豬精液-病毒傳播的載體（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比利時，梅勒爾貝克，根特大學，獸醫學院，畜群健康與產科學，繁殖科
a Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
b比利時，梅勒爾貝克，根特大學，獸醫學院，病毒學實驗室，免疫與寄生蟲學，病毒學科
b Department of Virology, Immunology and Parasitology, Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium
關鍵詞 Keywords：
豬精液、豬、人工授精、病毒、回顧/檢查
Semen, Pig, Artificial insemination, Virus, Review
接上文...
6. 預防措施
Preventive measures
人工授精中心需要采用經濟、有效且滿足生物安全的方法，生產出適量品質可控的精液。防止疾病通過精液傳播的最佳方法是，購買健康狀況優良的公豬，執行嚴格的生物安全措施，並對公豬進行健康監測。
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. 新進公豬的健康狀況
Health status of incoming replacement boars
人工授精中心每年通常要更換60％的公豬，但是新進公豬會帶來很大的疾病風險，因此對新進公豬實行隔離、馴化、充分的觀察和檢測，至關重要。首先，應掌握公豬來源豬群的健康狀況。其健康狀況應等同於，但最好高於人工授精中心的公豬。可通過獸醫之間的溝通，了解新進公豬的臨床診斷與實驗室檢查、疫苗接種計劃以及來源豬群的獸醫措施，通過這些來對其進行評估。其次，新進公豬應單獨隔離30至60天。隔離區最好位於場外。豬群隔離必須遵守“全進全出”原則。隔離期間，應評估豬群健康狀況，進行血液采樣以檢測是否有病毒血症或血清抗體，同時，對人工授精中心的公豬中出現的疾病進行疫苗接種
治療。感染危險病原體的公豬不得進入人工授精中心。不同的人工授精中心，隔離期、檢測方案和疫苗接種方案均不相同，主要是由於不同的因素，如引入公豬的頻率和原畜群動物的健康狀況所決定[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. 預防措施
Preventive measures
6.1. 生物安全措施
Biosecurity measures
人工授精中心最重要的生物安全措施涉及以下內容[3,88]：（1）人工授精中心的位置及通風設備--應盡可能遠離豬群。在豬隻密集區域使用空氣過濾係統，以保證人工授精中心避免病原通過空氣傳入[89]。確保通風設備不會將公豬站內的空氣排向實驗室。如果實驗室緊挨豬舍，則應采用正壓通風，從而最大程度地減少從豬舍到實驗室的空氣滲透。（2）對於進入人工授精中心的員工和來訪者，應有嚴格規定。員工需要隔離1至3天。隔離期間，不允許與豬隻接觸。此外，建議對所有來訪者和員工施行人員進出淋浴措施。（3）精液拿取地點及遞送——精液遞送人員拿取精液的地點，應位於人工授精中心的外麵。精液拿取點應是獨立的房間，或至少是獨立於實驗室和公豬站的房間。把精液拿取點單獨分開，可以最大程度上避免精液冷卻器、靴子、衣物等的交叉汙染。每個母豬舍的精液放置點應位於辦公室外。任何送入人工授精中心的物品（例如，物資、設備），在這之前都不應該接觸過豬。應在送入人工授精中心之前，將物品放入指定房間消毒。（4）滅鼠及昆蟲 - 老鼠及昆蟲可攜帶及傳播多
種豬病毒，因此應采取適當預防措施。（5）水質 – 應定期分析公豬的水源。如果質量不合格，應更換水源或對水進行適當處理。（6）執行嚴格的清潔消毒程序。
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. 預防措施
Preventive measures
6.3 公豬健康監測及疫苗接種
Animal health monitoring and vaccination
