猪精液-病毒传播的载体（一）

译者的话：“公猪好，好一坡”，同理，公猪坏，那也是坏一坡。对于几乎100%采用人工授精的国内规模化猪场，公猪及精液的健康状况对整个生产系统的影响不言而喻，本文从不同病毒性病原在精液中被监测到的时间、对繁殖性能的影响、常规预防及健康监测、生物安全系统（设施、物流等）等方面充分讨论精液做为病毒传播载体的可能性及对策，从深度、广度及实操性本文是此专项议题难得一见的雄文。伟大始于分享，强烈推荐养猪生产者、健康管理者、专业公猪站运营相关人士观阅、扩散乃至收藏。另，小声提醒：建立多个独立的公猪站--不将所有鸡蛋放在一个筐子内也是精液风险管理的常见策略。猪精液-病毒传播的载体（一）Porcine semen as a vector for transmission of viral pathogens-Part 1Dominiek Maesa,*, Ann Van Sooma, Ruth Appeltanta, Ioannis Arsenakisa, Hans Nauwynckba比利时，梅勒尔贝克，根特大学，兽医学院，畜群健康与产科学，繁殖科a Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgiumb比利时，梅勒尔贝克，根特大学，兽医学院，病毒学实验室，免疫与寄生虫学，病毒学科b Department of Virology, Immunology and Parasitology, Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium 关键词 Keywords：猪精液、猪、人工授精、病毒、回顾/检查Semen, Pig, Artificial insemination, Virus, Review摘要 ABSTRACT猪精液中可检测出各类病毒。其中一些已列入世界动物卫生组织（OIE）疫病名录，因此，这些病原对社会经济和/或公共卫生，以及国际动物及动物产品贸易有着重大影响。人工授精（AI）是全球养猪生产中最常用的辅助繁殖技术。与自然繁育相比，人工授精技术的广泛应用，能够帮助养猪生产者优化遗传体系，降低养殖成本。但是，用于人工授精的精液是经处理后大量分装而成，这样就增加了病毒在猪群中广泛传播的风险。精液中的病原可能来自被感染的公猪，也可能产生于精液采集、加工和储存过程。此类精液会导致精子质量下降、胚胎死亡，引起受体母猪子宫内膜炎、全身感染和/或发病。判断精液中是否存在病毒可以通过病毒活性检测、病毒核酸检测或对公猪进行血清抗体检测间接证明。避免疾病通过精液传播的最佳方法有：确保人工授精中心公猪无病，执行严格的生物安全措施，并对公猪进行常规健康监测。首要重点是预防病毒污染精液，这比清除精液中的病毒更容易。然而，新型精液处理方法（如单层离心）的研发仍在进行，或可在未来应用于精液净化。Different viruses have been detected in porcine semen. Some of them are on the list of the World Organization for Animal Health (OIE), and consequently, these pathogens are of socioeconomic and/or public health importance and are of major importance in the international trade of animals and animal products. Artificial insemination (AI) is one of the most commonly used assisted reproductive technologies in pig production worldwide. This extensive use has enabled pig producers to benefit from superior genetics at a lower cost compared to natural breeding. However, the broad distribution of processed semen doses for field AI has increased the risk of widespread transmission of swine viral pathogens. Contamination of semen can be due to infections of the boar or can occur during semen collection, processing, and storage. It can result in reduced semen quality, embryonic mortality, endometritis, and systemic infection and/or disease in the recipient female. The presence of viral pathogens in semen can be assessed by demonstration of viable virus, nucleic acid of virus, or indirectly by measuring serum antibodies in the boar. The best way to prevent disease transmission via the semen is to assure that the boars in Al centers are free from the disease, to enforce very strict biosecurity protocols, and to perform routine health monitoring of boars. Prevention of viral semen contamination should be the primary focus because it is easier to prevent contamination than to eliminate viruses once present in semen. Nevertheless, research and development of novel semen processing treatments such as single-layer centrifugation is ongoing and may allow in the future to decontaminate semen.© 2016爱思唯尔(Elsevier)公司版权所有            © 2016 Elsevier Inc. All rights reserved.1. 引言Introduction人工授精（AI）是全球养猪生产中最常用的辅助繁殖技术。此技术已有40多年历史，目前已广泛应用于集约化生产系统。二十多年来，大多数欧洲国家，超过90％的母猪采用人工授精繁殖[1]。同样，在北美和拉丁美洲等重要的养猪生产国，人工授精已覆盖约90％。在一些东亚国家，也有明显的增长趋势，如在菲律宾的现代化猪场，人工授精的母猪占50％，在泰国和中国台湾，目前已有超过70％的母猪采用人工授精[2]。