抗鸡球虫病的蛋白酶抑制剂药物的筛选及安全性评价

发布时间：2019-03-09 16:57

【摘要】：鸡球虫主要寄生于鸡肠道细胞内,引起以肠道病变为主要特征的鸡球虫病,严重危害养鸡业的发展。目前,由于预防鸡球虫病的疫苗大部分是活苗,接种活苗可能出现返祖、散毒现象。所以,药物防治鸡球虫病仍是最主要方式,例如盐霉素、马杜霉素、莫能菌素、海南霉素等。但随着球虫虫株抗药性问题的出现,使得临床防治陷入很大的困境。因此,有必要寻找新的药物来防治鸡球虫病。近来研究发现蛋白酶抑制剂药物能够抑制原虫侵入细胞。3,4-二氯异香豆素(3,4-dichloroisocoumarin,DCI)是一种丝氨酸蛋白酶抑制剂药物,能够抑制球虫子孢子侵入宿主细胞。吉非替尼、厄洛替尼及克里唑蒂尼是酪氨酸激酶抑制剂药物,可以抑制弓形虫入侵宿主细胞。但DCI、吉非替尼等蛋白酶抑制剂是否具有抗鸡球虫效果尚未见报道。因此,本试验拟通过DCI、吉非替尼、厄洛替尼和克里唑蒂尼预防和治疗鸡球虫病试验,筛选出能够有效防治鸡球虫病的药物,并通过临床观察和血液生化学等各项指标对筛选出的药物进行安全性评价。为鸡球虫病防治提供新的候选药物。1、预防鸡球虫病蛋白酶抑制剂药物的筛选试验选择体重相近的健康雏鸡380只,按照不同给药剂量分为19组,20只/组,(阳性对照组、药物对照组、空白对照组、DCI倍比剂量3个组,吉非替尼倍比剂量4个组,克里唑蒂尼倍比剂量4个组,厄洛替尼倍比剂量5个组)。空白对照组除外,其余组给药7天后,每只试验鸡经口服方式接种1.0×104个E.tenella孢子化卵囊。感染期间观察鸡的精神、食欲状况。接种球虫一周后,每组逐只称重、剖杀计算存活率、增重率、血便记分、相对增重率、盲肠病变记分,收集每组粪便,计算卵囊数量。结果表明:DCI综合评价其抗球虫指数为117.34-161.04;吉非替尼综合评价其抗球虫指数为87.83-110.55;克里唑蒂尼综合评价其抗球虫指数为98.94-132.79;厄洛替尼综合评价其抗球虫指数为68.54-97.49。表明DCI中剂量组预防鸡球虫病效果较好,抗球虫指数为161.04,即0.005mg/只,饮水给药,连用7天。2、治疗鸡球虫病蛋白酶抑制剂药物的筛选试验选择体重相近的健康雏鸡380只,按照不同剂量分为19组,20只/组,(阳性对照组、药物对照组、空白对照组、DCI倍比剂量3个组,吉非替尼倍比剂量4个组,克里唑蒂尼倍比剂量4个组,厄洛替尼倍比剂量5个组)。除空白对照组外,其余各组均给雏鸡接入球虫,每只试验鸡经口服方式接种1.0×104个E.tenella孢子化卵囊,接种球虫后即给药5天。接种球虫一周后,每组逐只称重,剖杀计算存活率、增重率、血便记分、相对增重率、盲肠病变记分等,收集每组粪便,计算卵囊数量。结果表明:DCI组综合评价其抗球虫指数为133.13-178.73;吉非替尼组综合评价其抗球虫指数为110.66-135.96;克里唑蒂尼组综合评价其抗球虫指数为85.51-125.17;厄洛替尼组综合评价其抗球虫指数为89.76-109.47。试验表明DCI中剂量组疗效较好,综合评价抗球虫指数为178.73,即0.005mg/只,饮水给药,连用5天。3、DCI对靶动物鸡的安全性评价将250只8日龄雏鸡,分成5组,每组50只,按照不同给药剂量分组为空白对照组、1倍剂量组(0.005mg/只)、3倍剂量组(0.015mg/只)、5倍剂量组(0.025mg/只)和10倍剂量组(0.05mg/只)。空白对照组饮用自来水,其余各组连续饮水给药21天。通过临床观察和对实验动物的血液学、血液生化学、组织病理学及尸体剖检各项指标进行药物的安全性评价。结果表明,与对照组相比,DCI各剂量组血液学参数差异不显著(P0.05);1倍剂量组用药血液生化学与眼观组织无病理变化,3倍及以上剂量组用药血液生化检测碱性磷酸酶、谷草转氨酶及胆红素与对照组相比差异显著(P0.05),个别鸡只出现肝脏黄染、肠道出血点或出血斑;组织病理学观察,镜下观察肝脏和肠组织切片,3倍剂量组肝脏有轻度的水肿和脂肪变性现象,肠道无病理变化;5倍剂量组脂肪变性严重,肠绒毛断裂;10倍剂量组有炎性细胞造成的网状病变,引起脂肪变性,肠绒毛断裂。安全性试验表明,3倍及以上剂量组对雏鸡有危害,主要对鸡的肝脏和肠道有损伤。结果表明,DCI 0.005mg/只给药临床用药安全。
[Abstract]:The coccidiosis of the chicken is mainly in the intestinal tract of the chicken, which causes the chicken coccidiosis which is the main characteristic of the intestinal pathological changes, and seriously harms the development of the chicken industry. At present, due to the prevention of the chicken coccidiosis, most of the vaccines are live seedlings, and the inoculation live seedlings may have the phenomenon of back-and-back. Therefore, the drug control and control of the chicken coccidiosis is still the most important way, such as, for example, the salinomycin, the maduramicin, the monensin, the hainan, and the like. However, with the emergence of the drug-resistance of the Eimeria strain, the clinical prevention and control are in great difficulty. Therefore, it is necessary to find new drugs to control the coccidiosis of the chicken. Recently, it has been found that the protease inhibitor drug can inhibit the invasion of the protozoa. Gefitinib, erlotinib, and criptinidine are tyrosine kinase inhibitor drugs, which can inhibit the invasion of host cells of Toxoplasma gondii. However, whether a protease inhibitor such as DCI or gefitinib has the effect of anti-coccidiosis has not been reported. Therefore, this test is to prevent and treat the chicken coccidiosis by DCI, gefitinib, erlotinib and Crinopini, and to screen out the drugs which can effectively prevent and control the coccidiosis of the chicken, and to evaluate the safety of the screened drugs through the clinical observation and the blood biochemical indexes. in order to provide a new candidate for the prevention and treatment of chicken coccidiosis, a screening test for preventing the preparation of the protease inhibitor of the chicken coccidiosis selected from the group consisting of 380 healthy chickens with similar body weight,19 groups according to the different administration doses,20/ group, (positive control group, drug control group, blank control group, The DCI-time-specific dose of 3 groups, the gefitinib-fold specific dose of 4 groups, the Kristinian-fold specific dose of 4 groups, and the erlotinib-fold specific dose of 5 groups). Except for the blank control group, after 7 days of administration of the rest of the group, 1.0 to 104 E. tenella sporulated oocysts were administered orally. The spirit and the appetite of the chicken