及时精准的实验室诊断监测助力大型母猪场的 伪狂犬病的控制与净化

秦立廷 博士 Dr. Qin Liting

新希望六和股份有限公司 New Hope Liuhe Co.,Ltd

Precisely and timely lab detection contribute to controlling and eradicating Pseudorabies

伪狂犬病(Pseudorabies)

“伪狂犬病” 是由伪狂犬病毒（Pseudorabies virus, PRV）引起一种以神经症状、奇痒、发热为主要症状的传染性疾病。 Pseudorabies (PR) ，caused by pseudorabies virus (PRV)， is a contagious disease characterized by neurological symptoms, itching and fever in piglets. 不同年龄段的猪都可以感染，主要引起仔猪神经症状、死亡，妊娠母猪流产、死胎、木乃伊胎，育肥及成年猪呼吸道症状。 Different age pigs can infected this disease. Infections with PRV result in piglet nervous disorders and death , pregnant sow abortion ，fattening pigs respiratory distress .

1813年，临床上第一次描述PRV是在牛上； 1849年，瑞士人使用伪狂犬对该病毒进行了命名； 19世纪上半叶，美国人用“疯痒”一词来形容该病毒； 1902年，匈牙利内科医生Aladar Aujeszky报道了在患病的公牛、犬和猫上分离到感染源，并证明这种病原

与狂犬病毒不同； 1931年，美国人Richard Shope确定该病原存在于家庭饲养的猪体内； 1933年，德国人Erich Traub第一次在体外移植器官中进行了PRV的培养； 1934年，将PRV归属到疱疹病毒组中； 1995年，ICTV列入疱疹病毒科α疱疹病毒亚科，水痘病毒属。

伪狂犬病（毒）(Pseudorabies Virus)

The clinical PRV was first described in cattle in 1813; In 1849, the rabies-like clinical picture in cattle prompted the use of the term “pseudorabies” in Switzerland; In the first half of the 19th century, “mad itch” was used to describe the disease in cattle in the United States; In 1902, the Hungarian physician Aladár Aujeszky reported the isolation of the infectious agent from a diseased

ox, a dog, and a cat, and differentiated it from Rabies. Richard Shope established that the agent of “mad itch” was also present in domestic pig holdings in the United

States in 1931; In 1933, Erich Traub in Germany was the first to cultivate PRV in vitro in organ explants. In 1934, PRVwas subdivided into the herpesvirus group. In 1995, According to the International Committee on Taxonomy of Viruses ，PRV was subdivided into the

genus Varicellovirus in the subfamily Alphaherpesvirinae of the family Herpesviridae.

伪狂犬病毒（PRV）

伪狂犬病病毒（PRV）：属于疱疹病毒科，α疱疹病毒亚科，水痘病毒属（Davision, 2010） PRV belongs to the genus Varicellovirus in the subfamily Alphaherpesvirinae of the family Herpesviridae (Davison 2010).

成熟的病毒粒子呈球形，直径约200-250nm;

The mature virions are spherical, PRV ranges in diameter from 200

to 250nm.

从内到外有线性双链DNA基因组、核衣壳、内膜和囊膜组

成；

The virions consists of the linear double-stranded DNA genome,

nucleocapsid, endometrial and capsule composition.

核衣壳呈二十面体对称，囊膜上有11个病毒糖蛋白；

There are 11 glycoprotein on the virus particle capsule and the

nuclear capsid of the virus is icosahedral symmetrical.

病毒持续存在，终身潜伏。

The virus exsit and latent infect the host all the time. （Pomeranz et al, 2005）

伪狂病毒的特性（Characteristic of PRV） 1、病毒持续存在，终身潜伏。在临床症状消失后病毒基因组可以持续的存在，在应激等作用下，

病毒可以活化，形成感染性病毒粒子，进行传播或者致病；

The virus exsit and latent infect the host all the time. After the clinical symptoms disappeared, the virus still exists and can be activated under stress.

2、猪是自然宿主，可以感染除了高等灵长类动物之外所有的哺乳动物，但是只有在猪体内可以进

行有效的感染；

Whereas PRV exhibits a wide host range capable of infecting basically all mammals except higher primates, only pigs are able to survive a productive infection and are thus considered the natural host.

3、传播途径主要是猪与猪的直接接触，或者接触PRV污染的物品，如饲料、水、动物尸体；也可

以垂直传播和精液传播；

The virus is spread primarily by direct contact between swine or by contact with PRV-contaminated fomites, for example, contaminated water, feed, animal carcasses. The virus also can be transmitted vertically and spread through semen.

