猪精液-病毒流传的载体（四）

猪精液-病毒流传的载体（四）

　　Porcine semen as a vector for transmission of viral pathogens-Part 4

　　Dominiek Maesa,*, Ann Van Sooma, Ruth Appeltanta, Ioannis Arsenakisa, Hans Nauwynckb

　　a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, Belgium

　　b比利时，梅勒尔贝克，根特大学，兽医学院，病毒学尝试室，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 

　　要害词 Keywords：

　　猪精液、猪、人工授精、病毒、回首/检查

　　Semen, Pig, Artificial insemination, Virus, Review

　　接上文...

　　6. 防范法子

　　Preventive measures

　　人工授精中心需要回收民生、有效且满足生物安详的办法，出产出适量品质可控的精液。防备疾病通过精液流传的最佳办法是，购置健康状况好的公猪，执行严格的生物安详法子，并对公猪进行健康监测。

　　Artificial insemination centers need to produce the appropriate quantity of quality-controlled semen in a cost-effective, efficient, and biosecure process. The best way to prevent disease transmission via semen is to purchase boars with a preferred health status, to maintain very strict biosecurity measures, and to monitor the health of the animals.

　　6.1. 新进公猪的健康状况

　　Health status of incoming replacement boars

　　人工授精中心每年凡是要改换60％的公猪，但是新进公猪会带来很大的疾病风险，因此对新进公猪实行断绝、驯化、充实的调查和检测，至关重要。首先，应把握公猪来源猪群的健康状况。其健康状况应等同于，但最优良高于人工授精中心的公猪。可通过兽医之间的相同，了解新进公猪的临床诊断与尝试室检查、疫苗接种打算以及来源猪群的兽医法子，通过这些来对其进行评估。其次，新进公猪应单独断绝30至60天。断绝区最优良位于场外。猪群断绝必需遵守“全进全出”原则。断绝期间，应评估猪群健康状况，进行血液采样以检测是否有病毒血症或血清抗体，同时，对人工授精中心的公猪中呈现的疾病进行疫苗接种治疗。传染危险病原体的公猪不得进入人工授精中心。差异的人工授精中心，断绝期、检测方案和疫苗接种方案均不沟通，主要是由于差异的因素，如引入公猪的频率和原畜群动物的健康状况所决定[88]。

　　Because new boar entry poses a great risk for disease, and because typically 60% of the boars in AI centers are replaced on a yearly basis, it is of paramount importance for incoming boars to implement proper isolation and acclimation procedures, with adequate time for observation and testing. First, the health status of the source herd should be known. The health status should be similar but preferably higher than the health status of the AI center. It could be evaluated through vet-to-vet communications of the diagnostics and laboratory examinations, vaccination programs, and veterinary involvement of the source herd. Second, replacement boars should be housed for 30 to 60 days in a separate isolation facility. Such a quarantine unit should be located preferably off-site. Isolation populations must always be flowed all-in/all-out. During this quarantine period, the health status should be evaluated, blood sampling could be performed to detect possible viremia or the presence of serum antibodies, and vaccinations can be applied against diseases present in resident boars of the AI center. Animals that are infected with pathogens of concern should not enter the AI center. The isolation period, the testing protocols, and the vaccination schemes vary between AI centers and depend on different factors such as the frequency of boar introduction and the health status of the animals of the origin herds [88].

