韋永芳,陳代雄,梁成結(jié),羅曉東,朱郇憫
廣州管圓線蟲(chóng)(Angiostrongyluscantonensis)是引起嗜酸粒細(xì)胞增多性腦膜炎和腦膜腦炎的重要病原體。人多因食生或半生含有廣州管圓線蟲(chóng)Ⅲ期幼蟲(chóng)的中間宿主(如褐云瑪瑙螺等)而感染。由該蟲(chóng)引起的廣州管圓線蟲(chóng)病已在多個(gè)國(guó)家先后暴發(fā)[1-4]。國(guó)內(nèi)外學(xué)者認(rèn)為長(zhǎng)爪沙鼠(Mongolian gerbil,Meriones unguiculatus)對(duì)多種原蟲(chóng)、線蟲(chóng)、絳蟲(chóng)和吸蟲(chóng)等實(shí)驗(yàn)性感染易感,現(xiàn)已成功建立了包括馬來(lái)絲蟲(chóng)、藍(lán)氏賈弟鞭毛蟲(chóng)、微小膜殼絳蟲(chóng)、類圓線蟲(chóng)等多種寄生蟲(chóng)的長(zhǎng)爪沙鼠感染動(dòng)物模型[5-9],本研究以廣州管圓線蟲(chóng)第Ⅲ期幼蟲(chóng)感染長(zhǎng)爪沙鼠,以探討廣州管圓線蟲(chóng)對(duì)長(zhǎng)爪沙鼠的致病性。
1.1材料
1.1.1褐云瑪瑙螺(Achatinafulica) 采集自廣州市番禺區(qū)。
1.1.2長(zhǎng)爪沙鼠 體重45~60 g之間,雌雄各半,由廣州醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心提供[SYXK(粵)2010-0104]。
1.2方法
1.2.1廣州管圓線蟲(chóng)第3期幼蟲(chóng) 參照李華等[10]報(bào)道的方法從褐云瑪瑙螺中分離。將褐云瑪瑙螺去殼,稱重后剁碎,每1 g螺肉加入20 mL人工消化液(配置方法:每1 000 mL消化液含3 g胃蛋白酶、7 mL濃鹽酸、993 mL水),37 ℃恒溫箱中消化2 h后經(jīng)篩網(wǎng)過(guò)濾,濾液靜置30 min后,置于方皿中,解剖鏡下檢查廣州管圓線蟲(chóng)第Ⅲ期幼蟲(chóng)(L3)。
1.2.2動(dòng)物感染 參照陳代雄等[11]方法進(jìn)行。長(zhǎng)爪沙鼠稱重后,隨機(jī)分為對(duì)照組20只,腹腔注射生理鹽水;感染組50只,腹腔注射廣州管圓線蟲(chóng)第3期幼蟲(chóng),10條/鼠。
1.3觀察指標(biāo)
1.3.1動(dòng)物一般行為觀察 觀察感染后長(zhǎng)爪沙鼠的生理學(xué)變化,記錄長(zhǎng)爪沙鼠的死亡情況。
1.3.2檢獲蟲(chóng)體數(shù)和蟲(chóng)體的組織分布 分別在感染后不同時(shí)間(第7、21、28、35、46 d)剖殺部分感染的長(zhǎng)爪沙鼠和檢查實(shí)驗(yàn)過(guò)程中瀕死或死亡的感染沙鼠,分離腦和心肺組織,先用肉眼及解剖鏡觀察,如有幼蟲(chóng)則檢獲,余下組織經(jīng)充分撕碎后加人工消化液置于37 ℃水浴箱消化2 h,再行過(guò)濾、沉淀,解剖顯微鏡下檢獲蟲(chóng)體。記錄各組織中檢獲的雌雄蟲(chóng)體數(shù)量,并觀察蟲(chóng)體形態(tài)結(jié)構(gòu)。
1.3.3組織病變觀察 分離感染后不同時(shí)間的腦和肺組織,肉眼觀察其病變特點(diǎn);福爾馬林固定后,進(jìn)行石蠟包埋、切片和HE染色。
