猪精液-病毒传播的载体（三）

猪精液-病毒传播的载体（三）Porcine semen as a vector for transmission of viral pathogens-Part 3Dominiek 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接上文...2.2 存在猪精液中，未列入OIE名录的病毒Viruses in porcine semen which are not in the OIE list2.2.4. 非典型猪瘟病毒Nonclassical swine fever pestiviruses猪对非典型猪瘟病毒易感，此类病毒包括牛病毒性腹泻病毒（BVDV）和边界病病毒；这些RNA病毒会导致牛羊罹患相关疾病。先天感染这些病毒的猪可能会大量散毒。Terpstra和Wenswoort [35]从先天感染及不育公猪的口咽液、尿液和精液中分离出BVDV。Pigs are also susceptible to non-CSF pestiviruses including BVDV and border disease virus; these are RNA viruses which are associated with disease in cattle and sheep, respectively. Pigs congenitally infected with these viruses may shed large amounts of virus. Terpstra and Wenswoort [35] isolated BVDV from oropharyngeal fluid, urine, and semen of a congenitally infected and infertile boar.Bungowannah病毒与猪瘟（CSF）病毒的基因不同，但是由于其分子特性，可将其纳入瘟病毒属。2003年，该病毒导致澳大利亚两个猪场爆发严重疾病，主要是死胎、木乃伊胎增多、断奶前死亡率升高[72]。尚未明确该病毒通过胚胎或精液传播的风险。Bungowannah virus is genetically distinct from CSF virus, but molecular characterization supports its inclusion in the genus Pestivirus. The virus caused a severe disease outbreak characterized by an increase in stillbirths, preweaning mortality, and mummified fetuses in 2003 in two pig herds in Australia [72]. The risk of transmission by embryos or semen has not yet been evaluated.2.2.5. 猪肠道小核糖核酸病毒Porcine enteric picornaviruses猪肠病毒(PEV)和猪捷申病毒均为无包膜RNA病毒，属于微小核糖核酸病毒科。这两类病毒在环境中存活力很强。病毒在猪之间的传染，主要是通过口腔接触受污染的粪便。精液有可能在采精过程中受到污染。公猪感染PEV可能导致精囊炎、精子异常、性欲下降[32]。除了妊娠期间发生感染，通常观察不到母猪表现出临床症状。若妊娠期间发生感染，可能会导致胚胎死亡、木乃伊胎、死胎和弱仔[73]。Porcine enterovirus (PEV) and porcine teschovirus are nonenveloped RNA viruses belonging to the family of Picornaviridae. They are highly resistant in the environment. Infections are transmitted between pigs mainly by oral exposure to contaminated feces. Contamination of semen during collection is possible. Infections with PEV in boars may lead to seminal vesiculitis, sperm abnormalities, and a decreased libido [32]. In the sow, usually no clinical signs are observed, except if infections occur during pregnancy. In the latter case, infections may cause fetal death and mummification, stillbirth, and weak-born piglets [73].已从公猪生殖道中分离出猪捷申病毒[32]，使用带毒精液对后备母猪授精，不会影响受精[74]。但Dunne等[44]提出，被PEV和猪捷申病毒感染的精液会导致胚胎和新生仔猪死亡。尚无后续研究证实这一发现。Porcine teschovirus has also been isolated from the male genital tract [32], but insemination of gilts with contaminated semen did not affect fertility [74]. Dunne et al. [44] however suggested that semen contaminated with PEV and porcine teschovirus could cause embryonic and neonatal death. There are no recent reports that confirmed this finding.2.2.6. 细环病毒Torque teno virus已经在猪身上发现两种细环病毒（TTV）：细环病毒1型（TTsuV1）及2型（TTsuV2）。 均为DNA病毒，属于新创的指环病毒科、细环病毒属。据估计，猪细环病毒（TTsuVs）在全球猪群中普遍存在 [75]。目前，感染猪细环病毒（TTsuV）无相关临床症状出现。但TTsuV与猪圆环病毒（PCV）相关疾病有关[76,77]。细环病毒呈垂直和水平传播。可在胎儿组织和血液、精液和初乳中检测到该病毒[36]，由此证明其存在垂直传播的可能。从人工授精中心收集100头公猪的独立精液样本，用常规PCR检测TTsuV1和TTsuV2[37]。其中四份样本呈TTsuV1 PCR阳性，五份呈TTsuV2阳性，合并感染后，两种TTsuVs均被检测到。胎儿感染的途径尚不明确，但相关显示证明可能是由于精液带毒或者胎盘传播。