 支气管肺泡灌洗液 ELISA 酶联免疫吸附

17 M. hyopneumoniae strains are not clonal 猪肺炎支原体菌株不是无性繁殖 Genetic heterogeneity 遗传异质性 : Protein profile 蛋白质表达谱 PCR Restriction enzymes digestion 限制性内切酶消化 MLST Sequencing of repeats 重复序列 (p146) Strain variability 菌株变异 : - Natural infection 自然感染 : - Clinical presentation 临床表现 - Experiments 实验 : - In vitro generation time 体外生成时间 - Transmission rate 传播速率 Meyns et al., 2007

18 More than one variant can circulate in a herd 不止一个变异在猪群流通 1 North American production system 一个北美生产系统 Two clonal complexes 两个克隆复合物 Several clonal variants 几个克隆变异体

19 Control of M. hyopneumoniae 猪肺炎支原体的控制 Prevention and control of M. hyopneumoniae can be achieved by integration of housing conditions, management practice, medication programs and vaccination 可通过整合的猪舍条件, 管理实践, 用药方案和疫苗接种实现猪肺炎支原体的预防和控制 (Maes et al., 1996; Thacker, 2006)

20 Vaccination 疫苗 Bacterins 菌苗 Intramuscular delivery 肌注 One or two doses 一剂或两剂 Effects 效应 : Improvement of average daily gain 提高平均日增重 Feed conversion ratio 饲料转化率 Days to slaughter weight 达屠宰体重天数 Reduced clinical signs and lung lesions 减少的临床症状和肺部病变 Lower treatment costs 较低的治疗费用 ~ Mortality 较低死亡率

21 Vaccination strategies 疫苗接种策略 Pig vaccination 猪疫苗接种 : Very early 非常早 Early 早 Late 晚 : Based on weaning age and disease presentation 基于断奶年龄和疾病表现 Induction of immunity prior to infection 感染前免疫诱导 Interference with maternal antibodies 干扰母源抗体 Meaning 意味着 One or two doses 一剂或两剂 : Similar benefits 类似的益处 Management 管理 Labor 人工 Vaccine compliance 疫苗合规

22 Vaccination strategies 疫苗接种策略 Sow vaccination 母猪疫苗接种 : Pre-farrow 分娩前 : Few studies 少有研究 Decreased shedding 减少排毒 Transfer of maternal immunity 传输母体免疫力 Lower number positive piglets at weaning 较低的断奶期阳性仔猪数量

23 Vaccination strategies 疫苗接种策略 Gilt vaccination 后备母猪疫苗接种 : Incoming gilts 新入后备母猪 Stable sow herds 稳定母猪群 : Protect incoming gilts 保护新入后备母猪 Protect sows 保护母猪 To maintain the stability of positive herds 维持阳性猪群稳定

24 Commonly used antimicrobial drugs in M. hyopneumoniae infections 猪肺炎支原体感染常用抗菌药物 Tetracycline 四环素 Clortetracycline 金霉素 Tiamulin 泰妙菌素 Tylosin 泰乐菌素 Valnemulin 沃尼妙林 Tilimicosin 替米考星 Tulathromycin 托拉 Lincomycin 林可霉素 Enrofloxacin 恩诺沙星 Marbofloxacin 麻保沙星 Danofloxacin 达氟沙星 Effect 效应 : Decreased bacterial load 减少细菌负荷 Decreased clinical signs 减少临床症状 Decreased lung lesions 减少肺部病变 Improved production parameters 改进的生产参数 Decreased mortality 死亡率下降

25 Medication strategies 用药策略 Preventive 预防 Therapeutic 治疗 Pig medication 猪用药 Gilt and sow medication 后备母猪和母猪用药

26 Sow medication 母猪用药 : Sow shedding 母猪排毒对照组治疗组产前分娩期断奶期

27 Sow medication 母猪用药 : Piglet colonization 仔猪定殖对照组治疗组出生断奶期

28 Different uses of diagnostics for Mycoplasma hyopneumoniae 猪肺炎支原体诊断的不同用途

29 Diagnostics for M. hyopneumoniae 猪肺炎支原体诊断 DISEASE INVESTIGATION 疾病调查 Clinical signs, Necropsy, Histopathology, lung PCR 临床症状, 剖检, 病理学, 肺 PCR MONITORING INFECTION IN POSITIVE FARMS 监测阳性农场的感染情况 ELISA 酶联免疫 nasal PCR?, oral fluid PCR? 鼻腔液 PCR? 口腔液 PCR? SURVEILLANCE IN NEGATIVE FARMS 监控阴性农场 ELISA 酶联免疫