2.1動(dòng)物一般行為變化 長(zhǎng)爪沙鼠在感染后第8 d即有不同程度食欲減退、萎靡、活動(dòng)減少等;約第16 d部分沙鼠出現(xiàn)被毛散亂、閉眼、脊柱側(cè)彎等癥狀;感染后第17~18 d,部分長(zhǎng)爪沙鼠出現(xiàn)步態(tài)不穩(wěn),打轉(zhuǎn)和癱瘓等神經(jīng)系統(tǒng)癥狀,隨著時(shí)間推移,出現(xiàn)這些癥狀的長(zhǎng)爪沙鼠數(shù)量增加,癥狀也逐漸加重,出現(xiàn)不同程度進(jìn)食和飲水困難。感染后第23 d開(kāi)始出現(xiàn)死亡,23~30 d為死亡的高峰時(shí)間,瀕死或死亡的沙鼠共28只;除了剖殺和死亡外,最終有12只能生存至感染后第46 d。
2.2檢獲蟲(chóng)體數(shù) 對(duì)照組長(zhǎng)爪沙鼠腦組織均未檢獲到廣州管圓線蟲(chóng)。感染后第7 d未能從腦組織中檢獲蟲(chóng)體;感染后第23~46 d檢查鼠35只(包括活的、頻死的和死的),共檢獲蟲(chóng)體127條(♀62/♂65),蟲(chóng)體收獲率約為36.29%(127/350),平均蟲(chóng)數(shù)為(3.63±1.46)/鼠。感染后第21 d長(zhǎng)爪沙鼠腦組織中即檢獲出第Ⅴ期幼蟲(chóng),感染后腦中的蟲(chóng)體數(shù)量隨時(shí)間呈現(xiàn)出不規(guī)則上升,23~30 d達(dá)峰值后逐漸下降;而第27~30 d后,心肺組織中呈現(xiàn)出不規(guī)則上升,檢獲的蟲(chóng)體大部分已發(fā)育成Ⅴ期幼蟲(chóng)或成蟲(chóng),至41 d后發(fā)育為性成熟的成蟲(chóng),感染后第46 d從存活的12只沙鼠的肺動(dòng)脈中均檢獲到雌雄蟲(chóng)寄生和單性寄生蟲(chóng)體,6只為雌雄蟲(chóng)寄生,其中4只糞便中查到廣州管圓線蟲(chóng)Ⅰ期幼蟲(chóng)((圖1 A1-A2);6只為單性蟲(chóng)體寄生,糞便中均未查到Ⅰ期幼蟲(chóng)。長(zhǎng)爪沙鼠感染廣州管圓線蟲(chóng)L3不同時(shí)間后,其腦和心肺內(nèi)蟲(chóng)體的分布見(jiàn)表1。
表1 感染后不同時(shí)間長(zhǎng)爪沙鼠的腦和心肺組織檢獲的蟲(chóng)體數(shù)
Note: * Brain and lung of other 10 infected gerbils were used for pathological examination.
2.3病理變化
2.3.1肉眼觀察 長(zhǎng)爪沙鼠感染廣州管圓線蟲(chóng)后第7 d腦組織病理變化不明顯。感染后第23 d,長(zhǎng)爪沙鼠以腦部病變?yōu)橹?,可?jiàn)腦組織表面有不同程度的血管擴(kuò)張與充血、出血及粘連;而肺部呈彌漫性充血改變。感染后第38~46 d,以肺部病變?yōu)橹?,肺組織表現(xiàn)為不同程度局灶性充血腫脹,表面及切面質(zhì)硬粗糙呈灰白色顆粒狀,還可見(jiàn)到點(diǎn)狀小片狀出血灶;腦組織病變較輕,只有局限性血管擴(kuò)張和充血(圖2 A1-B3)。
2.3.2病理切片觀察 感染后第25 d蛛網(wǎng)膜下腔可見(jiàn)蟲(chóng)體,蟲(chóng)體周圍有出血。第38 d,蛛網(wǎng)膜下腔仍可見(jiàn)蟲(chóng)體,肺內(nèi)可見(jiàn)多個(gè)橢圓形蟲(chóng)卵;第45 d,肺內(nèi)可見(jiàn)多個(gè)橢圓形蟲(chóng)卵,蟲(chóng)卵為多細(xì)胞期或幼蟲(chóng)期,部分肺泡輪廓消失和大量炎癥細(xì)胞浸潤(rùn)。
圖1檢獲的廣州管圓線蟲(chóng)
Fig.1WormrecoveryofA.cantonensis
A: Adult worm; B:A.cantonensis1st-stage larvae in stools (10×10)
