臺灣博碩士論文加值系統

豬離乳後多系統消耗性症候群（Post-weaning multisystemic wasting syndrome; PMWS）於1991年首次在加拿大爆發，不過近來已成為各養豬產業國家重要的問題。豬環狀病毒二型（Porcine circovirus type II; PCV2）已被證明為造成PMWS的主要病原，但是一些共同因子，如其他病原的混合感染也被認為是造成PMWS的必要條件。本研究的目的為調查台灣PMWS的發生情況，並研究其他病原的混合感染是否為造成PMWS的條件。我們判定PMWS病例的標準是遵照之前的研究，共有三點：（1）出現相符的臨床症狀、（2）出現特徵性的組織病變、（3）這些變病區能以PCR偵測到PCV2。並且我們使用RT-PCR或PCR，針對Porcine circovirus typee I (PCV1), Pseudorabies virus (PRV), Porcine parvovirus (PPV), Classic swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella作檢測。本次調查62隻豬中，55隻PCV2陽性（88.71％）、29隻PPV陽性（46.77％）、25隻PRV陽性（40.32％）、6隻PCV1陽性（9.68％）、36隻Mycoplasma hyopneumoniae陽性（58.06％）、11隻APP陽性（17.74％）、42隻PRRSV陽性（67.74％）、10隻CSFV陽性（16.13％）、0隻Salmonella陽性。在各種檢體的PCV2檢出率方面，扁桃腺PCV2陽性率為80.65％（50/62）、肺門淋巴PCV2陽性率為82.26％（51/62）、肺臟PCV2的陽性率為75.81％（47/62）、肝臟PCV2陽性率為67.74（42/62）、脾臟PCV2陽性率為82.26％（51/62）、腸間淋巴結PCV2陽性率為80.65％（50/62）、腎臟的PCV2陽性率為79.03％（49/62）、鼠蹊淋巴結PCV2陽性率為85.48％（53/62）、血清PCV2陽性率為33.87％（21/62）。PMWS病例共有30隻，經由統計分析，發現感染CSFV (p=0.04)、PRRSV (p=0.0027)和PPV (p=0.0047)時，皆能增加造成PMWS的機率。另外將本研究的8個PCV2分離株作定序之後，與發表在GenBank上的PCV2台灣分離株作比較，相同度有93.1%到99.8%。若比較ORF2轉譯出之胺基酸，台灣各分離株相似度有88.5%到100%。而與全世界PCV2分離株作核酸序列比較，則相同度有93.4%到99.8%。

Post-weaning multisystemic wasting syndrome (PMWS), first described in Canada in 1991, has been now reported in most swine-producing countries and has become a significant problem for the pig industry. Porcine circovirus type 2 (PCV2) is the necessary cause of PMWS in swine; however, a variety of co-factors, including other infectious agents, are thought to be necessary in the full expression of disease. The aim of this study is to find out if other co-infection is necessary in the PMWS cases in Taiwan. The PMWS cases were diagnosed by three criteria described in previous study: (1) the presence of compatible clinical signs, (2) the presence of the characteristic microscopic lymphoid lesions, and (3) the detection of PCV2 within these tissues by PCR. Also, we detected Porcine circovirus typee I (PCV1), Pseudorabies virus (PRV), Porcine parvovirus (PPV), Classic swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae, Salmonella from different biological specimens of growth retarded pigs by RT-nPCR or PCR. In 62 pigs examined, 55 pigs were PCV2 positive (88.71%), 29 pigs were PPV positive (46.77%), 25 pigs were PRV positive (40.32%), 6 pigs were PCV1 positive (9.68%), 36 pigs were Mycoplasma hyopneumoniae positive (58.06%), 11 pigs were APP positive (17.74%), 42 pigs were PRRSV positive (67.74%), 10 pigs were CSFV positive (16.13%), 0 pigs were Salmonella positive and there were 30 PMWS cases. After statistic analysis, we found that co-infection with CSFV (p=0.04), PRRSV (p=0.0027), and PPV (p=0.0047) can increase the probability to induce PMWS. The nucleotide sequences of full-length genome of 8 Taiwan isolates were also analyzed by PCR and sequencing. After comparing nucleotide sequences of Taiwan strains published on GenBank, the PCV2 strains shared 93.1% to 99.8% identity. Comparing the amino acid sequences translated from ORF2 of PCV2 among the Taiwan strains, their amino acid sequences shared 88.5% to 100% similarity. The nucleotide sequences of PCV2 isolates shared 93.4% to 99.8% identity with other isolates world wide.