4、鼻腔黏膜和口腔是主要的入侵部位。

The mucosae of the nasal and oral cavities are the main entry points.

伪狂病毒流行情况（Prevalence status of PRV）

• 目前，主要流行于欧洲东部和东南部、拉丁美洲、非洲和亚洲；

The disease is prevalent in southeast and eastern Europe, Latin America, Africa and Asia; • 欧洲大部分国家，如德国、荷兰、丹麦、瑞典、匈牙利、英国等，以

及加拿大、美国、新西兰等国家和地区已在饲养环境的猪群中消灭了伪狂犬病；

The disease in raising pigs has been eradicated in most European countries, such as Germany, the Netherlands, Denmark, Sweden, Hungary, Britain, etc., and Canada, the United States, New Zealand and other countries. • 野猪种群广泛带毒。

PRV exists widely in wild boar populations.

伪狂病的危害 The harm of PR 猪是PRV唯一的自然宿主，猪的年龄和免疫状态以及感染途径和毒株的毒力决定其发病率、死亡率和临床症状。 The incidence, mortality and clinical symptoms of PRV are determined by the virulence of strains, infection approach and the age and immune status of the pigs. And the pig is the only natural host.

年龄(age) 症状(symptom) 死亡率(mortality)

＜7 days 无明显的临床症状，突然性死亡 Suddenly death and no obvious clinical symptoms 100%

2-3 weeks

严重的中枢神经系统症状，如颤栗、共济失调、惊厥、震颤、运动失调、无力

Severe neurological symptoms, such as thriller, ataxia, convulsions, tremor,dyskinesia, weak

100%

4-6weeks 神经症状neurological symptoms 50%

育肥及成年Growing and fattening pigs

呼吸道症状，如咳嗽、打喷嚏、呼吸困难Respiratory symptoms such as cough, sneeze, difficulty breathing ＜5%

妊娠母猪（妊娠阶段）pregnant sows

胚胎死亡、吸收胎、木乃伊胎、流产或死胎Embryonic death, absorb the tyres, mummy foetus, miscarriage or stillbirth ——

与其他病毒性（如PRRSV、PCV2、SIV）混合感染的情况下，断奶后仔猪可形成严重的增生性坏死性肺炎（Morandi等，2010）

The PRV can infect postweaning pigs forming severe hyperplastic necrotizing pneumonia under the circumstances of mixed infections with other viruses (such as PRRSV and PCV2 and sivs) (Morandi, etc., 2010) .

伪狂病的危害与损失(The harm and loss)

引自Jeffrey J. Zimmerman J

伪狂犬阳性场与阴性场免疫程序差别 The difference of immunization program difference positive farm and negative farm

猪群 Swine 阴性场Positive farm 阳性场Negative farm

公猪 Boar 3 times /year 4 times /year

母猪 Sow 3 times /year 4 times /year

初生24h仔猪 Neonatal piglets none 滴鼻免疫1头份Intranasal

immunization ,1 dose

以2000头规模的猪场为例， 疫苗费用：母猪为2000*2=0.4万元，仔猪为（11.5*2000*2.3）*2=10.58万元，阳性场需多投入10.98万元； 生产损失：按照增加2%的流产率，PSY=24计算，每年减少断奶猪2000*24*2%=960头，每头盈利500，减少盈利48万元。 For example(2000 pigs): Vaccine costs: sow 2000*2=¥4000 ， Neonatal piglets（11.5*2000*2.3）*2=¥105,800 ,The positive farm need to invest ¥ 109,800 more than negative farm; Production loss: if the abortion rate increase 2%,PSY=24, the weaning pigs will reduce 960 per head each year, 500 profit per head, finally, we will reduce profit ¥.480,000

伪狂病的危害与损失(The harm and loss)

防控措施-净化 Prevention and Control Measures – Eradication

• 严重危害养猪业(Serious harm to pig industry)； • 病毒持续存在，终身潜伏(Virus persistent exist and latent)； 潜伏期的PRV具有潜在的活化和排出感染性病毒的危险，潜在带毒的动物是疾病控制的主要威胁。 The latent PRV has dangers of potential activation and discharge of infectious virus. The potentially carriers are a major threat to the control of the disease. • 成功的净化经验(Successful eradication experience)； 欧洲大部分国家，如德国、荷兰、丹麦、瑞典、匈牙利、英国等，以及加拿大、美国、新西兰等国家和地区已在饲养环境的猪群中消灭了伪狂犬病； The disease in raising pigs has been eradicated in most European countries, such as Germany, the Netherlands, Denmark, Sweden, Hungary, Britain, etc., and Canada, the United States, New Zealand and other countries.