Artificial insemination (AI) is one of the most commonly used assisted reproductive technologies in pigs. It has been carried out for more than 40 years and is currently widely practiced in almost all intensive pig production systems. For more than 2 decades, more than 90% of the sows have been bred by AI in most European countries [1]. Also in North America and in important pig producing countries of Latin America, AI has reached levels of approximately 90%. A significant increasing trend is also discernible in several East Asian countries, e.g., 50% of the sows are artificially inseminated in modern farms in the Philippines. In Thailand and Taiwan, more than 70% of the sows are currently artificially inseminated [2].人工授精技术的广泛应用有以下几方面原因[3]。与自然交配相比，人工授精可加速繁殖、充分利用优质基因、节约成本，繁殖管理更容易进行。另外，公猪与母猪无交配，也无直接身体接触，能够降低由公猪引入或传播病原的风险。然而，就人工授精而言，单次射出的精液会被处理并稀释成多份精液，随后售往不同的母猪场。如果精液中含有病原体，则会感染母猪，从而加速病原的广泛传播。因此，人工授精虽然可以极大降低疾病的传播风险，但牵涉大量母猪时，含有病原体的精液会造成巨大的影响。There are several factors that may explain the broad application of AI [3]. Compared to natural mating, there is an accelerated propagation and amplification of genetic merit, there are economic savings, and the reproductive management is easier. In addition, as there is no copulation and no direct physical contact between the boar and the sow, the risk for introduction or transmission of boar pathogens in the sow herd is lower. However, in the case of Al, one ejaculate is processed and diluted to produce several insemination doses that are subsequently sold to many different sow farms. If pathogens are present in the semen, they may initiate infection of the sow, and consequently, AI is associated with an increased risk for quick and widespread transmission of these pathogens. Consequently, although the risk for disease transmission after Al may be minimal, the impact of semen that is contaminated with pathogens can be enormous, especially if a large number of sow herds are involved.众所周知，猪精液中可能含有不同的微生物。这些微生物来自动物源或非动物源[4]。动物源微生物污染可能来自公猪的全身或局部感染，病原体通过睾丸和其他生殖道组织排散。精液的采集与处理过程中，污染也可能源于包皮积液、呼吸道分泌物和粪污颗粒。非动物源的污染主要来自采精人员（头发、皮肤、呼吸道分泌物）；来自精液加工过程中的用水、通风系统、气溶胶；以及水槽/排水沟等[5]。It is well known that ejaculated porcine semen may contain different microbes. They can be either from animal or nonanimal origin [4]. Contamination by microbes of animal origin can be due to general or local infections within the boar, and shedding through the testes and other tissues of the genital tract. Contamination can also originate from preputial cavity fluids, respiratory secretions, and fecal particles during collection and processing. Contamination of nonanimal origin mostly originates from the person collecting the semen (hair, skin, respiratory secretions); from the water used during semen processing, the ventilation system, and aerosols; and from sinks/drains [5].微生物类污染物包括可引起具体疾病的病原体，或不被视为病原体的微生物。公猪单次射出的精液中，通常每毫升含有104至105个细菌[6]。其中大多数不致病，但可能会影响精子质量[5]。猪精液中，特定的细菌性病原体包括布鲁氏杆菌、钩端螺旋体、分支杆菌、衣原体和支原体。猪精液中存在许多病毒性病原体，其有别于细菌性病原体。本文将阐述在人工授精时，猪精液中发现的一些重要病毒，包括其可能产生的影响、诊断方法，以及可用于减少或预防精液污染的管控措施。Microbial contamination may include pathogens causing specific disease conditions, or microorganisms that are not considered as pathogens. Boar ejaculates usually contain 104 to 105 bacteria/ mL [6]. Most of them are nonpathogenic for the animal, but they may however impair sperm quality [5]. Specific bacterial pathogens in porcine semen include Brucella suis, and Leptospira spp., Mycobacterium spp., Chlamydia spp., and Mycoplasma spp. Unlike bacterial pathogens, there are numerous viral pathogens that can be found in porcine semen. The present article will review the most important viruses found in porcine semen used for Al, the impact they may have, diagnostic procedures, and control measures that can be applied to limit or prevent contamination of semen used for AI.2. 