錦鯉感染錦鯉疱疹病毒病原與抗體之監測

Abstract

中文摘要 錦鯉疱疹病毒(koi herpesvirus, KHV)，現今被歸類在鯉魚疱疹病毒第三型(Cyprinid herpesvirus 3, CyHV-3)，為感染錦鯉魚(Cyprinus carpio koi)及鯉魚 (Cyprinus carpio carpio)並造成高死亡率之高度傳染性疾病病原。自1998年以色列和美國首先出現錦鯉感染KHV的病例後，許多國家皆相繼有病例報告。至今已造成各國鯉魚產業嚴重的經濟損失。台灣於2002年確定有錦鯉感染KHV的病例。本實驗試著模擬魚隻感染KHV的情形。將20隻鯉魚於KHV好發溫度下，分成兩組不同病毒劑量進行浸泡攻毒。並在魚隻進入急性期開始死亡後將水溫提高到好發溫度範圍之外(32℃)，使病毒無法繼續複製造成魚隻死亡。過程中持續以聚合酶連鎖反應(Polymerase chain reaction, PCR)和巢式聚合酶連鎖反應(Nested Polymerase chain reaction, Nested-PCR)進行感染KHV之錦鯉魚病原的檢測，並以血清中和試驗和間接酵素免疫連結反應(Indirect Enzyme link immunosorbent assay, indirect ELISA)持續監測魚隻體內抗KHV抗體的情形。實驗結果顯示，魚隻死亡時間集中在攻毒後十到二十天。血清抗體力價約於攻毒後十天開始出現，並在攻毒後二十天到三十天到達高值，持續到攻毒後七十天左右才開始下降。病原檢測方面，當魚隻處於KHV好發溫度範圍外時，PCR方式便幾乎無法檢測到KHV病原，但Nested-PCR仍可以檢測出陽性，說明了這些存活的魚隻可能為潛伏感染或持續感染。

Abstract Koi herpesvirus (KHV), recently designated Cyprinid herpesvirus 3 (CyHV-3), is the etiological agent of an emerging and mortal disease in common carp (Cyprinius carpio carpio) and koi (Cyprinus carpio koi). KHV was first identified in 1998 as the cause of mass mortality among juvenile and adult koi and among common carp in Israel, and United State. Since its emergence in the late 1900s, this highly contagious pathogen has caused severe financial and economic losses in both koi and common carp culture industries worldwide. In Taiwan, KHV was first reported in December 2002, and occurred continuousely among many areas. According to previous researchers, temperature has effect on KHV disease outbreak, which can occur only at permissive temperature (18-28℃), the highest mortality rates occur 8-12 days postinfection (dpi). Antibody titer began to rise at 10 dpi and plateau at 20-40 dpi. And highly sensitive diagnostic test must be used to identify latent or persistently KHV infected carrier, when fish exposed KHV under non-permissive temperature condition. In this study we tried to imitate the detectable pathogen and antibody produced condition when carp were infected with KHV. 20 Koi carps exposed to the virus at permissive temperature, and then transferred to non-permissive temperature (32℃) in disease acute phase. PCR and Nested-PCR methods was used to identify KHV virus, and neutralization test and ELISA was used to monitor serum antibody. Our results showed that death occurred at 10-20 dpi. Antibody titer began to rise at 10dpi, reach a peak about 20-30 dpi, and maintained at plateau until 70 dpi. When koi became latent infection under non-permissive temperature, KHV can’t identify by PCR method, while Nested-PCR still had positive result.