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译者的话:

By fei:

  非洲猪瘟让大量的猪场被动清群，在目前猪价大涨的情况下，所有受非洲猪瘟影响的猪场都在积极复场，然而目前国内尚未报道有成功复场的案例，而全球范围内可供参考的经验也是少之又少。在此情况下，借鉴其他疾病清群和复场的经验就成了最好的选择，因为疾病爆发后的清群、清洗消毒、检测、加强生物安全等步骤都是可以复制的。

      ASF has caused many farms to be depopped, with the pig price keeps going up, these farms are actively trying to restock. However, there is so far no report of any farm being successfully restocked in China, and globally there is little experience to draw from. Under this situation, instead of constant trial and error, the best option would be to learn from depop and repop experience of other diseases, because procedures such as depop, wash & disinfection, testing, improving biosecurity after outbreak can be copied.  

  本文详细介绍了一项来自美国的PCV2成功清群和复场的研究。猪场在自然爆发PCV2之后清群，并进行了彻底的清洗、消毒、设施改建、加强生物安全、实验室检测等，在清群63天之后复场成功，截止报告日期已维持PCV2阴性20个月。本文介绍了各个阶段的工作流程和详细步骤，希望对非洲猪瘟复场工作有所启发。

This article presents you a successful PCV2 depop and repop case in the US. The farm was depopped after a natural PCV2 outbreak and was completely washed & disinfected and partially renovated prior to restocking.  Biosecurity was improved and lab test performed regularly before and after the restocking to maintain the PCV2 negative status which had lasted for at least 20 months by the time the report was written. Detailed procedures and steps of each stage were illustrated in this report, hopefully it can provide some experience for ASF farm restocking. 

                                    PCV2成功清群和复场的启发（一）

          lmplications of Successful PCV2 De-pop and Re-pop (Part 1)

          在爆发猪圆环病毒(PCV2)相关疾病的猪场进行复场并维持PCV2阴性繁殖群

          Establishment and maintenance of a porcine circovirus type 2 (PCV2)-free breeding herd on a site that experienced a natural outbreak of PCV2-associated reproductive disease

作者 AuthorAbby R. Patterson, DVM, MS, PhD; Darin M. Madson, DVM, PhD; Shayleen D. Schalk, BS; Patrick G. Halbur, DVM, MS, PhD; Tanja Opriessnig, Dr med vet, PhD

摘要 

Summary

  2009年，爱荷华州一个封闭的、从未感染猪圆环病毒2型(PCV2)的无特定病原猪场爆发了PCV2相关的繁殖障碍疫情。本报告综述了该场从繁殖群中清除感染性PCV2的方法、复场后的结果，以及通过场外隔离培养PCV2阴性猪的尝试措施。本次疫情的临床症状仅限于木乃伊胎的发生率增加。在确认胎儿PCV2相关的病变和母猪PCV2病毒血症后，猪群被清群。     An outbreak of reproductive failure associated with porcine circovirus type 2 (PCV2) occurred in a closed, PCV2-naive, specific-pathogen-free herd in Iowa in 2009. Elimination of infectious PCV2 from the breeding-herd site, the outcome after repopulation, and the attempt to derive PCV2-negative animals by offsite segregation are summarized. Clinical signs were limited to an increased incidence of mummified fetuses. After confirmation of PCV2-associated lesions in the fetuses and PCV2 viremia in dams, the herd was depopulated.

  在复场之前，对猪场进行了彻底的清洗和消毒，包括清除肉眼可见的有机物质、把设备置于紫外线光下消毒、多次喷施消毒剂、用漆和密封剂填封有孔缝洞的表面等。在为期63天的清洗消毒期间，场内没有猪。之后执行了更为严格的生物安全措施。复场后，猪群一直保持PCV2阴性，在撰写本报告时，已保持阴性长达20个月。但通过场外隔离从PCV2 阳性猪群中培养阴性仔猪的尝试是失败的。本实验说明多重清洗和消毒步骤与严格的生物安全措施可以把爆发PCV2之后清群复场的猪场维持阴性。

Cleaning and disinfection of the premises prior to repopulation included removal of gross organic material, exposure of equipment to natural UV light, multiple applications of disinfectant, and application of paint or sealer to porous surfaces. During the 63-day clean-up period, no pigs were on the site. An improved biosecurity plan was implemented. The herd was repopulated and a PCV2-naive population had remained PCV2-negative for 20 months at the time of writing. Attempts to derive PCV2-negative pigs from the positive herd following offsite segregation were unsuccessful. The combination of a multistep cleaning and disinfection protocol with a strict biosecurity plan can result in the maintenance of PCV2naive animals on a previously contaminated site.

