豬第二型環狀病毒對兔化豬瘟疫苗效力之影響

The Adverse Effect of Porcine Circovirus Type 2 in the Efficacy of Lapinized Philippines Coronel Vaccine of Classical Swine Fever Virus

黃有良(Yu-Liang Huang)

指導教授 ： 鄭謙仁 ； 龐飛

國立臺灣大學/獸醫專業學院/獸醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.00274

摘要

從日據時代以來，豬瘟病毒（CSFV）始終嚴重困擾著國內豬隻畜牧業並造成嚴重經濟損失。此現象持續至李崇道及林再春博士研發出兔化豬瘟疫苗（LPC）後，方使整體豬瘟疫情獲得控制。由於此疫苗可藉由誘發體液性與細胞性免疫反應達到完全保護的功效，因此，LPC疫苗已被視為防治與控制CSFV的重要利器。近年，隨著豬第二型環狀病毒（PCV2）的入侵，國內養豬場幾乎皆已受到此病毒的侵襲，此病毒可導致離乳仔豬罹患離乳後多系統性消耗症候群（PMWS）與呈現嚴重的免疫抑制。由於PCV2主要感染25至120日齡之離乳仔豬，且此PCV2感受性年齡與國內推行的LPC疫苗免疫適期相互重疊，因此，本論文希望評估現行LPC疫苗效力是否會受到PCV2感染所影響。於動物試驗進行前，將先建立CSFV檢測法以提供後續研究使用，而為了讓此CSFV檢測法能夠廣泛被應用於田間樣品之檢測，本試驗發展單管多反轉錄反應之即時定量聚合酶反應（RT-MRT-PCR）以建立兼顧定量與基因型別鑑定之CSFV病毒檢測法。此CSFV RT-MRT-PCR並不會與其它豬隻病毒進行反應，且各基因型別間也無交叉反應，顯示其具有良好的特異性。於敏感性方面，其檢測極限可達1 viral copy/μl且與RT-nPCR具有相同的敏感性，此方法較病毒分離與RT-PCR敏感。另外，應用此檢測方法於豬瘟疫苗效力之測試，結果發現免疫E2次單位疫苗之豬隻，在暴露於具有強毒CSFV環境中，會出現CSFV病毒血症且較免疫LPC疫苗豬隻有較長的持續天數。此結果與先前其他學者之研究相似，顯示RT-MRT-PCR可被應用於豬瘟疫苗效力動物試驗之評估。在探索PCV2影響LPC疫苗效力的動物實驗研究結果顯示，於先有感染PCV2的LPC疫苗免疫豬隻，於強毒CSFV攻毒後會呈現短暫的發燒、病毒血症與從唾液與糞便排毒的現象。但在單純LPC疫苗免疫後接受野外強毒攻毒豬隻則無此現象。另外，藉由分析血液中不同淋巴球亞群與CSFV中和抗體力價，發現先前感染PCV2的LPC疫苗免疫豬隻，其IgM+、CD4+CD8-CD25+、CD4+CD8+CD25+、CD4-CD8+CD25+等亞群淋巴球數目與CSFV中和抗體力價均顯著低於單純免疫LPC疫苗豬隻。當進一步探討PCV2對CSFV特異性細胞性免疫反應之影響，以釐清PCV2干擾LPC疫苗效力的機制，結果顯示PCV2與UV-inactivated PCV2均具有干擾CSFV特異性周邊血液單核細胞增殖反應（CSFV-specific PBMC proliferation）的能力，且此具CSFV特異反應的PBMC其表面分子CD25的表現，也同樣受到PCV2所抑制。此種抑制CD25表現的干擾，同樣出現在先前曾感染PCV2的LPC疫苗免疫豬隻的淋巴球亞群。由於CD25是淋巴球活化與增生的重要指標，此結果顯示PCV2可藉由抑制淋巴球的活化與增殖來干擾疫苗所誘發的免疫反應。由於LPC是一種活毒減毒疫苗，其免疫接種後LPC必須於動物體內進行感染與複製，方可誘發完整的體液性與細胞性免疫反應，而達到完全保護的功效。為了釐清PCV2是否藉由干擾LPC疫苗病毒的感染與複製，來降低LPC疫苗誘發免疫反應的功效，本研究使用對PCV2與LPC具共同感受性的肺泡巨噬細胞來進行其相關實驗。結果顯示，PCV2具有干擾LPC病毒的感染與複製能力，但UV-inactivated PCV2並無此能力，顯示PCV2干擾機制可能與病毒複製過程使用的PCV2基因體，或與複製過程的病毒中間產物有關。在進一步測試PCV2基因體與病毒複製中間產物對LPC感染之影響，發現PCV2基因體中的C9 CpG-ODN與病毒複製中間產物（雙股全長PCV2 DNA）可以顯著抑制LPC的感染，然而此PCV2干擾LPC感染的機制與先前PCV2干擾CSFV-specific PBMC proliferation的機制不同，顯示不同的PCV2病毒成分具有不同的免疫干擾功能。綜合以上結果，PCV2確實可干擾LPC疫苗的效力，其主要經由干擾LPC感染與複製以降低LPC疫苗接種後所誘發的體液性與細胞性反應，最後導致LPC疫苗無法完全保護疫苗免疫豬隻。此結果在使用LPC疫苗防治CSFV的區域是一個重要的警訊，其可能導致現有使用LPC疫苗的防治CSFV計畫出現漏洞，並增加野外CSFV入侵豬場的風險與清除CSFV的困難度。