猪精液中的病毒Viruses in porcine semen猪精液中可检测出各类病毒。其中一些已列入世界动物卫生组织疫病名录（OIE 2015：表1和表2），因此，这些病原体对社会经济和/或公共卫生，以及国际动物和动物产品贸易有着重大影响。也就是说，会对被上述病原体污染的精液实行贸易限制。猪精液中其他未被列入OIE名录（表3和表4）的病毒，也可能给全世界或特定区域的养猪业带来疾病和重大经济损失。表1显示收入OIE名录中的病毒，以及采用不同诊断测试时，在精液中检测到此类病毒的时间，表3显示检测到非OIE名录病毒的时间。表2和表4分别显示这些病毒对精液质量的影响，以及人工授精后病毒传染对母猪的影响。Different viruses have been detected in porcine semen. Some of them are on the list of the World Organization for Animal Health (OIE 2015: Tables 1 and 2), and consequently, these pathogens are of socioeconomic and/ or public health importance within countries and are of major importance in the international trade of animals and animal products. This implies that major trade restrictions are in place for semen contaminated with pathogens in the OIE list. Other viruses found in porcine semen are not in the OIE list (Tables 3 and 4) but may also cause disease and major economic losses to the pig industry worldwide or in specific areas. The period during which viruses can be detected in the semen using different diagnostic tests is shown in Table 1 for viruses of the OIE list and in Table 3 for viruses not in the OlE list. The impact of these viruses on semen quality and transmission to the sow after AI is shown in Tables 2 and 4, respectively.因很难根据其重要程度对本文提到的病毒进行精确排名，故按其字母排序进行探讨。一些病毒对猪群影响不太大，如猪巨细胞病毒、牛病毒性腹泻病毒（BVDV）、边界病病毒、细环病毒和猪内源性反转录病毒（PERVs）等。最近发现的一些病毒影响程度未知，如雷斯顿埃博拉病毒[REBOV]，新型细小病毒（如4型猪细小病毒）。一些病毒仅在特定地区流行，如日本乙型脑炎病毒和风疹病毒。另外一些则在全球流行，如伪狂犬病毒（ADV）、猪瘟（CSF）病毒、口蹄疫（FMD）病毒以及蓝耳病毒（PRRSV）。The different viruses in the present article are discussed in alphabetical order as it is difficult to rank them precisely on the basis of importance. Some viruses such as porcine cytomegalovirus, bovine viral diarrhea virus (BVDV), border disease virus, torque teno virus, and pig endogenous retroviruses (PERVs) are not considered to be very important in pigs. The importance of some recently identified viruses (e.g., Reston ebolavirus [REBOV], novel parvoviruses such as porcine parvovirus type 4 [PPV4]) is not yet clearly established. Some viruses are only important in specific geographical areas, e.g., Japanese encephalitis virus and rubulavirus. The worldwide importance of other viruses such as Aujeszky's disease virus (ADV), classical swine fever (CSF) virus, foot-and-mouth disease (FMD) virus, and porcine reproductive and respiratory syndrome virus (PRRSV) is well known.表1：存在猪精液中且列入OIE名录（2015）的病毒类病原体；公猪感染类型及检测到病毒时间（试验用）。Table 1: Viral pathogens from the OIE list (2015) that have been found in porcine semen; boar infection type and timing of detection (test used).  缩写：DPI，接种后天数；PCR，聚合酶链反应；RT-PCR，反转录酶聚合酶链反应。Abbreviations: DPI, days post inoculation; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase polymerase chain reaction.表2：存在猪精液中且列入OIE名录（2015）的病毒类病原体：公猪生殖道病变，对精液质量的影响，以及人工授精（AI）后病毒传染对母猪的影响。Table 2: Viral pathogens from the OIE list (2015) that have been found in porcine semen: lesions in the genital tract of boars, impact on semen quality, and transmission to recipient sow on artificial insemination (AI).  缩写：NI，未调查研究。