were observed during the infection. After a week of coccidiosis, each group was weighed individually, the survival rate, the weight gain rate, the blood stool score, the relative weight gain rate and the cecum lesion score were calculated, and each group of feces was collected and the number of oocysts was calculated. The results showed that the anti-coccidiosis index was 117.34-161.04, the anti-coccidiosis index of gefitinib was 87.83-110.55, and the anti-coccidiosis index was 98.94-132.79, and the anti-coccidiosis index of erlotinib was 68.54-97.49. The results showed that the anti-coccidiosis effect of the dose group in the DCI is good, the anti-coccidiosis index is 161.04, that is, 0.005 mg/ min, the drinking water is administered for 7 days.2, the screening test for the treatment of the chicken coccidiosis protease inhibitor drug selects 380 healthy chickens with similar body weight, and is divided into 19 groups and 20/ groups according to different doses. (Positive control group, drug control group, blank control group, DCI-time ratio dose of 3 groups, gefitinib-time-ratio 4 groups, Kristitinix dose of 4 groups, erlotinib-times-specific dose of 5 groups). In addition to the blank control group, the other groups were inoculated with coccidiosis in the chicks, and 1.0 to 104 E. tenella sporozoites were inoculated orally, and 5 days after the inoculation of the coccidia. After a week of coccidiosis, each group was weighed individually, the survival rate, the weight gain rate, the blood score, the relative weight gain rate, the cecum lesion score, and the like were calculated, and each group of feces was collected and the number of oocysts was calculated. The results showed that the anti-coccidiosis index was 133.13-178.73 in the DCI group, the anti-coccidiosis index was 110.66-135.96 in the gefitinib group, 85.51-125.17, and the anti-coccidiosis index was 89.76-109.47 in the erlotinib group. The results indicated that the efficacy of the dose group in the DCI group was good, the comprehensive evaluation of the anti-coccidiosis index was 178.73, that is, 0.005 mg/ kg, the drinking water was administered for 5 days. The safety evaluation of the DCI on the target animal was estimated to be 250-day-old chicks, divided into 5 groups, each group of 50 chickens. The dose groups were grouped into the blank control group, the 1-fold dose group (0.005 mg/ day), the 3-fold dose group (0.015 mg/ day), the 5-fold dose group (0.025 mg/ day) and the 10-fold dose group (0.05 mg/ day) according to the different dose groups. The blank control group was treated with tap water and the rest of the other groups were treated with continuous drinking water for 21 days. The safety evaluation of the drug was carried out by clinical observation and the hematology, blood chemistry, histopathology and the body cross-section of the experimental animals. The results showed that there was no significant difference in the hematology parameters in the DCI group compared with the control group (P0.05). The blood biochemical and ocular tissue of the 1-fold dose group did not change the pathological changes, and the blood biochemical test of the alkaline phosphatase was detected in the 3-fold and higher dose group. Compared with the control group, the difference was significant (P0.05). The liver and intestinal tissue sections were observed in individual chickens, and the liver and intestinal tissue sections were observed under the microscope. The liver of the 3-fold dose group had mild edema and fat degeneration. There were no pathological changes in the intestinal tract; the fatty degeneration of the 5-fold group was severe, and the intestinal villi were broken; the reticulopathy caused by the inflammatory cells in the 10-fold group resulted in the degeneration of the fat and the break of the intestinal villi. The safety test shows that the three times and above dose groups are harmful to the chicks, and the liver and the intestinal tract of the chicken are mainly damaged. The results showed that the DCI 0.005 mg/ only was safe for clinical medication.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

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