净化是伪狂犬防控最有效的措施 PRV Eradication is the most effective measure

for prevention and control of PRV

PRV净化工作的基础条件 Basic Conditions of PRV Eradication

核心技术Core Technology：

gE基因缺失疫苗

gE gene-deleted vaccine

鉴别诊断ELISA方法

Differential diagnosis ELISA

成功案例 （Successful Cases）

美国伪狂犬病根除计划 PRV Eradication Program of USA 第一阶段：1989-1998年，建立、实施根除伪狂犬病项目。 The first stage: establishment and implementation of PRV Eradication Program, 1989-1998; 第二阶段：1999-2001年，加速根除伪狂犬病项目。 The second stage: acceleration of PRV Eradication Program, 1999-2001;

荷兰伪狂犬病根除计划 PRV Eradication Program of Netherlands 第一阶段、猪场净化措施，强制免疫； The first stage: eradication measure, and compulsory immunization; 第二阶段、区域免疫、净化，严格限制猪只流动 The second stage: regional eradication and immunization, strict restriction in the flow of pigs; 第三阶段、全国性净化措施 The third stage: national eradication measure; 第四阶段、控制阶段，强制免疫。 The fourth stage: the control stage, compulsory immunization; 第五阶段、强制淘汰，所有被感染的繁殖母猪一经检出即予淘汰。 The fifth stage: compulsory elimination, all of the infectious reproductive sow should be eliminated when diagnosed; 第六阶段、监测阶段，即无伪狂犬病阶段，将停止免疫程序。 The sixth stage: monitoring stage, known as PRV-free stage， Stop immune program.

成功案例 （Successful Cases）

国内某公司 One Chinese company

前提：使用gＥ缺失疫苗；猪群伪狂犬阳性率调查：

Precondition: immunization with gE gene-deleted vaccine;The survey of PRV gE Ab

positive rate

1、阳性率大于１０％的猪场，采取不断引入阴性后各群，逐步淘汰阳性群的方法。

In pig farm where PRV positive rate is higher than 10%, people continuously brought the

PRV-gE Ab negative groups in, and gradually knock out the PRV-gE Ab positive groups.

２、阳性率低于１０％的猪场，检测野毒抗体，找出阳性猪只进行隔离或淘汰。

In pig farm where PRV positive rate is lower than 10%, people detected the PRV gE antibody, and found

out the positive pigs to isolation or elimination.

成功案例 （Successful Cases）

中国伪狂犬控制情况 The conditions of PRV control in China

Before 2010 20 世纪70 年代从匈牙利引进了伪狂犬疫苗Bartha-k61 株，90 年代以来国内规模化猪场普遍使用该基因缺失疫苗，使猪伪狂犬病得到了很好的控制。

In 1970s, the PRV Bartha-k61 vaccine was introduced from Hungary. Since 1990s, PR had been well controlled in domestic large-scale pig farms due to the widely used of Bartha-k61 vaccines. 2011 to now 一种由伪狂犬病毒变异毒株引起的猪伪狂犬病在我国暴发，造成母猪繁殖障碍和初生乳猪死亡，给我国养猪业造成了巨大的经济损失

The outbreak of PR, leading to Sow reproduction disorder and death of newborn piglets ，caused by a new variant strain , has resulted in severe economic losses to the pig industry in our country

主要表现—大面积流产 Main Performance – a Large Area of Abortion

主要表现—gE抗体阳性率 Main Performance – Positive Rate of gE Ab

来自《2015年华中农业大学动物疫病诊断中心血清学分析》 From 《 serological analysis from animal disease diagnostic center ，

Huazhong agricultural university in 2015 》

传播速度快、流行范围广 Spread rapidly and prevalence widely

PRV在中国区域分布情况 Geographic distribution of PRV infections in China

A：1980前经典毒株流行情况；B：2011年PRV新毒株流行情况。 A:Classical PRV strains infections before 1980 B:PRV variants infections since 2011

（Sun et al 2016）

1、疫苗问题？Vaccines ？ 2、毒株毒力增强？Enhanced virulence of strain ……

疫苗情况分析（ The analysis of vaccines）

产地 Origin

编号 Serial

number

检测结果 Test Results （TCID50/头份 ）（TCID50/dose）

厂家标准（TCID50/头份 ） Manufacturer

s’standard（TCID50/dos

e）

兽药典标准（TCID50/头份）

Veterinary Pharmacopoeia

standard （TCID50/seam

heading）

结果判定 Results

进口 Import

1 106.0 106.3CCID50

≥5 X 103.0

合格qualified

2 106.33 ≥105.5 合格qualified 3 105.76 ≥105.2 合格qualified

4.1 105.5 ≥105.5 合格qualified 4.2 105.66

国产 Domestic

5 105.5 ≥104.5 合格qualified 6 105.5 ≥5 X 103.0 合格qualified 7 106.0 ≥5 X 103.0 合格qualified 8 105.66 ≥5 X 103.0 合格qualified 9 105.76 ≥5 X 103.0 合格qualified 10 104.66 ≥5 X 103.0 合格qualified 11 105.33 ≥5 X 103.0 合格qualified 12 105.61 ≥106.0 合格qualified