Two species of TTV have been described in pigs: torque teno sus virus types 1 (TTsuV1) and 2 (TTsuV2). They are DNA viruses that belong to the newly created family anelloviridae, genus Iotatorquevirus. It is considered that TTsuVs occur in pig populations worldwide [75]. At present, no clinical signs are associated with TTsuV infection. However, TTsuVs have been linked to PCV-associated disease [76,77]. Torque teno sus viruses are apparently transmitted both vertically and horizontally. They can be detected in fetal tissues and blood, semen, and colostrum [36], indicating the potential for vertical transmission. Single semen samples from 100 boars from an AI center were analyzed by conventional PCR for TTsuV1 and TTsuV2 [37]. Four samples were PCR positive for TTsuV1 and five for TTsuV2, whereas in a single coinfection, both TTsuVs were detected. The route of fetal infection is uncertain but may reflect virus-contaminated semen or transplacental transmission.2.2 存在猪精液中，未列入OIE名录的病毒Viruses in porcine semen which are not in the OIE list2.2.7. 埃博拉病毒Ebolavirus雷斯顿埃博拉病毒是一种有包膜RNA病毒，属于单分子负链RNA病毒目、丝状病毒科。 2008年，菲律宾猪群中爆发严重疾病，据报导疑似由蓝耳病毒（PRRSV）引起。但是，经详细的诊断后发现，并非PRRSV而是雷斯顿埃博拉病毒（REBOV）引起该病，而且在猪场工人身上检测到REBOV特异性抗体[30]。此次REBOV疫情爆发的深远影响尚待确定。随后的实验表明，尽管感染REBOV后，病毒在猪体内复制，但可能不表现症状。猪群间REBOV的传播机制尚不明确。也不清楚REBOV的散播途径。在非人灵长类动物模型中，REBOV会在包括精液在内的所有体液中排散[29]。尚不清楚REBOV是否可通过精液传染给母猪。与REBOV相比，已证实扎伊尔埃博拉病毒可导致5至6周龄猪产生严重肺部疾病，引起呼吸困难[42]。Reston ebolavirus is an enveloped RNA virus, classified in the order mononegavirales, family filoviridae. In 2008, severe disease outbreaks suspected to be caused by PRRSV were reported in swine herds in the Philippines. However, detailed diagnostic work revealed that not PRRSV but REBOV had caused the disease and that REBOV-specific antibodies were found in farm workers [30]. The broader significance of this outbreak of REBOV remains to be determined. Subsequent experimental challenge suggested that REBOV infection in pigs may be asymptomatic despite replication of the virus. The mechanism of transmission of REBOV to pigs or between pigs is unknown. Routes of REBOV shedding by pigs are also not known. In nonhuman primate models, REBOV is shed in all body fluids, including semen [29]. Whether REBOV is shed by boars in semen or transmitted to recipient females is unknown. In contrast with REBOV, Zaire ebolavirus has been shown to cause severe lung pathology in 5- to 6-week old pigs leading to respiratory distress [42].   2.2.8. 猪内源性反转录病毒Pig endogenous retroviruses猪内源性反转录病毒是一种有包膜RNA病毒，属于反转录病毒科、丙型反转录病毒属。高达8%的哺乳动物基因组DNA来源于反转录病毒[78]。在猪身上，已经发现了三种反转录病毒（PERV）亚型：PERV-A，PERV-B和PERV-C。PERV-A和PERV-B普遍存在于猪群中，而PERV-C在种间、种内分布程度各异 [38]。Pig endogenous retroviruses are enveloped RNA viruses of the genus Gammaretrovirus in the family retroviridae. Up to 8% of mammalian genomic DNA is believed to be retroviral in origin [78]. In pigs, three PERV subtypes have been identified: PERV-A, PERV-B, and PERV-C. Subtypes A and B are ubiquitous in all pig breeds, whereas PERV-C is variably distributed between and within breeds [38].尚未在猪中发现呈外源性水平传播的反转录病毒。然而，已经发现PERV-A和PERV-C之间的重组。PERV A/C重组体不以原病毒形式存在于宿主种系中，因此被认为是潜在外源病毒[79]。一些猪的体细胞中存在完整的A/C原病毒基因组，由此表明其可进行复制。尚未完全了解PERVs在猪之间传播的可能。然而，由于PERVs被嵌入基因组中，因此表明它们是通过精液传播的。Exogenous horizontally transmissible retroviruses have not been found in pigs. However, a recombination between PERV-A and PERV-C has been found. The PERV A/C recombinant is not present in proviral form within the host germ line and is therefore considered a candidate exogenous virus [79]. The presence of the full genomic A/C provirus in the somatic cells of some pigs indicates its potential for replication. Little is known regarding the potential for transmission of PERVs between pigs. However, as PERVs are embedded in the genome, they are transmitted via semen.2.2.9. 