关键词 

Keywords

养猪生产 Swine production，猪圆环病毒2型 PCV2，清洗消毒 Cleaning & disinfection，生物安全 Biosecurity，清群 Depopulation, 复场 Repopulation

  猪圆环病毒2型(PCV2)是一种小型、无包膜的单链DNA病毒，出现于20世纪90年代，是一种对经济影响极大的猪病原体。PCV2繁殖性疾病的第一起疫情发生在一个全部是1胎母猪存栏450头的商品场，感染的时间并没有确定，猪场的临床症状包括晚期流产(妊娠13周后)、分娩率下降、死胎和木乃伊胎增加，但母猪没有临床症状。在检查的9例胎儿中，有1例在胎儿心肌中发现了心肌炎和大量的PCV2抗原。未发现其他与繁殖疾病相关的常见病原体。随后，在血清阳性猪群中和PCV2血清学阴性的新建场和后备猪群中报道了多起与PCV2相关的繁殖障碍案例。常见的临床特征包括分娩时木乃伊胎和死胎增加，母猪不表现或极少表现临床症状，以及最初发现疫情后2到5个月临床症状消失。

Porcine circovirus type 2 (PCV2) is a small, nonenveloped, singlestranded DNA virus which emerged in the1990s as an economically important swine pathogen. The first report of PCV2 reproductive disease occurred in a commercial, 450head herd composed entirely of first- parity dams. The timing of infection was not determined in that case, and clinical signs on the farm included late-term abortions (losses after 13 weeks of gestation), decreased farrowing rates, and increased stillborns and mummified fetuses; no clinical signs were reported in dams. In one of nine examined fetuses, myocarditis and abundant PCV2 antigen were identified in fetal myocardium. Other common pathogens associated with reproductive disease were not detected. Multiple case reports on PCV2-associated reproductive failure have subsequently been described in seropositive herds or in start-up herds or gilt populations that may have been serologically naive to PCV2. Common clinical features included increased numbers of mummified fetuses and stillborns at parturition, no or low numbers of dams exhibiting clinical signs, and resolution of clinical signs between 2 and 5 months following initial detection. 

PCV2在环境中相当稳定，很多消毒剂无法完全消除环境中的病毒。据我们所知，目前没有关于建立和维持PCV2阴性猪群的猪舍消毒程序的文献资料。然而，爱荷华州立大学的研究人员最近在对PCV2接种动物的猪舍消毒方面取得了成功。本场健康度非常高，此前从未感染过PCV2.

Porcine circovirus type 2 is quite stable in vitro, and many disinfectants do not completely eliminate the virus under in vitro conditions. To our knowledge, no documented information exists on facility disinfection procedures for establishment and maintenance of a PCV2negative herd. However, a procedure for room decontamination used successfully by researchers at Iowa State University to disinfect rooms between PCV2 animalinoculation studies was recently described. It is a highhealth population previously known to be free of PCV2.

猪场布局 

Building descriptions

  该猪场位于爱荷华州，初建于20世纪70年代。场内有5栋22.8 m*15.2 m的猪舍，彼此相距很近（15-30米），共用一个污水池。2009年爆发该疫情并清群后，该场增设了一个连通各栋猪舍的露天连廊。2009年5月之后的猪场布局情况如图1所示。

   The source herd was housed in a facility located in Iowa, originally built in the 1970s. The facility was composed of five 22.8 m *15.2 m buildings in close proximity to each other (15 to 30 m) with an associated waste lagoon. An uncovered concrete walkway connecting the individual buildings had been installed after herd depopulation following the 2009 outbreak. A diagram of the facility after May 2009 is shown in Figure 1.

 

图1：PCV2爆发并彻底清场后（2009年5月）的猪场布局

Figure 1: Diagram of a swine research breeding facility following complete depopulation in May 2009 after an outbreak of reproductive failure associated with porcine circovirus type 2.

综合用房：

第一栋猪舍（栋1）是综合用房，由一堵水泥墙分成两半。一侧作为入口房，有办公室、淋浴间、洗衣房和更衣室等设施，另一侧是饲料和设备物资的库房以及工具间。栋1有1道门通往入口登记区，1道门通向办公区域，还有1道门进入饲料和设备库房。

General services building. 