　　6. 防范法子

　　Preventive measures

　　6.1. 生物安详法子

　　Biosecurity measures

　　人工授精中心最重要的生物安详法子涉及以下文章[3,88]：（1）人工授精中心的位置及通风设备--应尽可能远离猪群。在猪只密集区域使用空气过滤系统，以担保人工授精中心制止病原通过空气传入[89]。确保通风设备不会将公猪站内的空气排向尝试室。如果尝试室紧挨猪舍，则应回收正压通风，从而最洪流平地减少从猪舍到尝试室的空气渗透。（2）对付进入人工授精中心的员工和来访者，应有严格划定。员工需要断绝1至3天。断绝期间，不答允与猪只接触。别的，建议对所有来访者和员工施行人员进出淋浴法子。（3）精液拿取所在及递送——精液递送人员拿取精液的所在，应位于人工授精中心的外面。精液拿取点应是独立的房间，或至少是独立于尝试室和公猪站的房间。把精液拿取点单独分隔，，仅供最洪流平上制止精液冷却器、靴子、衣物等的交叉污染。每个母猪舍的精液安排点应位于办公室外。任何送入人工授精中心的物品（例如，物资、设备），在这之前都不该该接触过猪。应在送入人工授精中心之前，将物品放入指定房间消毒。（4）灭鼠及昆虫 - 老鼠及昆虫可携带及流传多种猪病毒，因此应采纳适当防范法子。（5）水质 – 应按期阐明公猪的水源。如果质量不及格，应改换水源或对水进行适当处置惩罚。（6）执行严格的清洁消毒措施。

　　The most important biosecurity measures for AI centers relate to the following items [3,88]: (1) location of the AI center and ventilation -- the AI center should be located as far from pigs as feasible. Filter systems for incoming air could be used in pig-dense areas to safeguard AI centers against entry of airborne pathogens [89]. The facilities should be oriented so that exhaust air from the boars does not exhaust toward the laboratory. If the laboratory is connected to the animal facility, there should be positive pressure ventilation to minimize air infiltration from the animals into the laboratory. (2) Employees and visitors -- strict regulations should be in place for persons entering AI centers. A downtime of 1 to 3 days may be required for persons entering the AI center. During this downtime period, persons are not allowed to have contact with pigs. In addition, it is recommended to have a shower-in/shower-out policy for all visitors and employees. (3) Semen pickup point and deliveries -- the semen pickup point for the courier delivery personnel should be located at the outside of the AI center. This pickup point should be in a separate room, or as a minimum, a separate room from both the laboratory and the stud population. By having a separate pickup point, cross-contamination of semen coolers, boots, clothing, and so forth is minimized. Drop-off points at each of the sow units should be located outside of the office. Any deliveries (e.g., supplies, equipment) on the AI center should not have been exposed to pigs previously. They should be delivered directly to a designated receiving chamber and properly disinfected before entering the AI center. (4) Rodent and insect control - rodents and insects are capable of carrying and transmitting several pig viruses, and therefore, proper prevention measures should be in place. (5) Water quality - water sources for boars should be analyzed regularly. If the quality is insufficient, the water source should be changed or the water should be treated properly. (6) Implementation of strict cleaning and disinfection procedures.

　　6. 防范法子

　　Preventive measures

　　6.3 公猪健康监测及疫苗接种

　　Animal health monitoring and vaccination

　　最普遍的办法是监测公猪的健康状况及临床表示。如果呈现临床问题，在动物康复之前不得收集精液。用自动化的办法丈量公猪的采食量和饮水量，从而更容易地在早期发觉抱病的公猪。每天有针对性的，对公猪的健康状况及表示进行多次监测，但这仍是一种很是根本的办法，并不能够独立监测病毒传染和/或精液中病毒的存在。因此，建议制定针对特定疾病的免疫、除虫及取样的尺度方案，并按期进行回首检查。公猪的免疫接种仅供很是有效地清除病毒或减少散毒，并降低人工授精流传病毒的风险。首先，人工授精中心需要遵守该国的动物卫生规矩，例如，强制性地接种ADV疫苗，因为是官方消灭病毒打算的一部门（例如东欧），而在其他已正式清除ADV的国度，可能禁止ADV的免疫接种（例如西欧，北美）。高效重组标志疫苗和多种精确的酶联免疫吸附尝试（ELISA）的结合，已使活着界上大范畴内（例如很多欧洲国度和北美）清除ADV成为现实可能[48]。《欧洲药典》要求，公猪接种打针用ADV疫苗后，其精液不能使用[90]。其他无法令规矩要求的疾病，人工授精中心可自行决定使用哪种疫苗。接种PPV和PCV2疫苗，可能有助于减少传染后散毒[91]。就PRRSV而言，公猪接种弱毒疫苗后50天内，体内野毒的散毒能力可被降低或消除 [92,93]。不行忽视的事实是弱毒疫苗只能提供部门免疫掩护，导致疫苗病毒在精液中的散毒长达39天。[17,92]。对比而言，灭活疫苗不能明显减少精液中野毒的排散[92]。