目錄
表次 VII
圖次 VIII
第一章 緒言 1
第二章 文獻探討 3
第一節 豬離乳後多系統消耗性症候群 3
2-1-1 豬離乳後多系統消耗性症候群的歷史背景及概況 3
2-1-2 PMWS的臨床症狀及病理變化 3
第二節 PMWS的病因探討 5
2-2-1 豬環狀病毒 5
2-2-2 其他共同因子 8
2-2-3 PMWS與豬隻品系關係 11
第三節 豬離乳後多系統消耗性症候群的診斷 12
第三章 材料與方法 13
第一節 病材收集與豬隻記錄 13
3-1-1 病材 13
3-1-2 豬隻基本資料、臨床症狀及肉眼、顯微病變的記錄 13
3-1-3 乳劑的製作 13
第二節 病原診斷 14
3-2-1 核酸萃取 14
3-2-2 病原的偵測 15
第三節 PMWS的診斷與各項因子相關性之分析 19
3-3-1 PMWS的診斷 19
3-3-2 PMWS與各項因子的相關性分析 19

第四節 PCV2基因體序列分析比對 20
3-4-1 PCV2基因體的定序 20
3-4-2 PCV2核酸序列分析 20
第四章 結果 21
第一節 病材收集及豬隻臨床記錄 21
第二節 豬隻病變記錄 21
第三節 各種病原檢測 22
4-3-1 PCR檢測PCV2 22
4-3-2 PCR檢測PPV 22
4-3-3 nPCR檢測PRV 22
4-3-4 PCR檢測PCV1 23
4-3-5 PCR檢測Mycoplasma hyopneumoniae 23
4-3-6 PCR檢測 Actinobacillus pleuropneumoniae 23
4-3-7 RT-nPCR檢測PRRSV 23
4-3-8 RT-nPCR檢測豬瘟病毒（CSFV） 23
4-3-9沙氏桿菌檢測結果 24
4-3-10各種病原檢測結果 24
第四節 PMWS的判定與相關因子分析 24
4-4-1 PMWS的診斷 24
4-4-2 PMWS與各項因子的相關性分析 24
第五節 PCV2基因體序列分析比對 25
第五章 討論 27
參考文獻 32
附錄一 81
附錄二 99

Allan GM, McNeilly F, Cassidy JP, Reilly GA, Adair B, Ellis WA, McNulty MS. Pathogenesis of porcine circovirus; experimental infections of colostrum deprived piglets and examination of pig foetal material. Vet Microbiol 44(1):49-64, 1995.

Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG, Ellis JA, Haines DM, Meehan BM, Adair BM. Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest 10(1):3-10, 1998.

Allan GM, McNeilly F, Ellis J, Krakowka S, Botner A, McCullough K, Nauwynck H, Kennedy S, Meehan B, Charreyre C. PMWS: experimental model and co-infections. Vet Microbiol 98(2):165-8, 2004.

Bolin SR, Stoffregen WC, Nayar GP, Hamel AL. Postweaning multisystemic wasting syndrome induced after experimental inoculation of cesarean-derived, colostrum-deprived piglets with type 2 porcine circovirus. J Vet Diagn Invest 13(3):185-94, 2001.

Calsamiglia M, Segales J, Quintana J, Rosell C, Domingo M. Detection of porcine circovirus types 1 and 2 in serum and tissue samples of pigs with and without postweaning multisystemic wasting syndrome. J Clin Microbiol 40(5):1848-50, 2002.

Chae C. Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Vet J 168(1):41-9, 2004.

