臺灣病豬副豬嗜血桿菌分離株血清型、基因型與潛在毒力相關因子之關聯性分析

副豬嗜血桿菌（Haemophilus parasuis, H. parasuis）為豬隻上呼吸道常在菌之一，普遍存在於各年齡層豬群中，為格氏病（Glässer’s disease）之病原，可造成豬隻急性暴斃、多發性漿膜炎、關節炎及腦膜炎，導致飼料換肉率下降，對養豬產業造成嚴重經濟損失。不同H. parasuis菌株具有之毒力相關因子並不一致且其致病力各有差異。為了解H. parasuis血清型、基因型及毒力相關因子之關聯性，以利致病機轉研究、疫苗開發與疾病防治，本研究蒐集從2013年8月至2017年3月之間自疑似H. parasuis感染之豬隻之病變組織分離細菌及病理學檢查，並將分離之H. parasuis菌株進行分子血清分型、基因分型及潛在毒力相關因子基因檢測。將取得之133株H. parasuis菌株利用分子血清分型配合序列分析，發現台灣盛行之血清型主要為第5/12型（37.6%）、第4型（27.8%）、第13型（15%）及無法分型菌株（13.5%）。腸道細菌基因間重複序列聚合酶鏈鎖反應（Enterobacterial repetitive intergenic consensus polymerase chain reaction, ERIC-PCR）結果可見眾多群集，顯示其基因遺傳具有多變性，但部分菌株之基因型與血清型、致病型間存在關聯性，且同一豬場存在多種致病基因型。多數H. parasuis感染之病例可見肺炎病變，且多數肺臟分離菌株與漿膜分離菌株之血清型與基因型相同。H. parasuis於肺臟病變之分離率最高（30.4%），顯著高於胸膜（20.1%）、心囊膜（16.2%）、腹膜（13.7%）、腦膜（10.3%）及關節囊膜病灶（9.3%）。血清型第5/12型、第4型與第13型之致病型別分析結果可見同時導致漿膜炎及肺炎病灶病例比例最高，僅導致漿膜炎病灶病例比例次之，兩者皆顯著高於僅導致肺炎病灶病例。然而，血清型無法分型菌株同時導致漿膜炎及肺炎病灶病例比例顯著高於僅導致漿膜炎病灶。經由血清分型與基因分型，12個送檢豬群（9.2%）可自不同豬隻、同一個體甚至同一病灶分離出一株以上菌株，且ERIC-PCR之菌株鑑別效果優於分子血清分型。與菌株侵犯能力相關之第一群毒力相關三聚體自轉運蛋白（virulence associated trimeric autotransporters, vta）基因普遍存在於台灣H. parasuis致病菌株中（98.6%）。綜上所述，台灣H. parasuis菌株之莢膜生合成基因座（capsule locus）存在基因序列變異，可能進一步使原有之血清型抗原多樣性更加複雜化，因而影響不活化死菌疫苗成效。藉由基因型高度識別力可辨別菌株並追朔來源以研究流行病學。肺臟病灶可做為H. parasuis分離之良好標的。決定血清型別之莢膜與致病型間可能存在部分關聯性。豬場中普遍存在多種不同血清型與基因型之致病性H. parasuis菌株，未來需研發可提供良好交叉保護力之廣泛性疫苗以利疾病防治。第一群vta基因可做為台灣H. parasuis感染症之疾病防治、監控之指標與疫苗候選抗原。

關鍵字

副豬嗜血桿菌；格氏病；血清型；基因型；毒力相關因子

並列摘要

Haemophilus parasuis (H. parasuis), a part of normal upper respiratory microbiota, is the etiological agent of Glässer’s disease which induces sudden death, polyserositis, polyarthritis, meningitis and poor production performance, resulting in severe economic losses in the swine industry. Virulence-associated factors (VAFs) of different H. parasuis strains are inconsistent and then vary the pathogenicity. For beneficial studies on pathogenesis, vaccine development and disease prevention and control, the correlation between serovar, genotype and potential VAFs of H. parasuis were investigated. Since August 2013 to March 2017, H. parasuis have been isolated from meninges, pleura, pericardium, peritoneum, synovial cavities and lung lesions of diseased pigs. H. parasuis isolates were serotyped by molecular molecular assay, genotyped by enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) and potential VAFs genes detected. Of 133 H. parasuis strains, the prevalent serovars are serovars 5/12 (37.6%), 4 (27.8%) and 13 (15%) fallowed by molecular serotyping non-typable strains (13.5%). The genotypic dendrogram of ERIC-PCR revealed heterogeneous genetic diversity with many clusters. Partial correlation between the ERIC-PCR clusters of different strains, serovars and lesion patterns was observed, and various pathogenic H. parasuis genotypes existed in pig farms. Most Glässer’s disease cases had pneumonia, and both serovars and genotypes of most isolates from lung and serosal lesions of same individuals were consistent. The isolation rate of lung lesions (30.4%) was significantly higher than that of pleura (20.1%), pericardium (16.2%), peritoneum (13.7%), meninges (10.3%) and synovial cavities (9.3%). Necropsied animals infected serovars 5/12, 4 and 13 H. parasuis with both serosal and pulmonary lesions were the most frequent, followed by those with only serosal lesions and those with only pulmonary lesions were significantly lesser than both of them. However, Necropsied animals infected molecular serotyping non-typable strains with both serosal and pulmonary lesions were significantly more frequent than those with only serosal lesions. According to serovars and genotypes, 12 herds (9.2%) were infected with more than one H. parasuis strains which were isolated from different pigs, same individuals and even same lesions. ERIC-PCR had better discriminatory capability than molecular serotyping assay. Group 1 virulence associated trimeric autotransporters (G1vta) genes associating with invasion ability are common in Taiwanese pathogenic H. parasuis isolates (98.6%). In summary, there may be deletions and/or unknown sequences in capsular polysaccharide biosynthesis loci which may result in enhancing antigenic variation then impairing inactivated vaccine effectiveness. Genotyping with better discriminatory capability can differentiate isolates and be applied for epidemiologic surveillance. Pulmonary lesions are important for H. parasuis isolation. There may be some correlation between pathotypes and serovars defined by capsule. Various pathogenic H. parasuis serovars and genotypes exist in pig farms. Therefore, it is crucial to develop universal vaccines with good cross-protection for disease prevention and control. G1vta can be selected as indicators for Taiwanese H. parasuis disease prevention, control and monitoring and vaccine candidates.

並列關鍵字

Haemophilus parasuis ； Glässer’s disease ； serotype ； genotype ； virulence-associated factors ； Glaesserella parasuis

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