30 Diagnostics for M. hyopneumoniae 猪肺炎支原体诊断 DISEASE INVESTIGATION 疾病调查 Clinical signs, Necropsy, Histopathology, lung PCR 临床症状, 尸体剖检, 病理学, 肺 PCR MONITORING INFECTION IN POSITIVE FARMS 监测阳性农场的感染情况 ELISA 酶联免疫吸附 nasal PCR?, oral fluid PCR? 鼻腔 PCR? 口腔液 PCR? SURVEILLANCE IN NEGATIVE FARMS 监控阴性农场 ELISA 酶联免疫吸附

31 Disease investigation 疾病调查 : a field case 案例 Clinical Signs 临床症状 : acute onset of respiratory disease with increased mortality. Suspect Influenza virus. 急性发作的呼吸系统疾病伴死亡率增加疑似流感病毒 Gross Lesions 肉眼病变 : Cranioventral lung consolidation, in some cases in a multifocal pattern 靠腹部肺变实, 有时为多点模式? Samples 样品 : Tissues from four pigs, 20 nasal swabs and 7 oral fluids samples 四头猪的组织,20 个鼻拭子和 7 个口腔液样本

33 Disease investigation 疾病调查 : RESULTS 结果 Histopathology 组织病理 : bronchopneumonia characterized by marked peribronchiolar lymphoid hyperplasia and alveoli filled with numerous macrophages and edema fluid 以细支气管周围淋巴组织增生和充满大量巨噬细胞和水肿液的肺泡为特征的支气管肺炎

34 Disease investigation: RESULTS Histopathology: bronchopneumonia characterized by marked peribronchiolar lymphoid hyperplasia and alveoli filled with numerous macrophages and edema fluid

35 Disease investigation 疾病调查 : RESULTS 结果 Histopathology 组织病理 : bronchopneumonia characterized by marked peribronchiolar lymphoid hyperplasia and alveoli filled with numerous macrophages and edema fluid 以细支气管周围淋巴组织增生和充满大量巨噬细胞和水肿液的肺泡为特征的支气管肺炎 Lung PCR 肺 PCR : POSITIVE for M. hyopneumoniae (4/4) and PCV2 (2/4) 猪肺炎支原体 (4/4) 和猪圆环病毒 (2/4) 阳性 Lung PCR 肺 PCR : negative for SIV and PRRSV 猪流感病毒和猪蓝耳病病毒阴性 Oral fluids PCR 唾液 PCR: 5/7 POSITIVES for M. hyopneumoniae 5/7 猪肺炎支原体阳性 Nasal swab PCR 鼻咽拭子 PCR: All 8 pools negative for M. hyopneumoniae 所有 8 组猪肺炎支原体阴性 All oral fluids and nasal swabs negative for SIV 所有唾液和鼻咽拭子猪流感病毒阴性

36 Disease investigation 疾病调查 : RESULTS 结果 Histopathology 组织病理 : bronchopneumonia characterized by marked peribronchiolar lymphoid hyperplasia and alveoli filled with numerous macrophages and edema fluid 以细支气管周围淋巴组织增生和充满大量巨噬细胞和水肿液的肺泡为特征的支气管肺炎 Lung PCR 肺炎 PCR : POSITIVE for M. hyopneumoniae (4/4) and PCV2 (2/4) ) 猪肺炎支原体 (4/4) 和猪圆环病毒 (2/4) 阳性 Lung PCR 肺炎 PCR : negative for SIV and PRRSV 猪流感病毒和猪蓝耳病病毒阴性 Oral fluids PCR 唾液 PCR : 5/7 POSITIVES for M. hyopneumoniae 5/7 猪肺炎支原体阳性 Nasal swab PCR 鼻咽拭子 PCR : All 8 pools negative for M. hyopneumoniae 所有 8 池猪肺炎支原体阴性 All oral fluids and nasal swabs negative for SIV 所有唾液和鼻咽拭子猪流感病毒阴性