　　The most general method is to monitor the health of the boars and the presence of clinical signs. In case of clinical problems, no semen should be collected until the animal has recovered. Feed and drinking water intake of the boars may be measured in an automated way, allowing to identify diseased boars more easily and at an early stage. Monitoring the health and performance of the boars, even if applied accurately and more than once a day, remains a very basic approach that is insufficient as a stand-alone measure to detect viral infections and/or the presence of viral pathogens in semen. Therefore, it is recommended to develop standard protocols for vaccinations, deworming, and sampling for specific diseases and to review these on a regular basis. Vaccination of boars can be highly effective in eliminating or decreasing shedding of viruses and decreasing the risk of virus transmission by AI. First of all, AI centers need to adhere to the animal health regulations of the country, e.g., vaccination against ADV may be mandatory because it is part of an official eradication program of the virus (e.g., Eastern Europe), whereas in other countries, vaccination may be forbidden because the country is officially free of ADV (e.g., Western Europe, North America). The combination of highly efficacious marker vaccines and accurate differential ELISAs has made eradication of ADV from large areas of the world practical and feasible (e.g., many European countries, North America) [48]. According to requirements of the European Pharmacopoeia, ADV vaccines for parenteral use must not be transmitted by semen [90]. For other diseases, no legal regulations are in place and the AI center can decide which vaccines to use. Vaccination against PPV and PCV2 may help to reduce shedding of the virus after infection [91]. In the case of PRRSV, the use of an attenuated vaccine shortened or eliminated virus shedding in boars challenged with wild-type virus for 50 days after vaccination [92,93]. Apart from the fact that only partial protection is conferred against infection, vaccination with an attenuated vaccine led to semen shedding of the vaccine virus for up to 39 days [17,92]. In contrast, an inactivated vaccine did not clearly reduce subsequent shedding of wild-type virus in semen [92].

　　因为需尽早发觉可能存在的病毒（如PRRSV）传染，健康监测对人工授精至关重要。 一般环境下，需要检测血液和精液样品中是否存在病毒和/或抗体。差异人工授精中心的检测措施相差很大。因为有时检测血液和精液会呈现差异的功效，且存在不确定因素，如最佳检测频率、样本数量、诊断办法以及检测功效的解读，虽然检测措施很庞大[87]。

　　Health monitoring programs are critically important in AI centers, as possible infection with pathogens (e.g., PRRSV) should be detected as soon as possible. Mostly, both blood and semen samples are tested for the presence of virus and/or antibodies. Monitoring programs can very between AI centers. They can be complex because blood and semen tests can sometimes generate different information, and uncertainties exist regarding optimal testing frequency, numbers of animals to test, diagnostic test to choose, and how to interpret the test results [87].

　　6. 防范法子

　　Preventive measures

　　6.4 无病毒精液收集技术

　　Technologies to obtain virus-free semen

　　对精液进行处置惩罚并添加抗菌剂可稀释精液，且减少细菌污染，但这些法子无法清除病毒[4,5]。目前，尚无有效的抗病毒剂仅供担保精液无病毒。精液中存在很多卵白质，以及类固醇激素、酶以及其他物质，可在必然水平上防备病毒传染，但是，这些物质同时也可能损害精子。因此，需要保持微妙平衡[94]。

　　Semen processing and addition of antimicrobials may have a possible dilution effect and decrease bacterial contamination, but these measures do not eliminate viruses [4,5]. Effective antiviral agents to render semen virus-free are currently not (yet) used in practice. Many different proteins found in semen along with steroid hormones, enzymes, and other substances may provide some protection against viral infections, but at the same time, these substances could damage sperm. Therefore, a delicate balance of these substances needs to be maintained [94].