风疹病毒（蓝眼病）Rubulavirus (blue eye disease)猪风疹病毒是一种有包膜RNA病毒，属于副黏病毒科、腮腺炎病毒属。该病毒可导致蓝眼病[80]。此病毒流行于墨西哥，会造成种猪繁殖障碍。感染后，与其他成年动物一样，公猪会罹患严重附睾炎和睾丸炎，性欲减退，通常不表现临床症状。尚未通过实验证明该病毒通过精液传播；但是，在接种后长达49天之内，已经从精液、睾丸和生殖道的其他组织中检测到该病毒[39]。近期数据表明，在接种后5至48天可从精液中分离出该病毒，接种后64至142天可从睾丸和附睾中分离出该病毒。接种后2到64天，血清中可检测到病毒RNA，接种后142 天，仍可在精液中检测到此病毒[40]。由此证明，猪风疹病毒RNA持续存在于精液中，且长时间感染生殖道。精液检查显示，感染病毒的猪群中，约30%的公猪出现暂时性或永久性不育、精子浓度下降、形态异常增加、精子的活力和存活能力降低。一些公猪出现无精症[40]。Porcine rubulavirus is an enveloped RNA virus that belongs to the genus Rubulavirus in the family paramyxoviridae. The virus is associated with blue eye disease in pigs [80]. It is an important pathogen in Mexico and causes reproductive problems in breeding pigs. Boars, like other adult animals, generally do not show clinical signs, except for epididymitis and orchitis and in severe cases, loss of libido. Transmission of the virus through semen has not been proven experimentally; however, virus has been recovered from semen, the testis, and other tissues of the reproductive tract for up to 49 days after inoculation [39]. More recently, isolation of the virus from semen was achieved between 5 and 48 days post inoculation (DPI) and from the testicles and epididymides between 64 and 142 DPI. Viral RNA was detected in the serum between 2 and 64 DPI and in the semen until 142 DPI [40]. These results confirm that the RNA of the porcine rubulavirus persists in the semen and that this virus remains in the reproductive tract for prolonged periods of infection. Semen evaluation demonstrated that about 30% of boars in herds infected with the virus showed temporary or permanent infertility, with a decrease in concentration, an increase in abnormalities in morphology, and a decrease in motility and viability of spermatozoa. In some boars, there was azoospermia [40].3.人工授精将病毒传染给母猪的风险Risk for transmission of viruses by AI to the recipient sow通常，在发病时期，精液中存在病毒的可能性最高。但是，现实情况中，不会从临床受影响的公猪中采精，这样可以降低病毒传染母猪的风险。无临床症状、出现轻微临床症状之前，散毒就已经开始，加之严重感染猪只未被发现，在这些情况下，如果不采取特别控制措施，会增加病毒传播风险。In general, the risk of virus to be present in semen is the highest during the stage of clinical disease. Under practical circumstances, however, no sperm collection will take place from clinically affected boars, and consequently, the risk of pathogen transmission to the sow is low in this case. Although virus shedding can start before the development of clinical signs, which can be mild or absent, acutely infected boars can remain unnoticed. In these situations, the risk of virus transmission is much higher as no special control measures will be taken.实验证明，CSFV [10] PRRSV [61]可通过精液传染母猪，在精液中加入PPV [45]、ADV [52]、PRRSV[18,21]和PCV2[41]后，亦可。Habu等 [13]，Guérin和Pozzi [57]提出，虽然没有找到科学的文章证实，但是如果使用含有日本乙型脑炎病毒的精液进行授精，则后备母猪极易被感染。若将被病毒污染的精液应用于人工授精，母猪乃至母猪群都会有病毒传播的风险。Nathues等[81]以瑞士引入公猪精液导致PRRSV爆发为例，对此加以说明。