Chae C. A review of porcine circovirus 2-associated syndromes and diseases. Vet J 169(3):326-36, 2005.

Caron J, Ouardani M, Dea S. Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes. J Clin Microbiol 38(4):1390-6, 2000.

Chen SP, Weng YJ, Tsai CP, Chang WF. Case report: Identification of pathogenic porcine circovirus in pigs with post-weaning multisystemic wasting syndrome in Taiwan. J Chin Soc Vet Sci 27:251-55, 2001.

Choi C, Chae C. In-situ hybridization for the detection of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J Comp Pathol 121(3):265-70, 1999.

Choi C, Chae C. Distribution of porcine parvovirus in porcine circovirus 2-infected pigs with postweaning multisystemic wasting syndrome as shown by in-situ hybridization. J Comp Pathol 123(4):302-5, 2000.

Choi C, Chae C. Colocalization of porcine reproductive and respiratory syndrome virus and porcine circovirus 2 in porcine dermatitis and nephrology syndrome by double-labeling technique. Vet Pathol 38(4):436-41, 2001.

Christopher-Hennings J, Nelson EA, Nelson JK, Hines RJ, Swenson SL, Hill HT, Zimmerman JJ, Katz JB, Yaeger MJ, Chase CC, et al. Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR. J Clin Microbiol 33(7):1730-4, 1995.

Dahle J, Liess B. A review on classical swine fever infections in pigs: epizootiology, clinical disease and pathology. Comp Immunol Microbiol Infect Dis. 15(3):203-11, 1992.

Done JT, Harding JDJ. Kongenitaler tremor der schweine (zitterkrankheit der ferkel): veranderungen und ursachen. Deutsche Tierarztliche Wochenschrift 74:333–334, 1967.
Done S, Gresham A, Potter R, Chennells D. PMWS and PDNS–two recently recognised diseases of pigs in the UK. In Practice 23:14–21, 2001.
Duran CO, Ramos-Vara JA, Render JA. Porcine dermatitis and nephropathy syndrome: a new condition to include in the differential diagnosis list for skin discoloration in swine. Swine Health and Production 5:241–245, 1997.

Ellis J, Krakowka S, Lairmore M, Haines D, Bratanich A, Clark E, Allan G, Konoby C, Hassard L, Meehan B, Martin K, Harding J, Kennedy S, McNeilly F. Reproduction of lesions of postweaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diagn Invest. 11(1):3-14, 1999.

Ellis JA, Krakowa S, Allan G, Clark E, Kennedy S. "The clinical scope of porcine reproductive and respiratory syndrome virus infection has expanded since 1987": an alternative perspective. Vet Pathol 36(3):262-5, 1999.

Gilpin DF, McCullough K, Meehan BM, McNeilly F, McNair I, Stevenson LS, Foster JC, Ellis JA, Krakowka S, Adair BM, Allan GM. In vitro studies on the infection and replication of porcine circovirus type 2 in cells of the porcine immune system. Vet Immunol Immunopathol 94(3-4):149-61, 2003.

Hardge T, Gaumann H, Hasberg W, Lange S. The economic impact of PMWS in the nursery – review of a successful control program. 4th International Symposium on Emerging and Re-emerging Pig Diseases. Rome June 29th – July 2nd, 2003.

Harms PA, Sorden SD, Halbur PG, Bolin SR, Lager KM, Morozov I, Paul PS. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine reproductive and respiratory syndrome virus. Vet Pathol 38(5):528-39, 2001.

Hines RK, Lukert PD. Porcine circovirus as a cause of congenital tremors in newborn pigs. Proceeding of the American Association of Swine Practitioners 25:344–345, 1994.

Kamstrup S, Barfoed AM, Frimann TH, Ladekjaer-Mikkelsen AS, Botner A. Immunisation against PCV2 structural protein by DNA vaccination of mice. Vaccine. 22(11-12):1358-61, 2004

Kennedy S, Moffett D, McNeilly F, Meehan B, Ellis J, Krakowka S, Allan GM. Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. J Comp Pathol 122(1):9-24, 2000.