關鍵字

豬瘟病毒 ； 兔化豬瘟疫苗 ； 豬第二型環狀病毒 ； 即時定量聚合酶反應 ； 周邊血液單核細胞 ； 增殖反應 ； 肺泡巨噬細胞

並列摘要

The presence of classical swine fever virus (CSFV) in Taiwan had been recorded in the Japanese era which was more than 50 years ago. During these years, Lapinized Philippine Coronel (LPC) vaccine developed by Drs. R. Lee and TC Lin is routinely used as an important tool to prevent and control CSFV infection in swine herds in Taiwan. The worldwide endemic infection of porcine circovirus type 2 (PCV2) has been reported at the end of 1990’s and the virus is also commonly present in Taiwan since then. PCV2 is able to induce post-weaning multisystemic wasting syndrome (PMWS) in PCV2-infected pigs and the syndrome is often associated with immunosuppression. The susceptible age of PCV2 infection is between 25 and 120 days old and is overlapping with the vaccination schedule of LPC vaccine. The aims of the present study are to investigate whether PCV2 infection could affect the efficacy of LPC vaccine in the pigs. The development of reverse transcription multiplex real-time PCR (RT-MRT-PCR) to detect, quantitate and genotype CSFV was the first project to proceed in this study. The results showed that the established RT-MRT-PCR was specific to CSFV and was not cross-reacted with the other swine viruses and the assay was able to differentiate various CSFV genotypes in the field samples. The detected limit of RT-MRT-PCR was 1 viral copy/μl and the sensitivity was similar as the reverse transcription nested PCR (RT-nPCR) but was more sensitive than viral isolation and RT-PCR in the field cases. The intra- and inter-assay variations of RT-MRT-PCR were below 3%. This assay was used to investigate the viremia of wild-type CSFV in vaccinated exposed pigs. The duration of viremia was longer in pigs vaccinated with E2 subunit vaccine than in pigs with LPC vaccination. The results are similar as previous studies using other detection methods which indicating RT-MRT-PCR is a rapid, sensitive, specific and reproducible, genotyping tool for CSFV detection and quantization. This assay has been used to differentiate LPC vaccine strain and wild-type CSFV strain in the field cases of Taiwan. The second project of the present study was to evaluate whether PCV2 affected the efficacy of LPC vaccine. The LPC-vaccinated pigs pre-inoculated with PCV2 showed transient fever, viremia, and viral shedding in the saliva and feces after wild-type CSFV challenge. The number of IgM+, CD4+CD8-CD25+, CD4+CD8+CD25+, and CD4-CD8+CD25+ lymphocyte subsets and the level of neutralizing antibodies against CSFV were significantly higher in the pigs with LPC vaccination alone than in the pigs with PCV2 inoculation/LPC vaccination. Further to evaluate whether PCV2 affected the CSFV-specific cell-mediated immunity (CMI), the CSFV-specific cell proliferative response of peripheral blood mononuclear cells (PBMCs) was established in an ex vivo experiment. PCV2-derived inhibition of the CSFV-specific PBMC proliferation was demonstrated in both live and UV-inactivated PCV2. The CD25 expression of PBMCs with CSFV recall antigens was also interfered by PCV2 infection. This situation was also reflected to LPC-vaccinated pigs pre-inoculated with PCV2 had low CD25 expression of lymphocyte subpopulations. In addition, in order to investigate the possible mechanisms of the PCV2-derived interference, an in vitro model was established to study the interaction of LPC and PCV2 in porcine alveolar macrophages (AMs). The results showed that PCV2 reduced the LPC infection and replication in AMs and the levels of PCV2-derived interference were dose-dependent. Both C9 CpG-ODN and full-length PCV2 DNA were able to reduce the LPC infection in AMs but the UV-inactivated PCV2 and soluble factors including TNF-α, IFN-γ, IL-8, and TGF-β1 were not involved in this interference. The experimental results of PCV2-derived interference on LPC infection were contradictory to the PCV2-derived interference on the CSFV-specific PBMC proliferation. The UV-inactivated PCV2 but not CpG-ODN of PCV2 genome is able to reduce CSFV-specific PBMC proliferation. The conclusions of the present study indicate that PCV2 infection decreases the efficacy of LPC vaccine by blocking the LPC infection and replication; as well as down-regulating both of CSFV-specific humoral and CMI response. This PCV2-derived interference may not only allow the invasion of wild-type CSFV in pig farms but also increase the difficulty of CSF prevention and control in the CSF endemic areas.

並列關鍵字

classical swine fever virus ； Lapinized Philippine Coronel ； porcine circovirus type 2 ； real-time PCR ； peripheral blood mononuclear cells ； proliferative response ； alveolar macrophages

參考文獻

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