检测13种伪狂犬疫苗，病毒含量均符合兽药典标准，2种疫苗不符合厂家标准。

All of the virus concentration conforms to the standard of Veterinary Pharmacopoeia, 2 kinds of vaccines do not conforms the standard of manufacturer， based on the detection of 13 kinds of PRV vaccine 12种疫苗为Bartha-K61毒株。3种疫苗病毒含量较高，TCID50在106.0-

106.33；8种略低，TCID50在105.33-105.76；1种最低， TCID50为104.66 。

12 kinds of vaccines （Bartha - K61 strain）， three kinds of vaccine virus content is higher， TCID50 from 106.0 to 106.33；eight kinds of vaccine virus content is lower， TCID50 from 105.33 to 105.76； One kind of vaccine virus content is the lowest， TCID50 is 104.66 。

疫苗情况分析（ The analysis of vaccines）

“病毒变异”、“毒力增强” “virus variation”“virulence enhancement”

PRV毒株毒力增强 Enhanced virulence of

PRV strain

常规免疫方案（3-4次活疫苗/年）??? Routine immunization schedule(live

vaccine，3-4 times/year)

疫苗不能完全保护 Vaccine could not provide complete

protection

新毒株疫苗研发 New strain vaccine development

• 上海兽医研究所 Shanghai Veterinary Research Institute • 哈尔滨兽医研究所 Harbin Veterinary Research Institute • 华中农业大学 Huazhong Agricultural University • 南京农业大学 Nanjing Agricultural University • 国家兽用药品工程技术研究中心

National Research Center For Veterinary Medicine • ……

• 申报周期3-5年？

Register period 3-5 years?

新希望六和PR防控成绩 Achievements on PR prevention and control in New Hope Liuhe

22

24

26.3

19

20

21

22

23

24

25

26

27

2014年 2015年 2016年

1、生产指标PSY数据 Production index -PSY

新希望六和PR防控成绩 Achievements on PR prevention and control in New Hope

0%10%20%30%40%50%60%70%80%90%

100%

0胎 1胎 2胎 3胎 4胎 5胎 6胎 7胎

2014年 2015年 2016年

2、PRV 阳性场母猪群gE阳性率 PRV gE Ab positive rate of positive sows group

PRV阴性场—维持 Maintain of the PRV negative farms

PRV阴性猪场 PRV negative farms

严密监控 Closely monitor 病原监测Pathogen monitoring gE抗体监测gE Ab monitoring

生物安全 Biological safety 人、物、动物流控制 People, things, animals, flow control 消毒、灭鼠 Disinfection, deratization

高效免疫 Efficient immune 优化免疫程序 Optimize immune procedure 疫苗效果评估 Assess the immune impact

种猪群(经产公母猪群/后备公母猪群)全面进行伪狂犬野毒（gE/gI）抗体监测

低阳性率≤10% 高阳性率＞10%

一次性淘汰所有阳性带毒种猪

免疫gE基因缺失苗

引进免疫合格且gE为阴性的种猪

引进免疫合格且gE为阴性的种猪

分批次淘汰所有阳性带毒种猪

1、监控伪狂犬免疫效果（gB） 2、检测伪狂犬野毒抗体（gE） 3、淘汰阳性带毒种猪； 4、加强生物安全管理

种猪群维持、监测

1、监控伪狂犬免疫效果（gB） 2、检测伪狂犬野毒抗体（gE） 3、加强生物安全管理

阳性率≤10%

一、准备

阶段

二、方案制定与实施

三、维持

PRV阳性场—净化 PRV Positive farms-Eradication

Swine Herd （ sows / gilts ）for detection of PRV gE antibody

Low positive ratio≤10% High positive ratio＞10%

Eliminate disposable all positive pigs

Immune gE gene-deleted vaccine

Introduction of qualified immunity and gE Ab

negative pigs

Introduction of qualified immunity and

gE Ab negative pigs

Batch Elimination of all positive pigs

1、 Assess the immune effect （gB） 2、 Detection of PRV wild strain antibody（gE） 3、 Eliminated infected positive pigs 4、 Strengthening biosecurity management