Transmission of viral pathogens by semen to the sow has been clearly proven for CSF virus [10] and PRRSV [61] on experimental infection of boars and for PPV [45], ADV [52], PRRSV [18,21], and PCV2 [41] after inoculation of the virus in the semen. Habu et al. [13] and Guérin and Pozzi [57] reported that Japanese encephalitis virus is easily transmitted to gilts if they are inseminated with contaminated semen, although we did not find a scientific article confirming these statements. Semen contaminated with viral pathogens used for AI does indeed poses a risk for transmission to the recipient sow and by extension the sow herd. This was illustrated by Nathues et al. [81] who described an outbreak of PRRSV in Switzerland after import of boar semen.但是，病毒并不总是能够形成传播（例如PRRSV）[18,61]。母猪被感染所需的条件很复杂，未能传播可能是由于母猪免疫、病毒特点以及未达到最小感染剂量。因此，针对精液传播PRRSV的风险，以及母猪感染病毒所需的最小剂量，开展了更多的研究。那么，与常规宫颈输精相比，小剂量精液的深部输精（40–50 mL对比80–90 mL），可降低病毒传染母猪的风险[82]。However, transmission will not always be successful (e.g., PRRSV) [18,61]. The conditions required for establishment of infection in the sow are complex, and lack of transmission might be explained by sow immunity, virus characteristics, and failure to reach the minimum infectious dose. In this regard, much research has been directed toward the risk of transmission of PRRSV by semen and the minimum dose necessary to establish infection in the sow. In this regard, it can be expected that postcervical insemination with smaller semen doses than conventional cervical insemination (40–50 mL compared to 80–90 mL) is associated with a lower risk for pathogen transmission to the sow [82].就群体而言，使用带毒精液进行配种的母猪数量，也决定了形成传播的可能性。此类母猪数量越多，形成病毒传播的可能性越高。其他动物，如牛，人工授精时主要使用冻精，而非鲜精。这样，就有更多的时间和机会，检测公牛和/或精液是否含有特定病原，进而降低通过人工授精传播病原的风险。At a population level, also the number of sows inseminated with contaminated semen determines the likelihood of successful transmission. The more sows are inseminated with contaminated semen, the higher the likelihood of successful transmission of the virus. In other animal species such as cattle, AI is mostly practiced with frozen semen and not with fresh semen as in pigs. This allows more time and opportunities to verify whether the bulls and/or semen are free from specific pathogens, and consequently, the risk for pathogen transmission via AI is lower.4.病毒通过人工授精对母猪的影响Impact of viruses by AI to the recipient sow使用带毒精液对母猪进行人工授精，会出现多种结果。精子质量下降、胚胎早期死亡和/或子宫内膜炎、母猪群中的临床疾病，和/或感染有害病原导致母猪健康水平下降、阻碍病原净化或干扰监管措施，这些均可能导致受胎率降低[57] 。病原体可穿过透明带直接侵入胚胎，和/或病原体（例如，感染PPV）引起子宫上皮病变，造成胚胎早期死亡[83]。妊娠后的6至7天，透明带作为不可渗透屏障，围绕并保护胚胎，有助于其避免受诸如PPV，PCV2，ADV和PRRSV等病原体的入侵[65,84,85]。但是，病原体（如ADV）在囊胚期可穿过透明带，导致胚胎更容易感染[84]。The consequences of AI with semen contaminated with viral pathogens for the sow can be quite variable. It can result in reduced conception rates because of reduced semen quality, early embryonic death and/or endometritis, clinical disease in the sow herds, and/or infections with unwanted pathogens leading to reduced health status, stamping out, or regulatory measures [57]. Early embryonic death may result from direct invasion of the embryo by the pathogen after it has hatched from the zona pellucida, and/or by uterine epithelial alterations in response to the pathogen (e.g., with PPV) [83]. Until 6 to 7 days after conception, embryos are surrounded and protected by the zona pellucida, an impervious barrier, which helps the embryo to avoid invasion of pathogens such as PPV, PCV2, ADV, and PRRSV [65,84,85]. After hatching, however, blastocyst stage embryos may become susceptible to the infection as is the case, e.g., with ADV [84].5.