巴斯德桿菌去活化疫苗不同免疫策略對海鱺專一性免疫反應之影響

Abstract

巴斯德桿菌(Photobacterium damselae subsp. Piscicida, Pdp)歷年都是造成台灣海鱺苗在春夏季移入箱網期間大量死亡的主因。疫苗是預防細菌性疾病的重要防疫策略，本研究則在探討Pdp疫苗免疫策略中的一些因子，包括去活化藥劑、免疫劑量、佐劑、追加免疫及免疫激活劑等，對疫苗誘發海鱺專一性免疫力及抗 Pdp感染力之影響。實驗結果顯示，0.5%福馬林能完全去活化 Pdp，且比相同濃度之β-propiolacton對抗原性的破壞小。福馬林去活化之Pdp疫苗，以0.333 mg/g fish 免疫劑量所誘發之專一性抗體力價，明顯高於0.006 mg/g fish組。比較魚油、沙拉油或氫氧化鋁佐劑對Pdp疫苗誘發專一性抗體力價之影響，氫氧化鋁佐劑效果最好，但抗體力價持續時間相對短。對15 g重之海鱺苗進行一次及追加免疫，結果追加免疫後抗體力價高峰產生的時間縮短，且力價明顯升高，顯示15 g重之海鱺苗已有免疫記憶力，但高力價維持時間短。在Pdp菌體疫苗中添加入去活化之Pdp胞外產物，其免疫效果和未添加組比較並無顯著差異。比較魚用ISA 763A油性佐劑和水包油包水(water in oil in water, WOW)製備的Pdp疫苗在追加免疫後所引起的專一性抗體力價，ISA佐劑組的免疫時效比WOW組長，且ISA佐劑組攻毒後死亡率比WOW組低。Pdp疫苗中添加鰻弧菌(Vibrio anguillarum)及黃耆，一次免疫後，添加黃耆之疫苗組在浸泡攻毒後的保護效果最好，但在注射攻毒試驗中則低於無添加組，推測黃耆可能有助於非專一性黏液免疫而降低Pdp感染；去活化鰻弧菌的添加對 Pdp專一性免疫或保護效果則無促進效果，且可能有干擾的反效果。

Photobacterium damselae subsp. Piscicida (Pdp) has been a major pathogen causing serious mortality of cultured cobia in the past years in Taiwan, particularly when they are moved into sea cage. Vaccination is a fundamental strategy to prevent the outbreak of bacterial diseases. In this study, several immune program factors of Pdp vaccine, including inactivated agent, immune dosage, adjuvant, boost immune, and immunostimulant were investigated to verify the influences on inducing specific immunity and the ability against Pdp infection in cobia. Our results revealed that 0.5% formalin could completely inactivate Pdp and retain higher antigenicity than 0.5% β-propiolactone did. The specific antibody titer caused by high dosage antigen (0.333 mg/g) was higher than low dosage (0.006 mg/g). Compare the influences on inducing specific antibody titer of different adjuvants in Pdp vaccines, aluminum hydroxide exhibited the highest immune effect among all adjuvants, although with a relatively shorter duration. Due to the low memory of cobia immunity, a second vaccination was required to enhance the immunity efficacy and protection period. No significant differences were observed between Pdp vaccine with or without adding inactivated extracellular product. Compare the antibody titer induced by Pdp vaccine with ISA 763A adjuvant or with fish oil adjuvant embedded as water in oil in water (WOW group), the duration of ISA group was longer than WOW group after boost. Besides, the cumulative mortality of ISA group was 40% lower than negative control (100%) after intraperitoneal challenge. Cobia immunized by Pdp vaccine was supplemented in Vibrio anguillarum (V.ang) and Astragalus polysaccharide (APS) showed the best protection efficacy after immersion challenge. However, the cumulative mortality was higher than that without APS after intraperitoneal challenge. It is suggested that APS may lower Pdp infection by inducing non-specific immunity in mucus. There is no enhancement on specific immunity and protection effect when inactivated V.ang was supplemented in Pdp vaccine ; contrarily it seems to interfere Pdp vaccine protection efficacy.