長期保存的犬蛔蟲受孕卵於鼷鼠之感染及台灣山地原住民成人犬蛔蟲感染之研究

范家堃

doi:10.6342/NTU.2004.00931

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長期保存的犬蛔蟲受孕卵於鼷鼠之感染及台灣山地原住民成人犬蛔蟲感染之研究

Studies of Mice Infected with Long-Term-Maintained Embryonated Eggs of Toxocara canis and T. canis Infection among Mountain Taiwanese Aboriginal Adults

范家堃 , Ph.D　　Advisor：蘇霩靄　　

英文 DOI： 10.6342/NTU.2004.00931

長期保存；犬蛔蟲；受孕卵；鼷鼠；台灣；山地原住民；成人；犬蛔蟲感染； Mice ； Long-Term-Maintained ； Embryonated Eggs ； Toxoc

Abstract │ Reference (63) │ Altmetrics

Abstract 〈TOP〉

本研究探討儲存於2％福馬林液內，於4 oC下保存14個月之犬蛔蟲受孕卵，對鼷鼠的感染性、致病性、幼蟲的移行行為和TGF-β1、TG2、collagen和apoptosis於所引致的肉芽腫肝炎的表現情形。感染鼷鼠後第1、2、3、5天至第1、4、8、12、16、20、24、28、42與67週，麻醉後以心臟穿刺採血致死收取血清，另將每隻鼠一半的肝臟和肺臟(肝肺期)與肌肉和腦部(神經肌肉期)組織製成蠟塊，進行組織學與免疫化學染色；另一半的內臟組織以酸性的胃蛋白酶消化進行幼蟲回收實驗。選擇台灣山地原住民成人為血清流行病學的研究對象以瞭解台灣地區人羣感染犬蛔蟲症的情況。感染鼠整體的免疫反應類型主要朝向Th2，因血清中IgG1抗體力價於整個實驗中明顯較未感染控制鼠的高 (P < 0.05)；然而Th1免疫反應的指標，IgG3抗體力價於整個實驗中，除在第八週於感染組鼠中較未感染控制組鼠的高 (P < 0.05)外，其餘感染組鼠與未感染的控制組鼠間並無明顯差異 (P > 0.05)。於整個實驗中，肝臟與肺臟的病變為中度(2+)至重度(3+)，肌肉的病變為輕度(1+)至中度(2+)，而腦部則為正常(0+)至輕度(1+)。感染第8週後大部分的鼠膽管上皮細胞可見似apocrine的特殊病變。另外靠近肌肉區的柔軟組織可於數個感染組鼠發現包住幼蟲的肉芽腫。大部分的幼蟲於感染後7天內移行至肝肺期，此後，大部分的幼蟲轉為移行至神經肌肉期。綜合而言，不僅保存14個月的犬蛔蟲受孕卵的感染性、致病性與孵化的幼蟲於小鼠體內的移行行為皆保留不變，孵化的幼蟲亦能於小鼠體內引起一些特殊的病變。TGF-β1主要被偵測於病灶處的浸潤細胞與膽管上皮細胞，分別於感染後第12週與第24週有最高的表現量。幼蟲本身亦有相當強的TGF-β1的表現。TG2主要被偵測於膽管上皮細胞與於病灶內或圍繞病灶周圍的肝細胞，分別於感染後第1週與第28週有最高的表現量。肝細胞、膽管上皮細胞與浸潤細胞罕見凋亡的發生。TGF-β1可能與TG2合同刺激膠原蛋白的生成，因TGF-β1和TG2的表現分別與膠原蛋白的表現間有統計學上的相關性。表現TG2而圍繞於病灶週邊的肝細胞形成一屏障似地，似乎可限制幼蟲移行甚或阻止病灶擴增，然而持續且不正常的膠原蛋白的堆積和罕見浸潤細胞凋亡的表現，可能造成肝臟不可逆性的纖維化損傷和持續性的發炎。本結果或可提供發展有效的控制藥物或疫苗的策略，藉調控TGF-β1與TG2的表現，達到治療由其他微生物所造成的類似肝病。5個原住民族群的成人整體血清抗體陽性率(46.0%, 247/537)明顯高於漢族(30.2%, 13/43) (P = 0.04)。年齡而非性別，似乎是與血清陽性有關的一個因子。有攝食生豬肝經歷(Odds Ratio (OR) = 1.65, P < 0.01)，眷養狗經驗(OR = 1.76, P < 0.01)，或職業是勞工(OR = 1.78, P = 0.01)的原住民成人，似乎較那些沒有上述經歷者與無工作者易於受犬蛔蟲的感染。