Abbreviation: NI, not investigated.2.1 存在猪精液中且列入OIE名录的病毒Viruses in porcine semen which are in the OIE list2.1.1. 非洲猪瘟病毒African swine fever virus非洲猪瘟病毒是一种有包膜DNA病毒，属于非洲猪瘟病毒科、非洲猪瘟病毒属。病毒侵入后，首先在周围淋巴结的单核细胞和巨噬细胞中复制，然后通过血液和/或淋巴系统扩散至全身。已从感染的公猪精液中分离出该病毒[7]。但尚未知其对精液质量和射精量的影响，且无有效治疗方法或疫苗。African swine fever virus is an enveloped DNA virus belonging to the family asfarviridae, genus Asfivirus. The virus replicates first in the monocytes and macrophages of the lymph nodes near the place of virus entrance and subsequently spreads through the blood and/or lymphatic system in the body. The virus has been isolated from semen after infection in boars [7]. Effect on semen quality and volume has not been reported. No treatment or effective vaccines are available.2.1.2. 伪狂犬病病毒Aujeszky’s disease virus (pseudorabies virus)伪狂犬病病毒是一种有包膜DNA病毒，属于疱疹病毒科中的α-疱疹病毒亚科、水痘病毒属。该病毒曾在全球范围内流行，但在欧洲、北美和新西兰部分地区的家猪中，已成功消除[48]。家猪中，病毒主要通过鼻-鼻传播，并在鼻粘膜和咽粘膜中复制。病毒也会在生殖道中复制，因此，在繁殖过程中，暴露于受感染的阴道粘膜或精液时，可导致病毒在猪之间传播[49]。野猪群中，伪狂犬病毒（ADV）主要通过性传播。Aujeszky’s disease virus is an enveloped DNA virus that belongs to the genus Varicellovirus in the subfamily alphaherpesvirinae of the family herpesviridae. The virus occurs worldwide but has been eradicated successfully from the domestic pig population in parts of Europe, North America, and New Zealand [48]. In domestic pigs, the virus is primarily transmitted by nose-to-nose contact, with viral replication in the nasal and pharyngeal mucosae. As viral replication also occurs in the genital tract, transmission among pigs can also occur during breeding from exposure to the contaminated vaginal mucosa or semen [49]. Within feral swine and wild boar populations, ADV appears to be preferentially transmitted by the venereal route.通过对睾丸进行实验感染，观察到睾丸白膜出现变性和坏死灶[50]。在鼻内、睾丸内和包皮内建立感染，发现公猪精子出现短暂性异常[50,51]。包皮自然感染[9]，以及实验感染[8]后，尿液、包皮膜或精液中不定期分离出病毒。After experimental infection by the intratesticular route, testicular degeneration and necrotic foci in the tunicae albugineae of the testicles were observed [50]. Transient elevations of sperm abnormalities were found in boars that had been infected by the intranasal, intratesticular, and intrapreputial routes [50,51]. The virus was irregularly isolated from urine, preputial membranes, or semen after both natural [8] and experimental infection through the prepuce [9].研究发现精液中病毒浓度很高，半数组织培养感染剂量（TCID50）/ mL可达103.7至109。通常在疾病的急性期，散毒可长达12天。不过，散毒并不直接影响临床疾病或降低精液质量，但公猪复发感染伪狂犬病毒（ADV）的潜伏期会造成长期危害。通常，临床上感染的公猪无法爬跨母猪。使用带毒精液输精则可能会引起母猪阴道炎或子宫内膜炎，进而导致胚胎死亡[52]。Very high viral concentrations ranging from 103.7 to 109 50% tissue culture infective dose (TCID50)/mL have been reported in semen. Transient virus excretion for up to 12 days usually takes place during the acute phase of the disease. However, virus excretion is not strictly associated with clinical disease or with a reduction in semen quality, and recrudescence of boars latently infected with ADV poses a permanent risk. Clinically infected boars are often unable to mount a dummy. Sows inseminated with contaminated semen may develop vaginitis or endometritis resulting in embryonic mortality [52].表3：存在猪精液中，未列入OIE名录（2015）的病毒类病原体：公猪感染类型及检测到病毒时间（试验用）。Table 3: Viral pathogens not on the OIE list (2015) that have been found in porcine semen: boar infection type and timing of detection (test used).  缩写：DPI，接种后天数；PCR，聚合酶链反应。Abbreviations: DPI, days post inoculation; PCR, polymerase chain reaction.未完待续...To be continued…