诊断  Diagnostics可以采用不同的诊断方法，如病毒活性检测、病毒核酸检测或病毒抗体检测，来判断人工授精中心的公猪是否感染病毒和/或精液中是否带毒。九十年代初期至今，在提高敏感性和特异性，以及同时检测不同病原和检测速度方面取得了重大进步。The presence of viral infections in boars of AI centers and/or the presence of viral pathogens in semen can be assessed using different diagnostic methods such as demonstration of viable virus, nucleic acid of the virus, or antibodies against the virus. From the early 1990s onward, major improvements have been made in terms of increased sensitivity and specificity, simultaneous testing of different pathogens and speed of testing.5.1. 病毒活性检测Detection of viable virus通常，公猪精液采集后几天内，即会被稀释使用，应在短时间内拿到检测结果。检测精液中病毒的常规方法，如病毒分离鉴定或生物鉴定，灵敏度不高，耗时且昂贵。由于细胞毒性、细菌污染，以及会干扰细胞培养系统、抗病毒因子会非特异性中和病毒，现已很少在精液内使用病毒分离技术[59]。基于现实条件和动物福利限制，无法对大量动物进行样本接种检测[64]。限于以上因素，病毒分离和猪生物鉴定于常规临床诊断而言并不实用。As diluted boar semen is mostly used within a few days after collection, the outcome of analysis should be available within a very short time. Conventional methods for virus detection in semen, such as virus isolation or conducting bioassays, are not very sensitive; they are time-consuming and very expensive. The application of the virus isolation technique is markedly reduced for semen because of cell toxicity, bacterial contamination, resulting in interference with cell culture systems, and antiviral factors that nonspecifically neutralize the virus [59]. Animal inoculations cannot be performed for testing large numbers of samples because of practical reasons and also because of animal welfare reasons [64]. Given these limitations, virus isolation and swine bioassays are not useful techniques for routine diagnosis.5.2. 病毒核酸检测Detection of viral nucleic acidPCR是一种灵敏度高、特异性强，能够快速检测基因组序列（如，病毒）的检测技术。已经有多种PCR技术可用于检测公猪精液中存在的病毒。Van Rijn等[86]研发出的实时荧光定量PCR，可以对精液中5种能够严重影响经济效益的病毒（ADV，CSFV，FMDV，SVDV，PRRSV）进行检测。实时PCR技术是将扩增和扩增产物的检测合二为一。从而来自环境中的污染可能大大降低。通过实验发现，相比病毒分离，采用实时PCR检测带毒精液，可以在感染后更早、更快发现病毒。 Rovira等[87]研究了反转录酶PCR针对不同生物样品的灵敏度，这些样品分别为3个样品为一池和5个样品为一池。用低毒PRRSV分离株接种了29头公猪。2周内，每2到3天从每头公猪获取血清、血液拭子和精液样本。得出的数据显示，急性感染期间，血清是检测PRRSV的最佳样品，血液拭子亦可。但是大多数精液样本中，未能检测到PRRSV感染。以3个和5个样品为一池会造成反转录酶PCR的灵敏度下降。当分别以5个样品为一池检测血清或血液拭子，其灵敏度分别降低6％和8％。The PCR technique is known as a sensitive, specific, and rapid tool for the detection of genomic sequences of, e.g., viruses. Different PCR techniques have become available for detecting viral pathogens present in boar semen. Van Rijn et al. [86] developed quantitative real-time PCR tests for detecting five economically important viruses in semen (ADV, CSFV, FMDV, SVDV, PRRSV). The real-time PCR technique combines amplification and detection of amplified products in one closed tube. Therefore, possible contamination from the environment is strongly reduced. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by real-time PCR tests than by virus isolation. Rovira et al. [87] investigated the sensitivity of reverse-transcriptase PCR on different biological samples run individually, in pools of 3 and in pools of 5. Twenty-nine boars were inoculated with a low-virulent PRRSV isolate. Serum, blood swab, and semen samples were obtained from each boar every 2–3 days for 2 weeks. Data showed that serum was the best sample to detect PRRSV during acute infection, with the blood swab sample performing almost as well. Semen samples failed to detect PRRSV infection in most of the cases. Pooling samples at pool sizes of 3 and 5 resulted in a decrease in the sensitivity of reverse-transcriptase PCR. Sensitivity was reduced by 6% and 8%, respectively, when serum or blood swab samples were run in pools of 5.5.3. 