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標題: 探討影響犬蛔蟲蟲卵傳播之因子及以糞內抗原診斷狗之消化道犬蛔蟲的感染
作者: 陳美君
關鍵字: 犬蛔蟲
出版社: 獸醫學系
摘要: 摘要 本論文分為三部分:(a)討論氧化還原劑對犬蛔蟲蟲卵發育之影響(b)探討麵包蟲二次性感染雞隻犬蛔蟲試驗(c)開發糞內抗原檢測方法作為犬之犬蛔蟲症之診斷。 第一部分,由成熟母蟲子宮前1/3段取得之蟲卵,以氧化劑2.5 % K2Cr2O7 與不同濃度之KMnO4及還原劑1.75 % FeSO4與0.8 % Na2SO3為培養液,進行蟲卵之培養試驗。結果以2.5 % K2Cr2O7於20℃和30℃培養蟲卵,有促進卵細胞分裂作用。1.75 % FeSO4於30℃培養蟲卵在最初的36小時,亦有促進卵細胞分裂作用。而不同濃度之KMnO4及0.8 % Na2SO3對蟲卵之孵化,則有不同程度之抑制作用。並使用穿透式電子顯微鏡觀察蟲卵卵殼及卵細胞之變化,結果以1.0 % KMnO4及0.8 % Na2SO3處理之蟲卵,蟲卵外面之蛋白鞘突起消失且卵殼變薄,而以saline, 2.5 % K2Cr2O7及1.75 % FeSO4處理之蟲卵,則無明顯變化。 此外進行犬蛔蟲蟲卵感染麵包蟲試驗,探討麵包蟲二次感染雞隻之病原性。犬蛔蟲蟲卵感染幼蟲期麵包蟲之試驗中,麵包蟲感染蟲卵2天後,40日齡麵包蟲平均蟲卵數為11.4 ±4.5顆,50日齡為39.2 ±9.2顆,60日齡為54.6 ±9.1顆,3天後分別為0、7.6 ±3.8、11.8 ±4.1顆,4日後則無蟲卵之檢出,且麵包蟲之糞便僅排出未含幼蟲之卵殼。另取感染蟲卵2天及4天後之60日齡麵包蟲,各感染3隻雞,每隻雞餵食20隻麵包蟲(每隻雞大約感染1100顆蟲卵),結果感染蟲卵2天後之麵包蟲可二次感染雞隻,其平均感染率為14.3±1.5 %(157±16.6/1100),此外取感染蟲卵4天後之麵包蟲,進行消化與製作組織切片,皆無發現有幼蟲移行,因此得知麵包蟲具有消化犬蛔蟲第2期幼蟲的能力,但在未完全消化之前,則具有二次感染其他動物的可能性。 建立犬之犬蛔蟲症之糞內抗原診斷方法,製備兔子抗犬蛔蟲成蟲體抗原之多源抗體,利用三明治ELISA方法,首先在microplate上coating多源抗體,結合感染動物糞內抗原,之後加入自行標定HRP (horseradish peroxidase)之多源抗體,以ABTS-H2O2作為呈色劑,波長410nm下讀出吸光值,進行檢測結果,抗成蟲體抗原的多源抗體與瓜實絛蟲之糞內抗原具有之交叉反應。而以抗成蟲體抗原的多源抗體先與瓜實絛蟲體抗原反應後,再進行三明治ELISA檢測糞內抗原,可將交叉反應排除,證實開發犬之犬蛔蟲症糞內抗原診斷方法是可行的,雖然需要進一步的純化抗體及排除交叉反應。
Abstract This thesis comprises of 3 parts, (a)elucidation of the effect of redox reagents on the development of Toxocara canis eggs, (b)studying the transmission of T. canis larvae to chicken through the predation of T. canis larvae infected mealworms and, (c)an attempt to develop the coproantigen detection method for the diagnosis of toxocariasis in dogs. In the first part of the thesis, T. canis eggs, obtained from the distal-third portion of the uterus of adult female worms, were cultured in oxidant such as 2.5 % K2Cr2O7 and different concentration of KMnO4 and, also in reductant such as 1.75 % FeSO4 and 0.8 % Na2SO4. Enhanced cellular division within the T. canis eggs was observed when cultured at 20℃and 30℃ in 2.5 % K2Cr2O7. Faster cellular division in the eggs was also seen in the first 36 hours culturing at 30℃ in 1.75 % FeSO4. Different degree of inhibition of egg development of was observed for different concentration of KMnO4 and also for 0.80 % Na2SO4. Ultrastructural study by transmission electron microscopy showed that outer most protein coat of the egg had disappeared after treatment with 1.0% KMnO4 and 0.8 % Na2SO4. The above structural change was not observed when the eggs were treated with saline, 2.5 % K2Cr2O7 and 1.75% FeSO4. In the second part of the thesis, experimental infection of chicken with T. canis larvae by feeding them infected mealworms(Tenebrio molitor)was carried out. When mealworms were experimentally fed with larvated eggs of T. canis, the average number of eggs recovered from its digestive tract was 11.4±4.5 in 40-day-old mealworm, 39.2±9.2 in 50-day-old mealworm and 54.6±9.1 in 60-day-old mealworm on Day 2, and 0, 7.6±3.8 and 11.8±4.1 on Day 3, respectively. No eggs were recovered from the mealworms on Day 4, and only T. canis eggs shell without larva was found excreted in the feces of mealworms. Two groups of three chickens were each fed with twenty 60-day-old mealworms that were infected with T. canis larvated eggs either 2 or 4 days before. From the above, infectivity experiment in mealworm, twenty infected mealworm were calculated to contain about 1100 larvae, an dthis was considered as the inoculating dose for the chicken. An average of 157±16.6(14.3±1.5 %)T. canis larvae was recovered from the chickens on day 2 PI that were fed mealworms which had been exposed to T. canis eggs before. No larvae were recovered from chickens fed with mealworms that had been exposed to T. canis eggs 4 days before. It is concluded that mealworms can completely digest L2 larvae of T. canis in 4 days, but may serve as a paratenic host for other animals if being eaten by the predator before the larvae could be completely digested. To establish coproantigen detection as a diagnostic method for toxocariasis in dogs, polyclonal antibodies against T. canis adult worms somatic antigen were produced in rabbits. A sandwich ELISA was performed for the detection of coproantigen in the feces of dogs by first adhering the polyclonal antibody to the microplate to capture the coproantigen. This was followed by the addition of the same antibody that was conjugated with horseradish peroxidase. ABTS-H2O2 was used as substrate for the expression of coloration and the optical density was read at 410 nm. Cross-reactivity was observed among feces of dogs infected with T. canis and those with Dipylidium caninum. However, this cross-reactivity could be reduced after absorbing the antisera with D. caninum somatic antigen prior to use in the sandwich ELISA. Although further refinement and cross-reactivity studies needs to be done, it was demonstrated that it is feasible to develop the coproantigen detection procedure into a diagnostic method for toxocariasis in dogs. Keywords: Toxocara canis, redox reaction, egg development, chicken, mealworm, Tenebrio molitor, coproantigen, diagnosis
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