Maintain and monitor of breeding herd

1、 Assess the immune effect （gB) 2、 Detection of PRV wild strain antibody （gE） 3、 Strengthening biosecurity management

Low positive ratio≤10%

一、 preparatory

stage

二、Program

formulation and

Implementation

三、maintain

PRV阳性场—净化 PRV Positive farms-Eradication

维持与净化的保障条件 Requirements for maintain and Eradication

条件保障 Requirements

充足的阴性后备猪群Enough PRV-gE Ab

negative pigs 确实、高效的免疫方案

Efficient immunization program

先进的实验室配套 Advanced laboratory

equipments

公司强有力的支持 The strong support from

company

PRV控制的具体措施 Concrete measures for the control of PRV

1. 高级别的生物安全控制措施 High level of biological safety control measures 2. 三点式生产模式调整为二点式 Set three site production mode to two site production 3. 阴性后备猪的引入与监控 The introduction and monitoring of negative reserve swine 4. 免疫程序优化与免疫效果评估 Optimize the immune procedure and assess the immune effect 5. 及时精准的实验室检测 Precisely and timely lab detection

载体：1. 活猪2. 生肉/血3. 车4. 器具5. 鞋衣6. 人7. 狗8. 猫9. 鼠10. 鸟11. 尘12. 空气

E人员设备器具进场

人通

道G转

猪栏

C中转栏

猪粪

B转

猪

A车辆消毒站

猪场附近区域

E人员设备器具进场

H尘

空气

猪场

猪舍1猪猪

猪舍1猪猪

一、高级别生物安全控制措施 High level of biological safety control measures

红橙黄绿区域划分/各级关口生物安全控

制策略的实施；

Area division / the implementation of biological safety control measures; 猪场洗车系统的建立完善：自建车辆清

洗、干燥、消毒配套设施；

Well established wash system : vehicle washing, drying and disinfection facilities; 后备猪隔离驯化时间的调整：2个月调整

4个月；; Adjust the isolation time of reserve swine from 2 months to 4 months

严格的消毒与灭鼠。

Strict disinfection and deratization.

二、三点式生产模式调整为二点式 Set three site production mode to two site production

断奶仔猪Weaning piglets

保育仔猪Nursery pigs

育肥猪Fattening pigs

断奶仔猪Weaning piglets

育肥猪Fattening pigs

父母代种猪场 Breeding Pig Farm

商品育肥场 Fattening pig farm

引自童光志等

Bucharest或Bartha-K61免疫血清交叉中和指数 Cross-neutralization index of Bucharest or Bartha-K61 immune serum

三、免疫程序优化与免疫效果评估 Optimize the immune procedure and assess the immune effect

类型 Type

公猪 boar

母猪 sow

产房仔猪piglets

保育猪Nursery

pigs

育肥猪 Fattening

pigs

以前previously

4次/年，2头份活疫苗 4 times/year， 2 doses of live vaccine

4次/年，2头份活疫苗 4 times/year，2 doses of live vaccine

0日龄滴鼻1头份活疫苗 0 day, Intranasal immunization, one dose live vaccine

70日龄，1头份活疫苗 70 days，one dose live vaccine

——

现在 Now

3次活疫苗+3次灭活疫苗/年，1头份+1头份，间隔3周 3 times/year , 1 dose live vaccine+1 dose inactivated vaccine

3次活疫苗+3次灭活疫苗/年，1头份+1头份，间隔3周 3 times/year , 1 dose live vaccine+1 dose inactivated vaccine

0日龄滴鼻1头份活疫苗 0 day, Intranasal immunization, one dose live vaccine

70日龄，1头份活疫苗 70 days，one dose live vaccine

98日龄1头份灭活疫苗 98days, one dose inactivated vaccine

三、免疫程序优化与免疫效果评估 Optimize the immune procedure and assess the immune effect

四、阴性后备猪的引入与监控 Introduction and monitoring of PRV gE Ab negative reserve swine

入场 ：gE抗体监测 Introduction

入群前：gE抗体监测 Before enter the group

入群后：批次跟踪监测 After entering the group

免疫抗体评估：中和/gB抗体 Assessment of the level of vaccine-induced antibodies

后备猪 Reserve swine

PRV净化进展—入群后备的批次监控 PRV Eradication progress-Batch monitoring of the entered reserve swine 猪场

Pig farm 批次编号 Batch No.