Parallel Abstract 〈TOP〉

Infectivity, pathogenicity, and larval migratory behavior as well as expression of TGF-β 1, TG2, collagen, and apoptosis in granulomatous hepatitis induced by embryonated eggs of Toxocara canis, which had been experimentally maintained in 2% formalin for 14 months at 4 oC, were evaluated in mice at day 1, 2, 3, 5 and week 1, 4, 8, 12, 16, 20, 24, 28, 42, and 67 weeks post infection (DPI or WPI). After sacrifice by heart puncture, serum were collected and the half part of the liver and lungs (hepatopulmonary phase) and the musculature and brain (myotropic-neurotropic phase) were processed for histological and immunohistochemical staining; the other half of the 4 organs were processed for acid-pepsin digestion for larva recovery assay. In addition, a seroepidemiological study was performed to understand the status of T. canis infection among mountain Taiwanese aboriginal adults.The overall immunological pattern of infected mice tended to be towards the Th2 type response. Serum IgG1 antibody titers in infected mice were significantly higher than that of the uninfected control mice throughout the trial (P < 0.05). While, no significant difference in titers of IgG3 antibody, an indicator for the Th1 type response, was observed between the infected and control mice, except at 8 WPI (P < 0.05). Inflammatory injury was moderate to severe in the liver and lungs, mild to moderate in the musculature, and only normal to mild in the brain throughout the trial. Apocrine-like change in epithelial cells of the bile duct was observed in most of the infected mice from 8 WPI onward. Moreover, larvae trapped by organized granuloma was found in soft tissue near the musculature at some groups of infection. Most 14-month-maintained T. canis larvae entered the hepatopulmonary phase of infection within 7 DPI; afterwards, larvae migrated throughout the body and principally exhibited the myotropic-neurotropic phase of infection. In conclusion, not only were the infectivity and pathogenicity as well as larval migratory behavior of the 14-month- maintained T. canis embryonated eggs retained, the hatched larvae were also capable of eliciting some special pathological changes in the murine host. TGF-β1 was detected mainly in cholangiocytes and infiltrating cells in lesions, with a peak at 12 and 24 WPI, respectively. Larvae per se also exhibited strong TGF-β1 expression in the trial. TG 2 was detected predominantly in cholangiocytes and hepatocytes which surrounded the lesions, reaching a peak at 1 and 28 WPI, respectively. Apoptosis was rarely detected in hepatocytes, cholangiocytes, and infiltrating cells in the trial. TGF-β 1 and TG 2 might collaborate to up-stimulate collagen synthesis due to a statistical correlation between TGF-β 1, TG 2, and collagen expression, respectively. Although a barrier constituted by TG 2-expressing hepatocytes encircling the lesions might limit larval migration and/or prevent the progressive expansion of the lesions, abnormally persistent collagen accumulation and rare inflammatory cells apoptosis may cause irreversible fibrotic injury and persistent inflammation in the liver. Present results may provide a strategy for the development of effective drug or vaccine to treat similar liver diseases caused by other microbes through manipulating both TGF-β1 cytokine and TG 2 expression. The overall seroprevalence (46.0%, 247/537) in the 5 aboriginal populations was significantly higher than that of ethnic Han population (30.2%, 13/43) (P = 0.04). Age, but not gender, seemed to be a factor related to positive serology. Aboriginal adults who had histories of eating raw pig liver (OR = 1.65, P < 0.01), raising dogs (OR = 1.76, P < 0.01), or whose occupation was laborer (OR = 1.78, P = 0.01) seemed to be more apt to be infected by T. canis than those without such histories and unemployed persons.

Reference ( 63 ) 〈TOP〉

Abo-Shehada MN, Herbert IV. 1984. The migration of larval Toxocara canis in mice. I. Migration through the intestine in primary infections. Vet Parasitol 17: 65-73.
連結：
Ajayi OO, Dunhlinska DD, Agwale SM, Njoku M. 2000. Frequency of human toxocariasis in Jos, Plateau State, Nigeria. Mem Inst Oswaldo Cruz 95: 147-149.
連結：
Akao N, Goto Y, Kondo K, Tsuda Y. 1993. Changing chemosusceptibility in the second-stage larvae of Toxocara canis by long-term incubation. J Helminthol 67: 145-150.
連結：
Anaruma FF, Chieffi PP, Correa CR, Camargo ED, Silveira EP, Aranha JJ, Ribeiro MC. 2002. Human toxocariasis: a seroepidemiological survey in the municipality of Campinas (SP), Brazil. Rev Inst Med Trop Sao Paulo 44: 303-307.
連結：
Badley JE, Grieve RB, Bowman DD, Glickman LT, Rockey JH. 1987. Analysis of Toxocara canis larval excretory-secretory antigens: physicochemical characterization and antibody recognition. J Parasitol 73: 593-600.
連結：

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