Kennedy S, Segales J, Rovira A, Scholes S, Domingo M, Moffett D, Meehan B, O''Neill R, McNeilly F, Allan G. Absence of evidence of porcine circovirus infection in piglets with congenital tremors. J Vet Diagn Invest 15(2):151-6, 2003.

Kim J, Choi C, Han DU, Chae C. Simultaneous detection of porcine circovirus type 2 and porcine parvovirus in pigs with PMWS by multiplex PCR. Vet Rec 149(10):304-5, 2001.

Kim J, Chung HK, Jung T, Cho WS, Choi C, Chae C. Postweaning multisystemic wasting syndrome of pigs in Korea: prevalence, microscopic lesions and coexisting microorganisms. J Vet Med Sci 64(1):57-62, 2002.

Kim J, Han DU, Choi C, Chae C. Simultaneous detection and differentiation between porcine circovirus and porcine parvovirus in boar semen by multiplex seminested polymerase chain reaction. J Vet Med Sci 65(6):741-4, 2003.

Krakowka S, Ellis JA, McNeilly F, Ringler S, Rings DM, Allan G. Activation of the immune system is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Vet Pathol 38(1):31-42, 2001.

Krakowka S, Ellis JA, Meehan B, Kennedy S, McNeilly F, Allan G. Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome in gnotobiotic swine by coinfection with porcine circovirus 2 and porcine parvovirus. Vet Pathol 37(3):254-63, 2000.

Ladekjaer-Mikkelsen AS, Nielsen J, Stadejek T, Storgaard T, Krakowka S, Ellis J, McNeilly F, Allan G, Botner A. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2). Vet Microbiol 89(2-3):97-114, 2000.

LeCann P, Albina E, Madec F, Cariolet R, Jestin A. Piglet wasting disease. Vet Rec 141(25):660, 1997.

Lipej Z, Segales J, Toplak I, Sostaric B, Roic B, Lojkic M, Hostnik P, Grom J, Barlic-Maganja D, Zarkovic K, Oraic D. Postweaning multisystemic wasting syndrome (PMWS) in pigs in Croatia: detection and characterisation of porcine circovirus type 2 (PCV2). Acta Vet Hung 53(3):385-96, 2005.

Liu Q, Wang L, Willson P, Babiuk LA. Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol 38(9):3474-7, 2000.

Lyoo KS, Park YH, Park BK. Prevalence of porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and porcine parvovirus from aborted fetuses and pigs with respiratory problems in Korea. J Vet Sci 2(3):201-7, 2001.

Madec F, Eveno E, Morvan P, Hamon L, Blanchard P, Cariolet R, Amenna N, Morvan H, Truong C, Mahe D, Albina E, Jestin A. Post-weaning multisystemic wasting syndrome (PMWS) in pigs in France: clinical observation from follow-up studies on affected farms. Livestock Production Science 63: 223-33, 2000.

Magar R, Larochelle R, Thibault S, Lamontagne L. Experimental transmission of porcine circovirus type 2 (PCV2) in weaned pigs: a sequential study. J Comp Pathol 123(4):258-69, 2000.

Mahe D, Blanchard P, Truong C, Arnauld C, Le Cann P, Cariolet R, Madec F, Albina E, Jestin A. Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes. J Gen Virol 81(Pt 7):1815-24, 2000.

Mankertz A, Domingo M, Folch JM, LeCann P, Jestin A, Segales J, Chmielewicz B, Plana-Duran J, Soike D. Characterisation of PCV-2 isolates from Spain, Germany and France. Virus Res 66(1):65-77, 2000.

McIntosh KA, Harding JC, Ellis JA, Appleyard GD. Detection of Porcine circovirus type 2 viremia and seroconversion in naturally infected pigs in a farrow-to-finish barn. Can J Vet Res 70(1):58-61, 2006.

Meehan BM, McNeilly F, Todd D, Kennedy S, Jewhurst VA, Ellis JA, Hassard LE, Clark EG, Haines DM, Allan GM. Characterization of novel circovirus DNAs associated with wasting syndromes in pigs. J Gen Virol 79:2171-9, 1998.

