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Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎支原体的断方法 Albert Rovira, DVM, MS, PhD 伯特·罗维拉博士 University of Minnesota 明尼达大学

Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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Page 1: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 2: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 3: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 4: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Mycoplasmas支原体Scientific classification科学

分类

Kingdom界: Bacteria 菌

Division门: Firmicutes 厚壁菌门

Class纲: Mollicutes 柔膜菌纲

Order目: Mycoplasmatales 支原体目

Family科: Mycoplasmataceae支原体科

Genus属: 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

Page 5: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Non pathogenic 非致病性M. Sualvi 猪肠枝原体

M. Hyopharyngis 舌咽枝原体

M. hyoarthrinosa??????

M. hyogenitalium 猪生殖道支原体

Page 6: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 7: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• 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

Clinical presentation 临床表现

Page 8: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 9: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 10: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Microscopic Lesions显微病变

Page 11: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality
Page 12: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Ultrastructural lesions超微结构病变

Page 13: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 14: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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?

Page 15: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Transmission chain of Mycoplasma hyopneumoniae猪肺炎支原体的传播链

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

Lateral spread横向传播

(transmission between farms农场

之间传输)

Page 16: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 17: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 18: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 19: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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)

Page 20: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 21: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 22: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 23: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 24: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 25: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 26: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 27: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Sow medication 母猪用药: Piglet colonization 仔猪定殖

对照组

治疗组

出生 断奶期

Page 28: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 29: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 30: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 31: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

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Page 33: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 34: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Disease investigation: RESULTS

• Histopathology: bronchopneumonia characterized by marked peribronchiolar lymphoid hyperplasia and alveoli filled with numerous macrophages and edema fluid

Page 35: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

Page 36: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

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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

Page 38: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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。

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Initial Scenario 初始情境

ADG平均日增重

(Kg)

0.582

FE料比

3.3

Culling 淘汰%

7.53

Clinical signs

临床症状

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12 months later 12个月后

ADG 平均日增重 (kg)

0.738

FE料比 2.82

Culling淘汰 %

4.65

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

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41

Slaughter evaluation 屠宰评价(Pneumonic Lesions 肺病变)

%

前 后

频率 肺病变评分

Page 42: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

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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

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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

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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 ELISAOxoid公司(Dako公司)猪肺炎支原体ELISA• Blocking ELISA 阻断ELISA• High specificity 特异性高

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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%

Page 47: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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?

Page 48: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

M. hyopneumoniae ELISA猪肺炎支原体ELISA

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

Positive sample阳性样品

Negative sample阴性样品

Page 49: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

M. hyopneumoniae ELISA猪肺炎支原体ELISA

Positive sample阳性样品

Negative sample阴性样品

Page 50: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

M. hyopneumoniae ELISA猪肺炎支原体ELISA

Positive sample阳性样品

Negative sample阴性样品

Page 51: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

M. hyopneumoniae ELISA猪肺炎支原体ELISA

Positive sample阳性样品

Negative sample阴性样品

Page 52: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

M. hyopneumoniae ELISA猪肺炎支原体ELISA

Positive sample阳性样品

Negative sample阴性样品

Page 53: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

M. hyopneumoniae ELISA猪肺炎支原体ELISA

Positive sample阳性样品

Negative sample阴性样品

Page 54: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

• Tools for surveillance (ELISAs) are available with good SE and SP• IDEXX M. hyopneumoniae ELISA

• Indirect ELISA间接ELISA

• Specificity = 98.6%• Oxoid (Dako) M. hyopneumoniae ELISA

• Blocking ELISA• High Specificity

M. hyopneumoniae ELISA猪肺炎支原体ELISA

Positive sample阳性样品

FALSE POSITIVE 假阳性!!!

Page 55: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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 ELISAOxoid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 expected-negative 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?

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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 = 99.99996 %

• 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%

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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

Page 58: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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 = 0.0196%• 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%

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One possible approach一个可能的方法

1. Indirect ELISA 间接ELISA2. Repeat indirect ELISA 重复间接ELISA + blocking ELISA阻断

ELISA3. 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

Page 60: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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?

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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

Page 62: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

M. hyopneumoniae infection猪肺炎支原体感染

Phases of Infection感染阶段

Acute 急性 Chronic 慢性

Clinical signs临床症状

0 ~ 70 254dpi

Experimental infection实验感染

Clearance清除

Pieters et al., 2009

Page 63: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

M. hyopneumoniae infection猪肺炎支原体感染

Acute 急性

0 10-16 ~ 70dpi

Experimental infection实验感染

Cough ceases咳嗽停止

• 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

Pieters et al., 2009

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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

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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 Intra-tracheal 10ml气管内

1x105 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

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Experimental design 实验设计

dpi -7 0 2 5 9 14 21 28

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

Page 67: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Nasal swabs鼻拭子

Sample collection 样品采集

Photo by Jon Ertl

Page 68: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Laryngeal swab喉拭子

Sample collection 样品采集

Page 69: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Sample collection 样品采集

Laryngeal swab 喉拭子

Page 70: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Sample collection 样品采集

Tracheo-bronchial lavage气管,支气管肺泡灌洗

Page 71: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

Sample collection 样品采集

Tracheo-bronchial lavage气管,支气管肺泡灌洗

Page 72: Diagnostic Approaches for Mycoplasma hyopneumoniae 猪肺炎 …€¦ · a field case案例 • Clinical Signs临床症状: acute onset of respiratory disease with increased mortality

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

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M. hyopneumoniae detection in experimentally inoculated pigs 实验接种猪猪肺炎支原体检测

* Real-time PCR接种后天数

阳性猪头数

鼻拭子喉拭子

气管,支气管肺泡灌洗

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M. hyopneumoniae detection in oral fluids猪肺炎支原体口腔液检测

dpi 0 2 5 9 14 21 28+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

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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

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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

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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

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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