　　猪精液胶体离心至少有20年历史，从早期的密度梯度离心，到近期成长的单层离心（SLC）[95]。与密度梯度离心对比，后者越发便捷。使用单层离心技术可消除病原（细菌和病毒），进而提高精液的生物安详性，以及减少抗菌剂的使用。 在Blomqvist等人[96]的研究中，可从精液中去除99％以上的PCV2，初步风行症毒滴度降为3.25至3.82 TCID50/50μL。

　　Colloid centrifugation of boar semen has been reported sporadically for at least 2 decades, beginning with density gradient centrifugation and progressing more recently to single-layer centrifugation (SLC) [95]. The latter method is easier to use and less time-consuming than density gradient centrifugation. The technique can be used to remove pathogens (bacteria and viruses), thus improving biosecurity of semen doses and potentially reducing the use of antimicrobials. In a study by Blomqvist et al. [96], it was possible to remove more than 99% of PCV2 from semen with an initial infectious virus titer of 3.25 to 3.82 TCID50/50μL.

　　Martinez-Alborcia等[97]提出，精液冷冻前，使用Androcoll-P的胶体对精液样品进行单层离心（SLC）可提高精子冷冻存活率，并改变冷冻生存精子的成果。随后的一项研究表白，使用Androcol-P对公猪单次射精精液进行SLC处置惩罚，能够全部处置惩罚富含精子部门，仅供提高精子的冷冻保留能力，以及精子解冻后的受精能力[98]。此技术可清除病原，改进精子的冷冻生存，这两点有利于使具有遗传优势的公猪冻精，在进行国际贸易时越发容易。不外，SLC需要进一步研究改进，如，使其越发快捷、可靠。

　　Martinez-Alborcia et al. [97] reported that SLC of semen samples using a pig-specific colloid called Androcoll-P before freezing enhances sperm cryosurvival rates and modifies the functionality of the cryosurviving sperm. A subsequent study showed that SLC processing of boar ejaculates using Androcoll-P is able to process the entire sperm-rich fraction of a boar ejaculate, improving both the sperm cryosurvival and the fertilizing ability of thawed spermatozoa [98]. The removal of pathogens coupled with improved cryopreservation are two important advantages of the technique allowing easier international trade of frozen–thawed semen doses from genetically valuable boars. However, further research is necessary to improve SLC, e.g., by making the technique less time-consuming and the results less variable.

　　7.结论

　　Conclusions

　　人工授精会将猪精液中的病毒快速引入母猪群，进而引起猪群传染发病，虽然带毒精液会给种猪群带来风险。公猪入场前的检测、在人工授精中心期间的通例监测，精液交付前的按期检测、执行严格的生物安详法子，这些均可增强出产者对人工授精技术的信任，相信其可提供大量安详且遗传特性好的精液。同时，防范病毒污染精液是重中之重，若精液一旦带毒，对比防范，清除病毒越发困难。不外，新型精液加工处置惩罚办法（如SLC）的发明，会逐步让人们对人工授精这一安详的养猪繁殖技术，更有信心。

　　Viral contamination of porcine semen poses a risk for breeding herds because AI may lead to fast introduction of viruses into sow populations and to loss of disease-free status for these pathogens. Pre-entrance testing of boars, routine health monitoring of boars during their stay in the AI center, regular testing of semen before delivery, and enforcement of strict biosecurity protocols at the AI center provide most producers with a high level of confidence that AI is a relatively safe means to offer unlimited exchange of desirable genetic characteristics. Prevention of viral contamination of semen should be a primary focus because it is easier to prevent contamination than to eliminate viruses once they are present in semen. Nevertheless, research and development of novel semen processing treatments such as SLC may even further instill confidence in the use of AI as a safe reproductive technology for pig production.

　　完

　　-The end-