Building 1, used as the general services building, was divided into two sides by a concrete wall. One side was used as the entrance to the facility with office space, shower, laundry, and locker room, and the second side provided storage for feed and equipment and tools for building maintenance. Building 1 had one door for entry into the sign-in area, a door that led out from the office area, and a garage door for entry into a feed and equipment storage area.

各栋猪舍：

栋2-5是全封闭猪舍，机械通风，半漏缝地板和粪塞排粪体系。栋2, 3和5中间各有一道水泥墙把猪舍分为两半。每一半都有独立的入口和地沟粪坑（见图1）。粪坑长3.4米，宽1.2米，深0.9米。其余的地面为倾斜的实心地面。栋4有一个地沟粪坑，长22.8米，宽1.2米，深0.9米。

Animal buildings. 

Buildings designated 2 through 5 were used for animal housing. The animal buildings were completely enclosed, power ventilated, and had partially slatted floors with pull-plug drains. Buildings 2, 3, and 5 each had a center concrete wall which divided the building into halves lengthwise; each half had a separate access point and a separate manure pit (Figure 1). The manure pits were 1.2 m wide, 3.4 m long, and 0.9 m deep. The remaining floor space had a sloped solid floor. Building 4 had one manure pit 1.2 m wide, 22.8 m long, and 0.9 m deep.

2009年5月清群前的猪群和圈舍情况 

Herd description and housing prior to depopulation in May 2009

该场在此次疫情爆发前约2年引入了阴性的经产母猪和后备母猪，这些猪都来自一个PCV2阴性猪群（血清学和PCR检测阴性），到场后立即进行PCV2血清学和PCR检测。这次进猪后（2007年12月）直至2009年5月清群，中间没有再引入其他猪。母猪是无特定病原的二元母猪，PCV2、蓝耳、猪流感、细小病毒都是阴性，会定期对不同胎次的母猪及其后代进行血清学（PCV2、蓝耳、猪流感、细小病毒）和PCR检测（PCV2、蓝耳）确定。母猪和后备母猪妊娠期间放在栋2和栋3，产前转入栋5。栋3是成年公猪和妊娠母猪。栋4主要作为保育用。在观察期间，场内一共有38头种猪（栋2有12头，栋3有13头，栋4有2头，栋5有11头）。

Naive sows and gilts were placed on site approximately 2 years prior to the described outbreak. All animals came from a PCV2- naive herd, as determined by PCV2 serological and polymerase chain reaction (PCR) testing, and all were immediately tested by PCV2 serology and PCR after arrival at the facility. No new animals entered the facility from the time of initial population in December 2007 until depopulation in May of 2009. Sows were cross-bred, specific-pathogen-free animals naive for PCV2, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and porcine parvovirus (PPV), as determined by periodically performed serological testing (PCV2, PRRSV, SIV, and PPV) and PCR testing (PCV2, PRRSV) on sows of mixed parities and their offspring. Sows and gilts were housed in Buildings 2 and 3 during gestation and moved into Building 5 prior to farrowing. Building 3 contained mature boars as well as gestating sows. Building 4 was primarily used as a nursery unit. During the observation period, a total of 38 breeding animals were onsite (12 in Building 2, 13 in Building 3, two in Building 4, and 11 in Building 5).

PCV2相关繁殖疾病爆发

PCV2-associated reproductive disease outbreak

疫情发现和确认 

  Outbreak detection and confirmation

图2总结了PCV2爆发的详细时间节点。2009年1月29日首次发现疫情，当时对栋4的部分断奶后两周的猪进行了常规筛查，使用前述的ELISA检测出抗IgG PCV2抗体呈阳性。随后，用间接免疫荧光法 (IFA)确认了样品中确实存在PCV2抗体，并用实时荧光定量PCR检测到了PCV2 DNA，检测极限为每毫升1 × 103 个拷贝数。对所有母猪采集了血清样本并检测，发现栋2, 3和5都有血清阳性无病毒血症、血清阳性有病毒血症和血清阴性有病毒血症的猪存在。栋4有2头单独饲养的后备母猪，血清中都没有检出PCV2抗体和PCV2 DNA。这些数据无法得出确切的感染时间。但是，栋2（12头中有4头，33.3%）和栋3（13头中有4头，30.8%） PCV2 DNA阳性但抗体阴性的母猪数量都大于栋5（11头中有1头，9.1%）。很明显，栋2和栋3为新近感染，而栋5感染时间更长。