37 Diagnostics for M. hyopneumoniae 猪肺炎支原体诊断 DISEASE INVESTIGATION 疾病调查 Clinical signs, Necropsy, Histopathology, lung PCR 临床症状, 剖检, 病理学, 肺 PCR MONITORING INFECTION IN POSITIVE FARMS 监测阳性农场的感染情况 ELISA 酶联免疫 nasal PCR?, oral fluid PCR? 鼻腔 PCR? 口腔液 PCR? SURVEILLANCE IN NEGATIVE FARMS 监控阴性农场 ELISA 酶联免疫吸附

38 Use of serology to monitor the efficiency of a medication program 使用血清学监测用药方案的效果 Herd 猪群 : 5,000 sows in a multi-site production system 多站点生产系统的 5,000 头母猪 Case description 案例介绍 : Increase of clinical manifestations associated to M. hyo in growing pigs (15 week old or older). Reduction in the ADG and increase in feed efficiency 生长猪 (15 周或以上 ) 肺炎支原体相关的临床表现增加平均日降低, 饲料转化效率增高 History 历史 : High proportion of young sows in the sow herd. Sow vaccination. No piglet vaccination. 母猪群内高比例的年小母猪母猪疫苗接种无仔猪疫苗接种 2 cross sectional studies (n=60 for each profile) with a 12 month interval, including pigs of 4, 8, 12, 15, 20 and 24 weeks of age. 两个以 12 个月为间隔的横断面研究 ( 样本量各为 60), 包括 4,8,12,15,20 和 24 周龄的生猪

39 Initial Scenario 初始情境 Clinical signs 临床症状 ADG 平均日增重 (Kg) FE 料比 Culling 淘汰 %

40 12 months later 12 个月后 Modification of the feed medication program, plus sow medication during lactation 饲料用药程序的修改, 再加上母猪哺乳期间用药 ADG 平均日增重 (kg) FE 料比 2.82 Culling 淘汰 % 4.65

41 Slaughter evaluation 屠宰评价 (Pneumonic Lesions 肺病变 ) % 前频率后肺病变评分 41

42 Diagnostics for M. hyopneumoniae 猪肺炎支原体诊断 DISEASE INVESTIGATION 疾病调查 Clinical signs, Necropsy, Histopathology, lung PCR 临床症状, 剖检, 病理学, 肺 PCR MONITORING INFECTION IN POSITIVE FARMS 监测阳性农场的感染情况 ELISA 酶联免疫吸附 nasal PCR?, oral fluid PCR? 鼻腔 PCR? 口腔液 PCR? SURVEILLANCE IN NEGATIVE FARMS 监控阴性农场 ELISA 酶联免疫

43 Mycoplasma hyopneumoniae in the US swine population 美国猪群中的猪肺炎支原体 The bad news 坏消息是 : 40% grow-finish sites reported M. hyopneumoniae disease 40% 生长 - 肥育猪场报道有猪肺炎支原体病 (NAHMS 2006) 81% large nursery sites vaccinated for M. hyopneumoniae (NAHMS 2006) 81% 的大型保育场接种猪肺炎支原体疫苗 The good news 好消息是 : M. hyopneumoniae eradication is feasible 根除猪肺炎支原体是可行的 Most genetic companies supply gilts negative for M. hyopneumoniae 大多数种猪公司提供肺炎支原体阴性后备

44 Towards a M. hyopneumoniae free population 得到无猪肺炎支原体猪群 Eradication 淸除 new negative populations 新阴性猪群 Risk of M. hyopneumoniae re-introduction 猪肺炎支原体重新引入风险 Incoming pigs 新入猪 Transport 运输 Aerosol 空气 Increasing importance of diagnostics for surveillance (vs. disease investigation) 监测诊断 ( 与疾病调查 ) 的重要性日益增加 Surveillance protocols based on detection of antibodies by ELISA 基于 ELISA 抗体检测的监控规划

