金門縣鼠形動物之恙蟲病病媒及病原體監測與分子鑑定

Surveillance and molecular identification on the vectors and pathogens of scrub typhus on murine-like animals in Kinmen County, Taiwan area

王錫杰(Hsi-Chieh Wang)

指導教授 ： 吳文哲

共同指導教授 ： 王重雄

國立臺灣大學/生物資源暨農學院/昆蟲學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.02049

摘要

1999年8月至2000年6月在金門縣金城鎮、金湖鎮、金沙鎮、金寧鄉及烈嶼鄉等五鄉鎮進行定期之捕鼠，並採集鼠身上恙蟎。發現金門縣的鼠形動物包括囓齒目 (Rodentia) 動物4種：黃胸鼠 (Rattus flavipectus)、屋頂鼠 (R. rattus)、溝鼠 (R. norvegicus) 及月鼠 (Mus caroli)；和食蟲目 (Insectivora) 動物1種--臭鼩 (Suncus murinus)。鼠形動物之總捕獲率為28.11%，其中以黃胸鼠數量最多 (91.67%)，臭鼩其次 (3.57%)。鼠形動物之帶蟎率以2000年4月最低為55%，2000年6月最高98%。金門縣鼠形動物恙蟎指數，以1999年8月最高為382，2000年4月最低為6。同一時期金門縣恙蟲病確定病例數與恙蟎指數呈現相關關係。鼠形動物之恙蟲病立克次菌抗體陽性率除2000年4月之檢出率為82.2%較低外，其餘月份均在90%以上。恙蟎種類鑑定結果，共發現5屬8種恙蟎，其中已知種類7種，未鑑定種1種，其各種與所佔比率分別為地里恙蟎 (Leptotrombidium deliense)：53.40%、小板恙蟎 (L. scutellare)：33.43%、中華無前恙蟎 (Walchia chinensis)：12.06%、于氏恙蟎 (L. yui)：0.70%、巨螯齒恙蟎 (Odontacarus majesticus)：0.28%、印度囊棒恙蟎 (Ascoschoengastia indica)：0.08%、合輪恙蟎屬一種 (Helenicula sp.)： 0.04%以及英帕恙蟎 (L. imphalum)： 0.01%。地里恙蟎於4月開始出現，至8月達高峰，是為金門縣明顯的夏季優勢種；小板恙蟎僅出現於11月至次年4月，為金門縣冬季優勢種。中華無前恙蟎全年皆可發現，但以夏季密度較高。金門縣之恙蟎帶病原情形以最小感染率 (minimum infection rate, MIR) 表示，地里恙蟎及小板恙蟎的MIR分別為12及5，此結果顯示地里恙蟎為可能夏季恙蟲病病媒，小板恙蟎為冬季自然儲主間恙蟲病病媒；由於中華無前恙蟎亦帶有立克次菌，MIR為8，其雖不叮咬人，但可能在動物間傳播病原有其重要性。經雙階聚合酶連鎖反應 (nested-PCR) 後之產物，以限制酶HhaI及SfaNI切割，發現金門縣恙蟎之恙蟲病立克次菌血清型型別以Karp及Gilliam為主，另發現有許多待定名型別的菌株。金門恙蟎鑑定過程中，發現許多恙蟎因形態變異而無法歸類，經將地里恙蟎飼養後，發現子代的形態變異範圍超過文獻之記錄，另建立以分子鑑定方法解決因形態變異而衍生的問題。以核醣體 DNA nested-PCR將金門縣所採集8種恙蟎之7種 (地里恙蟎、小板恙蟎、中華無前恙蟎、于氏恙蟎、巨螯齒恙蟎、印度囊棒恙蟎及合輪恙蟎屬一種) 之核醣體DNA ITS片段增幅定序後，所建立的親緣關係系統樹，與以形態特徵建立之系統樹完全相符。恙蟎核醣體DNA ITS片段在種內相似度超過97%，纖恙蟎屬 (Leptotrombidium) 屬內相似度為82-85%，以此檢驗形態變異之同種恙蟎，證實此片段可輔助恙蟎種之鑑定。另以nested-PCR 配合限制酶 (MspA1 I、Nla III、BsiHKA I 或 Tfi I ) 進行恙蟎核醣體DNA ITS片段RFLP分析，可將7種恙蟎清楚區分。以此建立的方法，使用同樣的DNA模板 (template)，除可鑑定恙蟲種類外，並可檢測其所攜帶立克次菌之血清型，將有助於防疫人員更迅速瞭解恙蟲病病媒種類與帶原情形。