第1次检测 （入群2月）

2 months

第2次检测（入群4月）

4 months

第3次检测 （入群6月）

6 months

第4次检测 （入群8月）

8 months

第5次检测 （入群10月）

10 months

A

LX031 0%（0/45） 0%（0/44） 0%（0/40） 0%（0/40） 0%（0/39）

LX032 0%（0/45） 0%（0/43） 0%（0/43） 2.33%（1/43）

LX033 0%（0/50） 0%（0/50） 0%（0/45）

LX034 0%（0/40） 0%（0/40） 0%（0/34）

LX035 0%（0/40） 0%（0/40）

LX036 0%（0/40） 0%（0/40）

B

GM16 0%（0/50） 0%（0/44） 0%（0/44） 2.23%（1/44）

GM17 0%（0/54） 0%（0/53） 0%（0/53） 0%（0/53）

GM18 0%（0/50）

五、先进的实验室配套 Advanced laboratory equipment

及时精准的实验室检测是PRV控制所必需的！ Precisely and timely laboratory detection testing is necessary for the control of PRV!

准确、快速的病毒检测 Accurate, rapid virus detection

病毒分离及病原性分析 Virus isolation and pathogenicity analysis

gE抗体的准确、稳定检测

免疫抗体的评估（gB/中和抗体）Assessment of immune antibody

专业的检测与分析人员

Professional personnel of detection and analysis

兽

医

实

验

室 Veterinary laboratory

Accurate and stable detection of gE antibody

gE抗体 gE Ab

免疫抗体（gB/中和）Immune antibody

（gB/neutralizing Ab） 病原pathogen

公猪boar 季度quarter 2次/年2 times / year 精液semen

生产母猪Production sow

季度、胎次 quarter、 parity

2次/年2 times / year 流产母猪abortion sows

后备猪 Reserve swine

引种后、 入群前 After introduction、 before entering the group

入群前 before entering the group ——

育肥猪（留种）Fattening pigs（seed stock）

抽查spot test 抽查spot test 抽查spot test

注：每一次引种的后备群作为一个管理批次。每个批次确定后备猪确定35头持续检测猪只。在入群后每2个月进行PRV gE抗体检测。

及时、精准的实验室检测 Precisely and timely laboratory detection

• 病毒感染后1-2天开始排毒、感染后2-5天达到高峰，可持续17天；

Pigs begin to excrete virus at 1-2 days post-infection, and virus excretion peaked at 2-5 days post-infection, and this will last 17 days；

• 在病毒排泄高峰期，1只感染猪在24小时内可以向空气中排泄1*105.3TCID50的病毒（Muller 等，2001；Wittmann，1991）；

One infected pig can excrete 1x105.3 TCID50 viruses within 24 hours during the peak of virus excretion ；

• 通常情况下，除了仔猪外（1*102TCID50），使青年猪、成年猪感染需要大于1*104-5TCID50，感染已经免疫的猪需要100-1000倍的野毒（Marika Genzow）；Normally, 1x104.5TCID50 viruses (except piglets 1x102TCID50) are enough to infect an adult pig or a growing pig, while it needs 100-1000 times of virus to infect the immune pigs；

1、准确、快速的病原检测 Accurate, rapid detection of pathogens

准确、快速的病原检测有助于阻止病毒的传播！ Accurate, rapid detection of pathogens contributes to prevent

the spread of the virus！

案例分析：PRV野毒感染 Case analysis:PR wild strain virus infection

2014.3 某PRV gE抗体阴性猪场，育肥猪（13周龄）表现不吃料，高烧，流脓鼻，随后，母猪群出现流产症状。 March of 2014, one pig farm with PRV gE antibody negative, the fattening pigs of 13 weeks showed loss of appetite, high fever and pus snot. And then the pregnant sows showed abortion.

实验室检测结果Lab detection results： PCR确诊伪狂犬抗原阳性。 PCR conformed PRV positive 8/28为伪狂犬gE抗体阳性，7/28为可疑。 8/28 are PRV gE antibody positive and 7/28 are PRV suspicious。

Marker pos 1 2 3

PRV-ge

编号 OD S/P Result

育肥1 0.531 0.506 Pos!

育肥2 1.01 0.963 Neg

育肥3 0.537 0.512 Pos!

育肥4 0.622 0.593 Pos!