圖2 長(zhǎng)爪沙鼠感染廣州管圓線蟲(chóng)后腦和心肺組織的變化
大鼠和小鼠是目前多數(shù)使用的廣州管圓線蟲(chóng)感染動(dòng)物模型,大鼠是廣州管圓線蟲(chóng)的適宜終宿主,而小鼠、豚鼠、兔、恒河猴和人類是其非適宜宿主,廣州管圓線蟲(chóng)可通過(guò)適宜宿主大鼠的中樞神經(jīng)系統(tǒng)(CNS)移行到肺動(dòng)脈,并在心肺發(fā)育成熟;非適宜宿主,大多數(shù)L3幼蟲(chóng)移行到腦,經(jīng)歷兩次蛻皮,未成熟的成蟲(chóng)移行到蛛網(wǎng)膜下腔,但在心肺未能發(fā)育為性成熟[12-15]。以往的研究證實(shí)長(zhǎng)爪沙鼠是一種對(duì)多種寄生蟲(chóng)易感的實(shí)驗(yàn)動(dòng)物,是馬來(lái)絲蟲(chóng)、微小膜殼絳蟲(chóng)(肥頭絳蟲(chóng)和多房棘球絳蟲(chóng))的終宿主,幼蟲(chóng)可在沙鼠中建立、生長(zhǎng)、發(fā)育到妊娠的成年階段[5,7],此外也用于線蟲(chóng)的感染,如棘唇線蟲(chóng)、類圓線蟲(chóng)、美洲板口線蟲(chóng)等[8,17-19]。長(zhǎng)爪沙鼠用于廣州管圓線蟲(chóng)感染研究很少見(jiàn)于報(bào)道[20-21]。過(guò)往的研究中長(zhǎng)爪沙鼠被認(rèn)為是廣州管圓線蟲(chóng)適宜宿主但不大易感,我們的研究結(jié)果顯示廣州管圓線蟲(chóng)在感染沙鼠后約3周開(kāi)始從腦移行至肺,在肺中發(fā)育至成蟲(chóng)并產(chǎn)卵,與大鼠的情況相似[22],符合適宜宿主特點(diǎn)。然而感染后第46 d,仍有部分蟲(chóng)體滯留于腦,且到達(dá)肺的蟲(chóng)體仍有個(gè)別未完全發(fā)育成熟;此外,與大鼠比較[12-15],長(zhǎng)爪沙鼠對(duì)廣州管圓線蟲(chóng)感染耐受性明顯較差,曾腹腔注射35條幼蟲(chóng)/鼠、15條幼蟲(chóng)/鼠廣州管圓線蟲(chóng)第3期幼蟲(chóng),第32 d 80%以上的沙鼠死亡,即使感染蟲(chóng)數(shù)減為10條幼蟲(chóng)/鼠,感染后第30 d 62%的沙鼠死亡。
長(zhǎng)爪沙鼠通常在感染2周后出現(xiàn)明顯的神經(jīng)系統(tǒng)癥狀,如步態(tài)不穩(wěn),打轉(zhuǎn)和癱瘓等,感染3周后出現(xiàn)死亡;在感染后第23~46 d,腦內(nèi)蟲(chóng)體數(shù)量逐漸減少,肺內(nèi)蟲(chóng)體數(shù)量逐漸增加(表2),提示蟲(chóng)體由腦向肺移行。肉眼和病理檢查結(jié)果提示長(zhǎng)爪沙鼠感染后30 d 內(nèi)以腦損害為主,表現(xiàn)為腦膜充血、出血及粘連等特點(diǎn)的嗜酸性粒細(xì)胞增多性腦膜炎。30 d后以肺部損害為主,成蟲(chóng)可在肺部產(chǎn)卵形成蟲(chóng)卵結(jié)節(jié),表現(xiàn)肉芽腫性肺炎[23],這與大鼠的病理?yè)p傷一致[24]。實(shí)驗(yàn)中發(fā)現(xiàn)感染后腦組織中未查出蟲(chóng)體的長(zhǎng)爪沙鼠的腦組織變化程度明顯輕微,出現(xiàn)癥狀的時(shí)間也推后,推測(cè)這與蟲(chóng)數(shù)多寡在腦組織中的移行造成腦組織的機(jī)械損傷,以及蟲(chóng)體的代謝、分泌產(chǎn)生的毒性產(chǎn)物導(dǎo)致了腦組織非特異性炎性病變反應(yīng)有關(guān)。
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