Mengeling WL, Lager KM, Vorwald AC. The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance. Anim Reprod Sci 60-61:199-210, 2000

Morozov I, Sirinarumitr T, Sorden SD, Halbur PG, Morgan MK, Yoon KJ, Paul PS. Detection of a novel strain of porcine circovirus in pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol 36(9):2535-41, 1998.

Nawagitgul P, Morozov I, Bolin SR, Harms PA, Sorden SD, Paul PS. Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein.
J Gen Virol. 81(Pt 9):2281-7, 2000.

Opriessnig T, Fenaux M, Thomas P, Hoogland MJ, Rothschild MF, Meng XJ, Halbur PG. Evidence of breed-dependent differences in susceptibility to porcine circovirus type-2-associated disease and lesions. Vet Pathol 43(3):281-93, 2006.

Opriessnig T, Thacker EL, Yu S, Fenaux M, Meng XJ, Halbur PG. Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2. Vet Pathol 41(6):624-40, 2004

Pallares FJ, Halbur PG, Opriessnig T, Sorden SD, Villar D, Janke BH, Yaeger MJ, Larson DJ, Schwartz KJ, Yoon KJ, Hoffman LJ. Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS). J Vet Diagn Invest 14(6):515-9, 2002.

Ramos-Vara JA, Duran O, Render JA, Craft D. Porcine dermatitis and nephropathy syndrome in the USA. Vet Rec 141(18):479-80, 1997.

Roca M, Balasch M, Segales J, Calsamiglia M, Viaplana E, Urniza A, Hattermann K, Mankertz A, Plana-Duran J, Domingo M. In vitro and in vivo characterization of an infectious clone of a European strain of porcine circovirus type 2. J Gen Virol 85(Pt 5):1259-66, 2004.

Rosell C, Segales J, Ramos-Vara JA, Folch JM, Rodriguez-Arrioja GM, Duran CO, Balasch M, Plana-Duran J, Domingo M. Identification of porcine circovirus in tissues of pigs with porcine dermatitis and nephropathy syndrome. Vet Rec 146(2):40-3, 2000.

Rovira A, Balasch M, Segales J, Garcia L, Plana-Duran J, Rosell C, Ellerbrok H, Mankertz A, Domingo M. Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2. J Virol 76(7):3232-9, 2002.

Stevenson GW, Kiupel M, Mittal SK, Choi J, Latimer KS, Kanitz CL. Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors. J Vet Diagn Invest 13(1):57-62, 2001.

Smith WJ, Thomson JR, Done S. Dermatitis/nephropathy syndrome of pigs. Vet Rec 132(2):47, 1993.

Soares RM, Durigon EL, Bersano JG, Richtzenhain LJ. Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1. J Virol Methods 78(1-2):191-8, 1999.

Thacker EL. Porcine respiratory disease complex – what is it and why does it remain a problem? The Pig Journal 48:66–70, 2001.

Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like particles in permanent pig kidney cell lines. Zentralbl Bakteriol [Orig A] 226(2):153-67, 1974.

Tischer I, Mields W, Wolff D, Vagt M, Griem W. Studies on epidemiology and pathogenicity of porcine circovirus. Arch Virol 91(3-4):271-6, 1986.

Thibault S, Drolet R, Germain MC, D''Allaire S, Larochelle R, Magar R. Cutaneous and systemic necrotizing vasculitis in swine. Vet Pathol 35(2):108-16, 1998.

Wang C, Huang TS, Huang CC, Tu C, Jong MH, Lin SY, Lai SS. Characterization of porcine circovirus type 2 in Taiwan. J Vet Med Sci 66(5):469-75, 2004.

Wang X, Jiang W, Jiang P, Li Y, Feng Z, Xu J. Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice. Vaccine. 24(16):3374-80, 2006.

Yoon HA, Eo SK, Aleyas AG, Cha SY, Lee JH, Chae JS, Jang HK, Cho EG, Song HJ. Investigation of pseudorabies virus latency in nervous tissues of seropositive pigs exposed to field strain. J Vet Med Sci 68(2):143-8, 2006.