45 M. hyopneumoniae ELISA 猪肺炎支原体 ELISA Tools for surveillance (ELISAs) are available with good SE and SP 监测工具 (ELISA 法 ) 具有良好的敏感性和特异性 IDEXX M. hyopneumoniae ELISA IDEXX 猪肺炎支原体 ELISA Indirect ELISA 间接 ELISA High specificity 特异性高 (98.6%) Oxoid (Dako) M. hyopneumoniae ELISA Oxoid 公司 (Dako 公司 ) 猪肺炎支原体 ELISA Blocking ELISA 阻断 ELISA High specificity 特异性高

46 M. hyopneumoniae ELISA specificity 猪肺炎支原体 ELISA 特异性 Assuming specificity 假设特异性 = 98.6% If we run 1 sample 如果我们做 1 个样本 : probability of false positive 假阳性的概率 = 1.4% If we run 30 samples 如果我们做 30 个样品 : probability of at least 1 false positive 至少 1 例假阳性的概率 = 34%

47 What are these false positives? 这些假阳性是什么? Laboratory error 实验室误差 Cross-reactions with antibodies against similar bacteria 对类似的细菌交叉抗体反应 Autoimmune diseases, allergies 自身免疫性疾病, 过敏 Contamination of antigen preparation in ELISA plates 抗原制备的酶标板污染 Acute infectious disease? Vaccination? 急性感染性疾病? 疫苗? Kidney/liver disease? 肾脏 / 肝脏疾病?

48 M. hyopneumoniae ELISA 猪肺炎支原体 ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品阴性样品 IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity Negative sample

49 M. hyopneumoniae ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品阴性样品 IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA 猪肺炎支原体 ELISA Negative sample Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity

50 M. hyopneumoniae ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品阴性样品 IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity 猪肺炎支原体 ELISA Negative sample

51 M. hyopneumoniae ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品阴性样品 IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity 猪肺炎支原体 ELISA Negative sample

52 M. hyopneumoniae ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品阴性样品 IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity 猪肺炎支原体 ELISA Negative sample

53 M. hyopneumoniae ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品阴性样品 IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity 猪肺炎支原体 ELISA Negative sample

hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.

54 M. hyopneumoniae ELISA Tools for surveillance (ELISAs) Positive are sample available with good SE and SP 阳性样品假阳性!!! IDEXX M. hyopneumoniae ELISA Indirect ELISA 间接 ELISA Specificity = 98.6% Oxoid (Dako) M. hyopneumoniae ELISA Blocking ELISA High Specificity 猪肺炎支原体 ELISA FALSE POSITIVE

55 M. hyopneumoniae ELISA 猪肺炎支原体 ELISA Tools for surveillance (ELISAs) are available with good SE and SP 监测工具 (ELISA 法 ) 具有良好的敏感性和特异性 IDEXX M. hyopneumoniae ELISA IDEXX 猪肺炎支原体 ELISA Oxoid (Dako) M. hyopneumoniae ELISA Oxoid 公司 (Dako 公司 ) 猪肺炎支原体 ELISA But we want excellent SE and SP! 但是我们需要出色的敏感性和特异性! What can we do when we get a positive from an expectednegative population??? 当我们从预期阴性的猪群得到了阳性结果时, 我们能做什么?

56 How can we deal with false positives? 我们如何处理假阳性? Confirmatory test (highly specific) 验证测试 ( 高度特异性 ): HIV testing by ELISA + Western Blot HIV 检测酶联免疫 + 蛋白印迹 by ELISA only 只用 ELISA 法 --- Specificity 特异性 = 98.5% by ELISA + WB 用 ELISA 法 + WB --- Specificity 特异性 = % PRRSV testing by ELISA + IFA 猪蓝耳病病毒用 ELISA 法检测 + IFA by ELISA only 只用 ELISA 法 --- Specificity 特异性 = 99% by ELISA + IFA 用 ELISA 法 + IFA- --- Specificity 特异性 = 99.9%

57 Confirmatory tests for M. hyopneumoniae 猪肺炎支原体的验证测试 Indirect ELISA 间接 ELISA + Blocking ELISA 阻断 ELISA Blocking ELISA 阻断 ELISA + Indirect ELISA 间接 ELISA ELISA + follow up ELISA 跟进酶联免疫 ELISA + nasal PCR ELISA+ 鼻 PCR ELISA + bronchial/lung PCR ELISA+ 支气管 / 肺 PCR