育肥5 0.973 0.928 Neg

育肥6 1.112 1.06 Neg

育肥7 0.796 0.759 Neg

育肥8 1.12 1.068 Neg

…… …… …… ……

采取措施： 育肥猪清群 Depopulation of

the fattening unit 全群检测、淘汰阳性母猪 Obsolete the positive sows 紧急免疫 Emergency immune 全场切底消毒 Sterilize the whole

place

案例分析：PRV野毒感染 Case analysis:PR wild strain virus infection

0 0 1 0 0 1 1 0 0 0

6

0 0

3

7

0

8

1 2

10

5 5

2

7

3

12

7

12

15

6

11

5 4

7

4 2 1 2 1 0 1 2 1 1 0 0 0

2 0 1 1 0 1 1 1 1 0 1 1 1

0

5

10

15

20日期-流产

流产头数 7 per. Mov. Avg. (流产头数)

34 34 40 45

10 3 1 0 0 0 0 0

20.3

6%

20.3

6%

23.9

5%

26.9

5%

5.99

%

1.80

%

0.60

%

0.00

%

0.00

%

0.00

%

0.00

%

0.00

%

0.00%

10.00%

20.00%

30.00%

0

10

20

30

40

50

0 1 2 3 4 5 6 7 8 9 10 11

胎次-流产

流产头数 比例%

背景： 2015.1.10，某PRV阳性猪场初生仔猪发生PED，检测返饲病料中无CSF、PRRS和PR病原。返饲后及时控制PEDV蔓延，母猪返饲后，2015.1.11-2.14出现流产高峰，流产猪妊娠日龄跨度较大，集中在42d-105d，且多为0-3胎母猪。 结果分析：2015.1.21，从流产胎猪病料中检出2份（共4份）PRV阳性。 Background： 2015.1.10，Pig farm with PRV positive found the piglet occuring typical PED symptom. The back-feeding Sample did not detect the CSF, PRRS or PR viruses. 2015.1.11-2.14, pregnant sows showed peak level abortion with broad range of the gestation period, but focus on 42d-105d, and most are 0-3 parity sows. Result analysis：2015.1.21，1 of 8 serum samples from the abortion sows detected the PRRS virus, and 2 of 4 fetus samples detected to be PRV positive.

案例分析：“反饲”样品病原检测 Case analysis： Pathogen detection of back-feeding Sample

精液病原检测 Sperm- pathogen

detection

常规病毒监测 Routine virus monitoring

流产、神经症状病例检测Abortion and Neurological

abnormal

2、病毒分离与病原特性分析 Virus isoation and pathogeny analysis

PRV的宿主广泛，不同分离株的初始培养在许多细胞系上均可实现，通常用于病毒分离的细胞系有PK－１５、MDBK、Vero以及BHK２１等。PRV has a broad range of the host animals, many strains can be cultured in many cell lines, usually use PK-15, MDBK, Vero and BHK21 etc.

PRV野毒分离

正常细胞 Normal cell

病变细胞 CPE

Isolation of PRV wild strain

PRV分子特征分析 Molecular Genetic Analysis of PRV

毒株序列：gC、gE进化树中三株分离株与2012年以来的参考毒株处于同一分支，且与经典株相比与2012年以来分离的变异株具有相同的氨基酸插入的分子特征，说明这三株毒株仍为目前的流行变异株。 Sequence analysis: Three isolates and reference strains since 2012 are in the same branch in gC, gE phylogenetic tree. And mutants isolated since 2012 have the same molecular features of amino acid insertion compared with classic strains. This shows that the three strains is still current popular variants.

2、病毒分离与病原特性分析 Virus isoation and pathogeny analysis

种猪群(经产公母猪群/后备公母猪群)全面进行伪狂犬野毒（gE）抗体监测

低阳性率≤10% 高阳性率＞10%

一次性淘汰所有阳性带毒种猪

免疫gE基因缺失苗

引进免疫合格且gE为阴性的种猪

引进免疫合格且gE为阴性的种猪

分批次淘汰所有阳性带毒种猪

1、监控伪狂犬免疫效果（gB） 2、检测伪狂犬野毒抗体（gE） 3、淘汰阳性带毒种猪； 4、加强生物安全管理

种猪群维持、监测

1、监控伪狂犬免疫效果（gB） 2、检测伪狂犬野毒抗体（gE） 3、加强生物安全管理

阳性率≤10%

一、准备

阶段

二、方案制定与实施

三、维持

3、gE抗体的准确、稳定检测 Accurate and stable detection of gE antibody

Swine Herd （ sows / gilts ）for detection of PRV gE antibody

Low positive ratio≤10% High positive ratio＞10%

Eliminate disposable all positive pigs

Immune gE gene-deleted vaccine

Introduction of qualified immunity and gE Ab

negative pigs

Introduction of qualified immunity and

gE Ab negative pigs

Batch Elimination of all positive pigs

1、 Assess the immune effect （gB） 2、 Detection of PRV wild strain antibody（gE） 3、 Eliminated infected positive pigs 4、 Strengthening biosecurity management

