雞傳染性貧血的病毒學與致病性研究

Virological and pathogenic characteristics of chicken infectious anemia

金保存(Suttitas Tongkamsai)

指導教授 ： 連一洋

國立屏東科技大學/獸醫學院/獸醫學系所/博士(2020年)

https://doi.org/10.6346/NPUST202000160

摘要

雞傳染性貧血(chicken infectious anemia, CIA)是由雞傳染性貧血病毒(chicken anemia virus, CAV)所引起的一種具有高度傳染性的免疫抑制性疾病，常造成雞隻貧血及對於其他病原的易感性。雞傳染性貧血病毒屬於Anelloviridae科，為不具封套的二十面體病毒，直徑約25 nm。本研究檢驗45個野外分離出的CAV病毒株，其中26株在MSB-1細胞培養中發育良好，它們之間的抗原性並未觀察到有差異性。以1日齡小雞檢測8株CAV病毒株的致病性，發現除了05-19毒株表現較輕微之外，其他病毒株皆在雞隻表現出較明顯的臨床症狀。以即時定量PCR (real-time qPCR) 方法觀察CAV病毒株的核酸複製動力曲線，發現其中05-19毒株在相同感染時間下，其濃度顯著低於04-35和06-30毒株，推測可能是因為其VP1基因之5’端發生了無義突變 (nonsense mutation) ，進而弱化了病毒的複製能力。分子流行病學研究顯示CAV 06-09分離毒株的核酸序列和疫苗株極為相似。依據台灣CAV基因組序列親緣關係分析，顯示所有45個分離株之間具有94.2%-99.8%的相似度，且屬於全球廣為分佈的基因二型與基因三型。有鑑於近十年CAV造成的亞臨床感染病例增加情形，本研究進行了CAV免疫抑制特性對雞宿主的影響，從中發現雞隻不理想的抗體反應與CAV持續存在於胸腺有關。在模擬養禽場自然糞口傳播病毒的實驗中，得到病毒持續感染與抗體反應缺乏相關的證據。先前文獻指出CAV造成的臨床症狀具有年齡抵抗性，然而與在台灣養禽場的現狀卻有些出入。本實驗以高劑量CAV接種成雞後，發現能誘發短暫性貧血現象，但骨髓中的病毒基因Copy數卻未增加，反而在周邊血液紅血球中檢測到CAV的存在，顯示在較大雞齡的成雞，CAV侵襲的目標細胞已由骨髓中的Hemocytoblast轉移到周邊血液的成熟紅血球細胞，而所觀察到的貧血是由於這些細胞的凋亡所致。此外，在血清陽性雞隻的周邊血液單核球細胞中仍可檢測到CAV的存在，這代表單核球細胞是病毒持續性感染的目標細胞。雞傳染性貧血病毒感染可以促進行其他疾病的發展，本實驗也評估了CAV和柔嫩艾美球蟲 (Eimeria tenella) 合併感染時的病原性效應。透過觀察 E. tenella所引起的腸道出血症狀的加重以及卵囊排出量的增加，我們證實了CAV所造成的免疫抑制作用確可加重E. tenella的發病機制。

關鍵字

雞貧血病毒 ； 病毒學 ； 致病機序

並列摘要

Chicken anemia virus (CAV), the member of the family Anelloviridae, is a nonenveloped, icosahedral virus with about 25 nm in diameter. The virus causes chicken infectious anemia (CIA) in chickens, which is a highly contagious and immunosuppressive viral disease of chickens, causing anemia enhanced susceptibility to other pathogens. Forty-five CAV isolates were detected from tissue sample cases. Twenty-six CAV isolates grew well in MSB-1 cells and are not different in antigenicity among Taiwanese isolates. The pathogenicity of 8 CAV isolates was studied in day-old SPF chicks. The pathogenicity study indicated that all the CAV were able to develop clinical disease except isolate 05-19 which show lesser severe clinical disease than the other isolates. The CAV growth curve was performed using viral replication kinetics assays. The isolate 05-19 was detected significantly low concentration compared with isolate 04-35 and 06-30. The point-nonsense mutation was found on the 5’ of VP1 gene of the isolate 05-19, which may cause the reduction of viral replication. The epidemiology study of CAV showed that the nucleotide sequence of the isolate 06-09 is very similar to a vaccine strain. Phylogenetic analysis of Taiwanese CAVs from this study revealed that all the 45 isolates had 94.2 - 99.8 % homology among themselves and they were classified into two distinct genotypes II and III showing a worldwide distribution. Considering the increased subclinical infection of CAV cases in the last decades, we have examined the effects of immunosuppression in chicken host. Sub-optimal antibody responses were associated with CAV persistency in the thymus. The viral persistence and lack of antibody responses were investigated in the oral inoculated group, which was naturally occurred in the field. The age resistance to clinical diseases caused by CAV was experimentally demonstrated while it may not always be valid in the field. The transient anemia was showed in the high dose inoculated group, and even the viral genome copies did not increase in the bone marrow during the entire trial period. The symptoms of anemia in older chickens are due to apoptosis of the peripheral erythrocytes directly infected by CAV. In addition, CAV can still be detected in the mononuclear cell in seropositive chickens, indicating that the mononuclear cell is the target cell for persistent infection. CAV infection can facilitate the development of other diseases. The effects of pathogenic CAV and E. tenella coinfection were evaluated. The exposure to CAV had an effect on the pathogenicity of E. tenella induced severe bleeding. Due to their immune-deficient effects result in interfered with the development of immunity to E. tenella, which can be measured by increases of oocyst production. The findings of the present experiment revealed the immunosuppressive effects of CAV and aggravated the pathogenesis of E. tenella infection.

並列關鍵字

Chicken anemia virus ； Virology ； Pathogenicity

參考文獻

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