關鍵字

恙蟲病 ； 恙蟲 ； 雙階聚合酶連鎖反應 ； 恙蟲病立克次菌之血清型 ； 鼠形動物儲主 ； 分子鑑定

並列摘要

Five species of murine-like animals were obtained in five towns of Kinmen County, Taiwan area from August 1999 to June 2000. Four species were members of the Muridae of the Rodentia, Rattus flavipectus, R. rattus, R. norvegicus and Mus caroli, and one species was of the Soricidae of the Insectivora, Suncus murinus. The total capture rate of murine-like animals was 28.11%. Among these specimens, R. flavipectus was dominant, representing 91.67% of the collected animals, followed by S. murinus (3.57%). The percentage of animals infected with chiggers ranged from 55% to a peak of 98%, while the mean number of chiggers per animal ranged from six to 382. My study showed that the seasonal variation in the mean number of chiggers per animal was slightly correlated with the seasonal incidence of human infection. Except for April 2000, the seropositive rates of these animals against Orientia tsutsugamushi exceeded 90% in each survey. Eight species of chiggers were identified, including Leptotrombidium deliense (53.40%), L. scutellare (33.43%), Walchia chinensis (12.06%), L. yui (0.70%), Odontacarus majesticus (0.28%), Ascoschoengastia indica (0.08%), Helenicula sp. (0.04%), and L. imphalum (0.01%). My survey showed that L. deliense appeared from April to November, with a peak occurring in August, whereas L. scutellare appeared from November to April, with a peak occurring in December. Walchia chinensis occurred throughout the year, but was more abundant in summer. The state of O. tsutsugamushi infection was demonstrated by the minimum infection rate (MIR). The MIRs of L. deliense and L. scutellare were 12 and 5, respectively. These results indicate that L. deliense may be the vector in summer, while L. scutellare may transmit disease in winter. Walchia chinensis has not been reported to bite humans, but it was found to harbor O. tsutsugamushi with an MIR of 8, and it may transmit rickettsia among animal reservoirs. In this study, the products of nested polymerase chain reactions (nested-PCR) with special primer sets for O. tsutsugamushi 56 kDa gene and mite’s total DNA templates were digested with two restriction enzymes, HhaI and SfaNI, the resulted profiles could be used to identify serotypes of O. tsutsugamushi. Two serotypes, Karp and Gilliam, were dominant, but several unidentified local strains existed in Kinmen Country. During identification process, I found many chiggers could not be classified due to morphological variations. The offspring of reared L. deliense in laboratory showed the morphological variation that was over the descriptions of literatures. However, the difficulty could be solved by molecular discrimination instead. Using nested-PCR with same DNA templates and designated primer sets for mite’s ITS region, I had carried out the sequences of seven mite’s ribosomal DNA ITS regions from collected eight chiggers (L. deliense, L. scutellare, Walchia chinensis, L. yui, Odontacarus majesticus, Ascoschoengastia indica, Helenicula sp., except L. imphalum). The phylogenetic tree was constructed based on the sequences of mite’s ribosomal DNA ITS regions, the results were coincident with the tree constructed by morphological characters. The ITS sequences of intraspecific mites was over 97% identity, but only 82~85% was found in interspecies of the genus Leptotrombidiun. I examined the morphological variation of ambiguous chiggers, and proved that ribosomal DNA ITS region could help to differentiate morphological variations among chigger species. The products of nested-PCRs were digested with 4 restriction enzymes (MspA1 I, Nla III, BsiHKA I or Tfi I) and these seven examined mites could be discriminated by their RFLP profiles. This combination of nested-PCR and RFLP might be an available tool for detecting not only chigger species, in spite of the morphological variation, and also its harbored rickettsia with the same DNA template and special primer sets.

並列關鍵字

Orientia tsutsugamushi serotypes ； scrub typhus ； molecular identification ； chigger mites ； murine-like animal reservoirs ； nested polymerase chain reaction

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