Maintain and monitor of breeding herd

1、 Assess the immune effect （gB) 2、 Detection of PRV wild strain antibody （gE） 3、 Strengthening biosecurity management

Low positive ratio≤10%

一、 preparatory

stage

二、Program

formulation and

Implementation

三、maintain

3、gE抗体的准确、稳定检测 Accurate and stable detection of gE antibody

2015年第二季度，某PRV 阴性猪场季度健康监测时，在育肥猪中检测到1份gE抗体阳性： 采取措施： 1、母猪全群检测： 全群1258份样品共检测到2 份阳性样品，阳性率为0.16%(2/1258)，可疑样品33 份。母猪群PRV-gE 野毒抗体正处于转阳阶段。淘汰阳性、可疑猪只，后续每两周采血复查，全部为阴性。 2、育肥猪清群 3、母猪普疫PRV疫苗 4、保育猪与育肥猪完全分开 跟踪监测结果： 2015年第三季度至今未检测到PRV gE抗体阳性猪只，成功阻断猪群转阳。 Second season of 2015,one pig farm previously certified PRV gE antibody negative, detected one PRV gE antibody positive pig when doing the seasonal healthy detection. Strategy： 1、PRV detection toward the entire sow： 2 positive samples were detected among 1258 samples(positive rate:0.16%)；33 suspicious samples.PRV gE antibody is turning to positive stage. Obsolete the positive、suspected pigs, recheck using blood samples two weeks later till all pigs turning to negative ones. 2、Depopulation of the fattening unit 3、Immune the sows with PRV vaccine 4、Separate the fattening pigs and conserving pigs Track the detecting result： We did not detect the PRV gE positive pigs from the 3rd season of 2015 to the second season of 2016, informing that the pigs was successfully protected.

案例分析：准确、及时的检测 Case analysis：Detect precisely and timly

4、免疫抗体评估（gB/中和抗体） Assessment of immune antibody（gB/neutralizing antibody ）

免疫的猪致病性感染剂量是非免疫猪需要100-1000倍； The infection dose of immune pigs is 100-1000 times of the un-immune pigs；

——免疫是控制PRV的有效方法 Immunation is the effective way to control PRV

PRV新的流行毒株出现，使得现有的疫苗不能完全保护新流行毒株的感染，猪群患病的风险依然存在；

Current PRV vaccine can not protect newly occurred PRV strains, the risk of PRV disease still exist;

——免疫不能完全保护 Vaccine could not provide complete protection

猪场的免疫效果如何？immune effect ?

如何提高免疫效果？How to promote the immune effect?

4、免疫抗体评估（gB/中和抗体） Assessment of immune antibody（gB/neutralizing antibody ）

应用Application：

1、疫苗及免疫方式筛选

Screening of Vaccine and immune strategy

2、监控免疫效果（合格率、抗体水平……）

monitoring immune effect（pass rates、 antibody level……）

免疫抗体评估方法与应用 Application and evaluation of immune antibody

方法Method 时间Time 条件Condition 抗体类型Antibody species

ELISA 3-4h 低low gB抗体

中和试验Neutralization test 72-96h 高high

中和抗体Neutralizing

antibody

5、人员要求：专业、敬业 Laboratorian requirements:Professional、Responsibility

准确数字Precise data

编号 OD S/N ResultG2 1.152 1.005 Neg G3 1.036 0.904 Neg G4 1.102 0.962 Neg G5 0.994 0.867 Neg G6 1.168 1.019 Neg G7 1.167 1.018 Neg G8 1.146 1 Neg G9 1.221 1.065 Neg G10 1.146 1 Neg G11 1.025 0.894 Neg G13 0.966 0.843 Neg G14 1.014 0.885 Neg G15 1.202 1.049 Neg G16 1.243 1.085 Neg G17 1.076 0.939 Neg G18 1.086 0.948 Neg G19 1.106 0.965 Neg G20 1.173 1.024 Neg

PRV

可靠数据

Reliable result

新希望六和动保中心实验室位于青岛市崂山区株洲路3号，实验

室占地509m2，2013年5月开始运行。现有人员24名，博士2人，硕士

18人。Animal health center of New Hope Liuhe locates in No.3 Zhuzhou road,

Laoshan district, Qingdao. The lab is 509m2 in area and operated in May,2013.

We now have 24 lab members, including 2 doctors and 18 Masters.

新希望六和动保中心 Animal health center of New Hope Liuhe

功能研究室 Function department

Central Laboratory

Histopathology

Molecular biology

Serology

Microbiology

Cytology