Details on the timeline of the PCV2 outbreak are summarized in Figure 2. The outbreak was first noted on January 29, 2009, when a portion of weaned 2-week-old pigs from Building 4 were routinely screened and were positive for anti-IgG PCV2 antibodies when tested using a previously described ELISA. Subsequently, the presence of anti-PCV2 antibodies in the samples was confirmed by an in-house immunofluorescent antibody (IFA) assay, and PCV2 DNA was detected by using a quantitative real-time PCR with a detection limit of 1 × 103 copies per mL. Serum samples were collected from all sows, and a mixture of seropositive nonviremic animals, seropositive viremic animals, and seronegative viremic animals were detected in Buildings 2, 3, and 5. Neither anti-PCV2 antibodies nor PCV2 DNA were detected in the serum of the two gilts housed by themselves in one half of Building 4. The exact timing of infection was not deduced from this data. However, there were higher proportions of sows in which PCV2 DNA was detected without detectable anti- PCV2 antibody in Buildings 2 (four of 12; 33.3%) and 3 (four of 13; 30.8%) than in Building 5 (one of 11; 9.1%). Infection was apparently most recent in Buildings 2 and 3 and of longer duration in Building 5.

 

图2：PCV2爆发、清群及复场的时间线

Figure 2: Timeline of events after detection of an outbreak of reproductive failure associated with porcine circovirus type 2 (PCV2) in a swine research breeding facility and repopulation of the facility with a PCV2-naive research breeding herd. BSL-2 = Biosafety Level 2

为了进一步鉴定感染该猪群的PCV2毒株，用前述的嵌套式PCR从两头母猪和两头仔猪的血清样本中提取了DNA，并对其进行ORF2基因测序。PCR产品使用QIAquick PCR纯化试剂盒(Qiagen, Valencia, California)按照制造商的说明进行纯化，并在爱荷华州立大学DNA实验室内进行测序。序列分析采用序列扫描软件1.0版(Applied Biosystems, Foster City, California)，使用基本局部序列对齐比较搜索工具(BLAST) 与四种常见的PCV2a和PCV2b毒株进行比较，发现分离到的毒株为PCV2b(100%同源，GenBank收录号为EU340258)。

To further characterize the PCV2 strain that infected the herd, PCV2 open reading frame 2 (ORF2) sequencing was performed on extracted DNA from two sow serum samples and two piglet serum samples using a nested PCR as previously described. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Valencia, California) per manufacturers’ instructions and sequenced at the Iowa State University DNA facility. Sequences were analyzed with Sequence Scanner software version 1.0 (Applied Biosystems, Foster City, California) and compared with four common PCV2a and PCV2b strains using the basic local alignment search tool (BLAST), and the isolate was  identified as PCV2b (100% homology with GenBank Accession No. EU340258).

2008年5月到2009年4月繁殖群的繁殖性能和临床观察情况

Reproductive parameters and clinical observations in breeding animals from May 2008 to April 2009

爱荷华州立大学实验动物资源部的工作人员记录了2008年5月至2009年4月间38头母猪的分娩数据。2009年2月，一头母猪产下一窝13头木乃伊胎，其繁殖性能明显受到影响。显微镜下对木乃伊胎组织的评估显示多灶性、严重的心肌坏死并伴有矿化。免疫组化(IHC)检测胎儿心肌中含有大量的PCV2抗原。2008年5月，一只母猪因脊椎脓肿引起的四肢瘫痪而被安乐死。在此期间，繁殖群中没有发现其他临床症状。繁殖群感染PCV2的大致时间为2008年12月至2009年1月。10头妊娠中期的母猪(约57天，2009年2月3日)PCV2状态以及分娩情况(木乃伊胎、死胎和活仔数量)详见表1. 虽然PCR和血清学检测表明10头母猪中有9头感染了PCV2，但它们繁殖表现正常。

Farrowing data from the 38 sows between May 2008 and April 2009 were recorded by Iowa State University Laboratory Animal Resources personnel. Reproductive failure was evident in one sow that farrowed a term litter composed of 13 mummies in February 2009. Microscopic evaluation of the mummified fetal tissues revealed multifocal, severe myocardial necrosis with mineralization. Abundant PCV2 antigen was detected by immunohistochemistry (IHC) in the fetal myocardium. One sow was euthanized in May 2008 due to hind-limb paresis associated with a vertebral abscess. No other clinical signs were reported in the breeding herd during this time period. The approximate time of PCV2 infection of the breeding herd was between December 2008 and January 2009. The PCV2 status of 10 sows at mid gestation (approximately 57 days; February 3, 2009) and their litter characteristics at farrowing (number of mummified fetuses, stillborns, and born-alive piglets) are summarized in Table 1. Although nine of the 10 sows were exposed to PCV2 as determined by PCR and serology data, reproductive performance appeared normal.

