- 學位論文
魚奴卡氏菌之流行病學、致病性特徵及疫苗研發
Epidemiology, Pathogenicity Characterization and Vaccine Development against Nocardia seriolae Infection
摘要
魚類奴卡氏菌病 (Nocardiosis)是由奴卡氏菌 (Nocardia seriolae )引發， 是一種常見且嚴重感染水生動物的病菌，在許多國家都已蔓延開。 近年來， 世界各地發現奴卡氏菌可感染的魚種宿主越來越廣泛，顯示這種細菌的傳染 性和易感性是十分重要的。石斑魚是台灣水產養殖中高經濟價值的養殖魚類 ，報導指出石斑魚易受奴卡氏菌感染，但對該物種的奴卡氏菌病知之甚少。 儘管已針對這種臭名昭著的魚類疾病開發疫苗，但並未朝此類細菌進行流行 病學和致病性的研究，若要制定有效策略解決魚類的奴卡氏菌病，其流行病 學和致病性的研究是非常重要。首先，本研究透過基因型和酵素活性的鑑定 來了解台灣奴卡氏菌分離株的流行病學關係。 其次，評估點帶石斑魚 (orange-spotted grouper Epinephelus coioides) 感染奴卡氏菌後造成奴卡氏菌病 的致病性。 第三，以反向疫苗學(Reverse Vaccinology)預測細菌中潛在性抗原 開發 DNA 疫苗，並在點帶石斑魚中進行了評估。 以流行病學進行分析，從台灣五個地點進行取樣，總共有 14 種已知和 4 種未知宿主魚類，混和五種分析方法分離出 66 株菌株進行了基因型分析( 以脈衝式電泳 PFGE、rep PCR 和 natural plasmid 分離)和表型分析(以 API ZYM test 和 alpha-glucosidase test 分離)。66 個分離株中具有高度的遺傳多樣 性，以 PFGE 方法鑑定出 10 株 pulsotypes，另透過 rep PCR 擴增鑑定出 21 株 reptypes;然而，在這 66 個分離株中並沒有檢測到 natural plasmid。分別透過 PFGE 和 rep PCR 分析發現，台灣這五個地點在過去的 17 年間，pulsotypes A8 及 RI 占有主導地位。於酵素學研究，大多數分離株於亮胺酸芳基醯胺酶 (leucine arylamidase)、β-葡萄糖苷酶(β- glucosidase)和 α-葡萄糖苷酶(α- glucosidase)具有高度的酵素活性表現，但脂肪酶(lipase)、α-半乳糖苷酶(α- galactosidase)、β-葡萄醣醛酸酶(β-glucuronidase)、N-乙醯氨基葡萄糖苷酶(N- acetyl-glucosaminidase)、α-甘露糖苷酶(α-mannosidase)及 α-岩藻糖苷酶(α- fucosidase)等酵素活性皆呈陰性。 再者將點帶石斑魚感染努卡氏菌評估其所造成的致病性，挑選已鑑定 分離出的 9 種菌株注射入點帶石斑魚之腹膜內 (intraperitoneal, IP)進行了活體 試驗，並每天觀察，總共進行 35 天之臨床觀察。確定強毒株後，再透過不同 的攻毒模式來評估同一魚模型中努卡氏菌感染之致病性，分別為 IP 攻毒、肌 肉注射攻毒 (intramuscular, IM)、口服攻毒 (oral, OR) 和浸泡攻毒 (immersion, IS)，並每天觀察，總共進行 42 天之攻毒試驗。 結果顯示，在這 9 株感染點 帶石斑魚之努卡氏菌分離株間的毒性有輕微差異。根據統計的死亡率及動力 學分析，AOD107132-2K 和 OT103003-N11 菌株分別具有最高和最低的感染毒力。 以 IM 和 IP 的攻毒模式下最早引發致死性，攻毒試驗後 5 天即有 30- 100%的死亡率;而以 OR 和 IS 方法攻毒時奴卡氏菌病之動力學較慢，待攻毒 後 35 天死亡率才至 4-8%。攻毒後，從組織病理之臨床切片下觀察出典型的 肉芽腫，證實了點帶石斑魚中奴卡氏菌病的進展。 以反向疫苗學方法預測努卡氏菌潛在抗原用於開發及評估點帶石斑魚 的 DNA 疫苗。透過疫苗演算法 (In silico)，推測膽固醇氧化酶 (Cholesterol oxidase)、LD-轉肽酶 (LD-Transpeptidase)和糖基羥化酶 (Glycosyl hydroxylase)此三種蛋白具有高度抗原性可作為候選疫苗之開發。重要的是，相對於注射 PBS 之對照組，施打奴卡氏菌之 DNA 疫苗組在第 1 天或第 2 天 IL1b、IL17 及 IFNg 基因表達量即有顯著較高，免疫後 7 天除了血清抗體增加以外，其他 細胞免疫相關基因例如 MHCI、CD4 和 CD8 也有相對增加。 值得注意的是， 免疫具有膽固醇氧化酶之 DNA 候選疫苗 (pcD::Cho)能增加其免疫反應，增強 保護力免受高毒力之奴卡氏菌感染，可使點帶石斑魚存活率可達 73.3% 在目前的研究中，我們發現基因型和酵素活性之間存在很強的關聯性 ，原因歸咎於大多數所鑑定出的 pulsotypes 顯示出相同類型的酵素活性。 此 外，我們認識到點帶石斑魚之奴卡氏菌其基因遺傳型與致病性之間存在密切 關係，原始宿主中所鑑定出的 pulsotypes 解釋了分離株在體內的毒力。奴卡 氏菌透過不同的感染途徑是會誘發不同的奴卡氏菌病相關臨床症狀和引發相 對的死亡率，試驗結果顯示，高毒力之 AOD107132-2K 奴卡氏菌株透過 IP 的 感染途徑似乎是實驗環境中最有效的接種方式。研究結果表明，在疫苗開發 中，反向疫苗學方法可作為篩選候選疫苗的有效策略，pcD::Cho 是一種很有前途的候選疫苗，除了增強先天性和適應性免疫反應，也為控制魚類感染奴 卡氏菌提供相當大的保護。
並列摘要
Nocardiosis in aquatic animals caused by Nocardia seriolae is a serious infection that occurs frequently and has spread in many countries. An increasing number of aquatic animal species have been infected by this pathogen in recent years worldwide, highlighting the importance of this bacterium's infectiousness and susceptibility. Grouper is a highly valued production fish in Taiwan aquaculture and has been reported to be susceptible to N. seriolae infection, however, little is known about nocardiosis in this species. Despite numerous efforts to develop vaccines against this notorious disease, such studies have not yet concerned the bacterial epidemiology and pathogenicity, which is important to developing effective prevention strategies. This study first aimed to investigate the epidemiological relationship among N. seriolae isolates in Taiwan by investigating their genotype and enzymatic activities. Secondly, the pathogenicity of nocardiosis following N. seriolae infection was evaluated in the orange-spotted grouper Epinephelus coioides. Thirdly, DNA vaccines containing bacterial potential antigens predicted by the Reverse Vaccinology approach were developed and evaluated in orange-spotted grouper. For epidemiological analysis, a total of 66 strains isolated from 14 known