最普遍的方法是監測公豬的健康狀況及臨床表現。如果出現臨床問題，在動物康複之前不得收集精液。用自動化的方法測量公豬的采食量和飲水量，從而更容易地在早期發現患病的公豬。每天有針對性的，對公豬的健康狀況及表現進行多次監測，但這仍是一種非常基礎的方法，並不能夠獨立監測病毒感染和/或精液中病毒的存在。因此，建議製定針對特定疾病的免疫、除蟲及取樣的標準方案，並定期進行回顧檢查。公豬的免疫接種可以非常有效地清除病毒或減少散毒，並降低人工授精傳播病毒的風險。首先，人工授精中心需要遵守該國的動物衛生法規，例如，強製性地接種ADV疫苗，因為是官方消滅病毒計劃的一部分（例如東歐），而在其他已正式清除ADV的國家，可能禁止ADV的免疫接種（例如西歐，北美）。高效重組標記疫苗和多種精確的酶聯免疫吸附實驗（ELISA）的結合，已使在世界上大範圍內（例如許多歐洲國家和北美）清除ADV成為現實可能[48]。《歐洲藥典》要求，公豬接種注射用ADV疫苗後，其精液不能使用[90]。其他無法律法規要求的疾病，人工授精中心可自行決定使用哪種疫苗。接種PPV和PCV2疫苗，可能有助於減少感染後散毒[91]。就PRRSV而言，公豬接種弱毒疫苗後50天內，體內野毒的散毒能力可被降低或消除 [92,93]。不可忽視的事實是弱毒疫苗隻能提供部分免疫保護，導致疫苗病毒在精液中的散毒長達39天。[17,92]。相比而言，滅活疫苗不能明顯減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].
因為需盡早發現可能存在的病毒（如PRRSV）感染，健康監測對人工授精至關重要。 一般情況下，需要檢測血液和精液樣品中是否存在病毒和/或抗體。不同人工授精中心的檢測程序相差很大。因為有時檢測血液和精液會出現不同的結果，且存在不確定因素，如最佳檢測頻率、樣本數量、診斷方法以及檢測結果的解讀，所以檢測程序很複雜[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. 預防措施
Preventive measures
6.4 無病毒精液收集
技術
Technologies to obtain virus-free semen
對精液進行處理並添加抗菌劑可稀釋精液，且減少細菌汙染，但這些措施無法清除病毒[4,5]。目前，尚無有效的抗病毒劑可以保證精液無病毒。精液中存在許多蛋白質，以及類固醇激素、酶以及其他物質，可在一定程度上防止病毒感染，但是，這些物質同時也可能損害精子。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].
豬精液膠體離心至少有20年曆史，從早期的密度梯度離心，到近期發展的單層離心（SLC）[95]。與密度梯度離心相比，後者更加便捷。使用單層離心
技術可消除病原（細菌和病毒），進而提高精液的生物安全性，以及減少抗菌劑的使用。 在Blomqvist等人[96]的研究中，可從精液中去除99％以上的PCV2，初始感染病毒滴度降為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，精液冷凍前，使用Androcoll-P的膠體對精液樣品進行單層離心（SLC）可提高精子冷凍存活率，並改變冷凍保存精子的功能。隨後的一項研究表明，使用Androcol-P對公豬單次射精精液進行SLC處理，能夠全部處理富含精子部分，可以提高精子的冷凍生存能力，以及精子解凍後的受精能力[98]。此
技術可清除病原，改善精子的冷凍保存，這兩點有利於使具有遺傳優勢的公豬凍精，在進行國際貿易時更加容易。不過，SLC需要進一步研究改善，如，使其更加快捷、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.結論
Conclusions
人工授精會將豬精液中的病毒快速引入母豬群，進而引起豬群感染發病，所以帶毒精液會給
種豬群帶來風險。公豬入場前的檢測、在人工授精中心期間的常規監測，精液交付前的定期檢測、執行嚴格的生物安全措施，這些均可增強生產者對人工授精
技術的信任，相信其可提供大量安全且遺傳特性優良的精液。同時，預防病毒汙染精液是重中之重，若精液一旦帶毒，相比預防，清除病毒更加困難。不過，新型精液加工處理方法（如SLC）的研發，會逐步讓人們對人工授精這一安全的養豬繁殖
技術，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-
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