58 M. hyopneumoniae ELISA specificity 猪肺炎支原体 ELISA 特异性 Assuming specificity 假设特异性 = 98.6% If we run 1 sample 如果我们做 1 个样本 : probability of false positive 假阳性的概率 = 1.4% If we run 30 samples 如果我们做 30 个样品 : probability of at least 1 false positive 至少 1 例假阳性的概率 = 34% Using an alternative ELISA as a confirmatory test 使用替代 ELISA 作为确证试验 (testing in series 系列测试 ) If we run 1 sample 如果我们做 1 个样本 : probability of false positive 假阳性的概率 = % If we run 30 samples 如果我们做 30 个样品 : probability of at least 1 false positive 至少 1 例假阳性的概率 = 0.6%

59 1. Indirect ELISA 间接 ELISA One possible approach 一个可能的方法 2. Repeat indirect ELISA 重复间接 ELISA + blocking ELISA 阻断 ELISA 3. Euthanize positive pigs 阳性猪安乐死 Gross lesions 肉眼病变 Microscopic lesions 镜下病变 Bronchial/lung PCR 支气管 / 肺 PCR

60 Other approaches? 其他方法? What if we don t want to euthanize pigs? 如果我们不想给猪安乐死怎么办? What if we have time to re-bleed suspect and contact pigs? 如果我们有时间重新采集疑似和接触猪的血样呢? What if we are ready to take the risk? 如果我们准备好冒险了呢? What about other in-vivo samples (BALF, oral fluids)? 其它体内样本 (BALF, 口腔液 ) 如何? Should we look for alternatives to current diagnostic tests? 我们应该看看目前诊断试验的替代方案吗?

61 Early detection of Mycoplasma hyopneumoniae in live pigs 早期检测生猪肺炎支原体 : Comparison of current methods and development of new assays 目前方法的比较和新试验的开发

62 M. hyopneumoniae infection 猪肺炎支原体感染 Phases of Infection 感染阶段 Acute 急性 Chronic 慢性 dpi 0 ~ Clinical signs 临床症状 Experimental infection 实验感染 Clearance 清除 Pieters et al., 2009

63 M. hyopneumoniae infection 猪肺炎支原体感染 Acute 急性 dpi ~ 70 Experimental infection 实验感染 No clinical signs 无临床症状 No antibody production 无抗体产生 ~ Shedding 无排毒 Dry cough 干咳 Antibodies 抗体 Shedding 排毒 Lesions 病变 Cough ceases 咳嗽停止 Pieters et al., 2009

64 Objective 目标 To perform a side-by-side comparison of various sampling and testing tools currently available for detection of M. hyopneumoniae during the early stages of infection 做一次目前可用的在早期感染阶段检测猪肺炎支原体的不同采样和检测工具的并行比较

65 Materials and methods 材料与方法 Twenty three 8-week old M. hyopneumoniae naïve pigs 二十三只八周大的猪肺炎支原体阴性猪 : 2 control 2 只对照 mock inoculated pigs 模拟接种猪 21 experimentally inoculated pigs 21 只实验接种猪 : 10mL Intratracheal 10ml 气管内 1x10 5 CCU/mL Strain 232 菌株 232 Real time-pcr 即时 PCR (VetMax ABI- Strait el al., 2008) ELISA 酶联免疫 : Oxoid, Idexx & Biochek Statistical analysis 统计分析 : McNemar s paired Chi-square test McNemar s 配对卡方检验

66 Experimental design 实验设计 dpi Adaptation 适应 * * * * * ^ Experimental inoculation 人工接种试验 * - Nasal swab 鼻拭子 - Laryngeal swab 喉拭子 - Tracheo-bronchial lavage 气管, 支气管灌洗 - Serum 血清 - Oral fluids 唾液 (by room 室 ) ^ Bronchial swab 支气管拭子