抗体检测Detection of antibodies 常用血清学方法来调查公猪站中是否存在针对不同病原体的血清抗体。这是一种简单且经济的检测方法。检测结果适用于解释群体或公猪站内的健康水平，而非单个猪只。但是，此种检测方法重要的缺点是，病原体暴露与检测到抗体水平之间的时间间隔。许多血清检测间隔一般为一到几周，这意味着在此期间，未被确定感染的公猪，也可能散毒。成年公猪的颈静脉穿刺，对操作者的安全而言，有一定的局限性。因此，血液拭子更常用。为了采集血液拭子，在收集精液时用针刺其耳朵，并使用棉签收集血滴。检测PRRSV时，与精液样本相比，血液拭子中病毒量更多，可更快检出PRRSV[88]。Serology is frequently used to investigate the presence or absence of serum antibodies against different pathogens in boar studs. It is an easy and rather inexpensive method that is suitable for monitoring. The results should be interpreted at group or stud level, not at the individual animal level. An important disadvantage is the time period between pathogen exposure and detectable levels of antibodies. For many serologic tests, this time period ranges from one to several weeks, which implies that during that period, boars may shed the virus although they are not identified as being infected. Puncture of the jugular vein in adult boars has some practical limitations in terms of safety of the handler. Therefore, blood swabs have become popular. For blood swab collection, the ear is pricked with a needle during semen collection and a cotton swab is used to absorb the blood droplet. For PRRSV, increased amounts of PRRSV were shown in the blood swab as compared to semen samples, and infections could be detected earlier [88].5.4. 临床诊断的说明Interpretation of diagnostic testing显然，无法直接将精液归类为无病毒或带毒精液。许多病毒，如PRRSV，ADV，PCV2，在病毒血、精液中病毒的存在和血清中的抗体之间存在时间不一致性。就PRRSV而言，如成年公猪的病毒血症持续时间很短，且病症的结束早于精液散毒[59]。此外，在感染的初期，尽管精液已在散毒，但血清学结果仍为阴性。最后，当精液停止散毒后很长一段时间，公猪仍可能为血清阳性。因为精液通常是间断散毒，尤其是在感染后期，所以即便检测精液样品阴性，病毒随后可能还会继续排散。阴性结果仅表示所检测的精液样品不包含病毒，且仅指单次射精精液中可能不含病毒。并不能保证公猪随后射出的精液不带毒。因此，对待血清阳性，但精液检测阴性的公猪应更加谨慎。It is clear that categorizing semen as either virus free or virus contaminated is not straightforward. For many viruses, e.g., PRRSV, ADV, PCV2, temporal inconsistencies exist among viremia, the presence of virus in semen, and antibodies in serum. In the case of PRRSV, e.g., viremia in adult boars may be of short duration and end before virus shedding in semen ends [59]. Furthermore, in the initial phases of the infection, serologic results will be negative although the virus can be shed in the semen. Finally, boars may remain serologically positive long after the virus is no longer shed in the semen. Because shedding of virus in semen is often intermittent, especially in the later phases of the infection, a negative test result on a semen sample does not preclude subsequent shedding of the virus. A negative result only means that the tested sample of the ejaculate does not contain virus and that the particular ejaculate is likely to be virus free. It does not guarantee absence of contamination in subsequent ejaculates of a boar. Consequently, a negative semen test from a serologically positive boar should be interpreted with caution.未完待续...To be continued…