 

表1：妊娠期57天（2009年3月2日）母猪的PCV2抗体和病毒血症情况，以及之后的分娩情况

Table 1: Dam PCV2 antibody and viremia status at approximately 57 days of gestation (February 3, 2009) and characteristics of their litters at the time of farrowing in a swine research breeding herd naturally infected with PCV2

清除猪场环境中的PCV2病毒

Removal of PCV2 from the farm environment

猪场生物安全规定

Facility biosecurity protocols 

猪场入口大门，当员工不在场时，大门是锁着的。猪场外围设立了围栏，把猪场封闭，并张贴了生物安全标志。为了进一步限制进入猪舍的人员数量，所有猪舍都始终上锁。

Access to the facility was through a gated entrance that was locked when employees were not present. An additional perimeter fence enclosed the swine facility, where a biosecurity sign was posted. To further limit the number of personnel that had access, all animal buildings were locked at all times.

进入猪场时，需要换衣服。唯一获准进入猪场的车辆是一台除雪机和一台割草机，两者都不是猪场专用的。猪场所需的所有设备都在进入生物安全门之前用卫可(杜邦)消毒。进入猪场后，每栋猪舍入口处都有脚踏盆。每栋舍的进风口外侧都安装有空气过滤系统(3M Filtrete Micro Particle Reduction Filter 700; 3M Co, Ames, Iowa)，这个过滤系统(包括过滤器和外框)是为猪场定制的。

At entry into the facility, a change of clothes was required. The only vehicles granted access to the swine facility were a snow blower and a lawn mower, both nonspecific to the swine facility, which were used for property maintenance. All equipment needed in the facility was disinfected with Virkon S (Dupont, Pharmacal Research Laboratories, Inc, Naugatuck, Connecticut) prior to entrance through the biosecurity gate. Upon access to the facility, the use of foot baths was required between all animal buildings. A custom-fitted system for air filtration was installed on the outside of air inlets in each building using air filters (3M Filtrete Micro Particle Reduction Filter 700; 3M Co, Ames, Iowa) and custom frames.

地沟粪坑每周排空一次，流入污水池内。污水池由猪场员工每年清空一次，粪污施在邻近的田地里。啮齿动物控制方面，每栋舍有4个诱鼠盒，诱饵为溴鼠隆 (Havoc Rodenticide Bait Pack; Hacco, Inc, Randolph, Wisconsin)。屋顶和墙体之间的孔缝处用保温泡沫堵塞(Great Stuff insulating foam; Dow Chemical Company, Midland, Michigan)，以防止鸟类进入。PCV2爆发前和复场后猪场的生物安全规定的不同之处具体表现在猪和人在不同栋舍之间的转移和活动、精液的使用、饲料运送、设备消毒、访客入场程序、空气管理、粪坑管理和日产工作，具体情况详见表2.

Pits were emptied once per week into a nearby lagoon. The lagoon was emptied once per year in the fall by facility employees and contents were spread onto an adjacent field. For rodent control, four bait boxes per building were filled with a brodifacoum product (Havoc Rodenticide Bait Pack; Hacco, Inc, Randolph, Wisconsin). For bird control, foam insulation (Great Stuff insulating foam; Dow Chemical Company, Midland, Michigan) was placed into openings between the roof and walls. Specific differences in the biosecurity protocol concerning animal movements between buildings, personnel movements between buildings, semen usage, feed delivery, equipment disinfection, visitor entry procedures, air management, manure-pit management, and daily chores prior to the PCV2 outbreak and following repopulation of the herd are summarized in Table 2.

表2：PCV2 爆发前与用PCV2阴性猪复场后的生物安全规定的差异

Table 2: Outline of specific biosecurity protocols used at a research swine breeding facility site prior to an outbreak of reproductive failure associated with PCV2 infection and after repopulation of the facility with PCV2-naive animals

未完待续……

To be continued……

 

注：请关注明日第二部分：详细的清洗消毒流程，消毒结果监测，复场及检测，PCV2爆发期间用阳性母猪生产阴性仔猪的尝试，以及研究讨论等。

Note: Please refer to Part 2 tomorrow for detailed wash & disinfection procedure, monitoring of the disinfection success of the premises, re-pop and testing, attempt to derive negative animals from PCV2-infected sows during the outbreak, and research discussion, etc.