67 Sample collection 样品采集 Nasal swabs 鼻拭子 Photo by Jon Ertl

68 Sample collection 样品采集 Laryngeal swab 喉拭子

69 Sample collection 样品采集 Laryngeal swab 喉拭子

70 Sample collection 样品采集 Tracheo-bronchial lavage 气管, 支气管肺泡灌洗

71 Sample collection 样品采集 Tracheo-bronchial lavage 气管, 支气管肺泡灌洗

72 Results 结果 Negative controls remained negative to M. hyopneumoniae throughout study for all sample types 所有样品类型, 猪肺炎支原体阴性对照仍为阴性 21/21 experimentally inoculated pigs were diagnosed as positive to M. hyopneumoniae by parallel interpretation any of the following samples 通过以下例子中的任何一个平行解释, 21/21 实验接种猪诊断为猪肺炎支原体阳性 : Nasal/laryngeal swab 鼻 / 喉拭子 TBL 气管, 支气管肺泡灌洗 Bronchial swab 支气管拭子 Lung lesions 肺部病变

73 M. hyopneumoniae detection in experimentally inoculated pigs 实验接种猪猪肺炎支原体检测鼻拭子喉拭子气管, 支气管肺泡灌洗阳性猪头数接种后天数 * Real-time PCR

74 M. hyopneumoniae detection in oral fluids 猪肺炎支原体口腔液检测 dpi 阳性 / Tested 检测的 (0/1) (0/3) (0/3) (2/3)* (0/3) (0/3) (2/3)^ Real-time PCR 即时定量 PCR * Ct value >37 Ct 值 >37 ^ 1 sample Ct value >39 单样品 Ct 值 >39

75 M. hyopneumoniae-specific antibody detection in serum 猪肺炎支原体特异性血清抗体检测阳性猪头数接种后天数

76 Conclusions 结论 Laryngeal swabs showed the highest sensitivity for early detection of M. hyopneumoniae compared to other sample types 相比其他样品类型, 喉拭子表现出早期检测肺炎支原体的最高灵敏度 Tracheo-bronchial lavages were more sensitive than nasal swabs during the first 21 dpi 气管, 支气管灌洗在接种后前 21 天比鼻拭子更敏感 Nasal swabs showed lower sensitivity during the first 3 weeks post-inoculation, compared to laryngeal swabs and tracheo-bronchial lavage 相比喉拭子和气管支气管灌洗, 鼻拭子在接种后前 3 周表现出较低敏感性 Oral fluids showed low sensitivity for M. hyopneumoniae detection during the early stages of infection 口腔液在感染的早期阶段猪肺炎支原体检测表现灵敏度低 M. hyopneumoniae-specific antibodies were similarly detected by the 3 commercial kits used in this study at 21 and 28 dpi 猪肺炎支原体特异性抗体同样地在接种后 21 和 28 天, 被 3 个本研究中使用的商业试剂盒检测到

77 Implications 启示 Detection of M. hyopneumoniae using laryngeal swabs appeared to be the best tool for In vivo diagnostics during the early stages of infection 使用喉拭子检测猪肺炎支原体为感染早期阶段体内诊断的最佳工具 Laryngeal swabs were faster to collect than tracheo-bronchial lavages, but required snaring the pig, using a mouth gag and a laryngoscope 喉拭子相比于气管支气管灌洗收集更快, 但需要用开口器和喉镜挖开 Oral fluids may not be the best sample type for monitoring M. hyopneumoniae negative populations 口腔液未必是监测猪肺炎支原体阴性群体的最好样本

78 Diagnostic approaches for Mycoplasma hyopneumoniae 猪肺炎支原体的诊断方法 Albert Rovira, DVM, MS, PhD 阿尔伯特罗维拉博士 University of Minnesota 明尼苏达大学

Mycoplasma hyopneumoniae and its role in the Porcine Respiratory Disease Complex 猪肺炎支原体及其在猪呼吸系统综合疾病中的作用 Dr. Maria Pieters 玛丽亚皮特斯博士

Mycoplasma hyopneumoniae and its role in the Porcine Respiratory Disease Complex 猪肺炎支原体及其在猪呼吸系统综合疾病中的作用 Dr. Maria Pieters 玛丽亚皮特斯博士 Outline 纲要 Porcine Respiratory Disease Complex (PRDC) 猪呼吸道疾病综合征 (PRDC)

Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎支原体的诊断方法 Albert Rovira, DVM, MS, PhD 阿尔伯特 罗维拉博士 University of Minnesota 明尼苏达大学

Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎支原体的诊断方法 Albert Rovira, DVM, MS, PhD 阿尔伯特罗维拉博士 University of Minnesota 明尼苏达大学