and four unknown host fish from five sites in Taiwan were characterized using five combined methods for genotyping (by pulsed-field gel electrophoresis PFGE, rep PCR and natural plasmid isolation) and phenotyping (by API ZYM test and alpha-glucosidase test). High genotypic diversity was recognized among the isolates, with 10 pulsotypes being identified from the PFGE method and 21 reptypes from the amplification method; however, no natural plasmids were detected in this bacterial population. Pulsotypes A8 and RI analysed by PFGE and repPCR, respectively, were found to be predominant within five sites in Taiwan over 17 years of isolation. Enzymatically, the majority of isolates displayed high leucine arylamidase, β- glucosidase and α-glucosidase activities but were negative for lipase, α-galactosidase, β-glucuronidase, N-acetyl-glucosaminidase, α-mannosidase and α-fucosidase activities. In pathogenicity characterization, nine identified genetic isolates of N. seriolae were tested in vivo using the intraperitoneal method and observed daily for 35 days. The most virulent isolate was then used to evaluate transmission through different routes (intraperitoneal IP, intramuscular IM, oral OR, and immersion IS) in the same fish model and was observed daily for 42 days. The results showed mild variation in virulence among N. seriolae isolates. AOD107132-2K and OT103003- N11 strains displayed the highest and lowest risk virulence, respectively, based on the accumulation and kinetics of mortality. IM and IP administrations showed an early phase response with early mortality by 5 dpc (30–100%), while slower kinetics of nocardiosis occurred in the OR and IS routes with slow mortality at 35 dpc (4– 8%). Histopathology revealed typical granulomas, confirming the progression of nocardiosis in the diseased fish. DNA vaccines containing bacterial potential antigens predicted by the Reverse vaccinology approach were developed and evaluated in orange-spotted grouper. In silico analysis indicated that Cholesterol oxidase, LD-Transpeptidase, and Glycosyl hydroxylase proteins have high immunogenicity as vaccine candidates. Importantly, when compared to a control PBS injection, DNA N. seriolae-based vaccines showed significantly higher expression of IL1b, IL17 and IFNg at 1 day or 2 days, in line with higher serum antibody production and other cellular immune-related genes such as MHCI, CD4 and CD8 at 7 days post-immunization. Remarkably, enhanced immune responses and strong protective efficacy against a highly virulent strain of N. seriolae infection were recorded in DNA vaccine-Cholesterol oxidase (pcD::Cho) injected fish with a relative per cent survival value of 73.3%. Generally, in the present study, we identified a strong association between genotype and enzymatic activity since the majority of pulsotypes displayed the same type of enzymatic profile. Additionally, we recognized an intimate relationship between genetics and the pathogenicity of N. seriolae in orange-spotted grouper, and the original host of pulsotypes explained the virulence of isolates in vivo. Different routes of administration were sufficient in inducing variable nocardiosis-associated clinical signs and mortality rates following N. seriolae infection. the IP route of transmission of the AOD107132-2K strain appears to be the most effective means of host inoculation in an experimental setting. In vaccine development, our results demonstrate that the Reverse Vaccinology approach is a valid strategy for screening vaccine candidates; and the pcD::Cho is a promising candidate that can boost both innate and adaptive immune responses, and confer considerable protection for controlling N. seriolae infection.