枯草芽孢杆菌对猪疱疹病毒1型体内外拮抗作用研究
发布时间：2018-07-25 21:20
【摘要】：猪疱疹病毒1型是多种哺乳动物共患的伪狂犬病的病原。主要引起母猪生殖障碍、公猪不育、仔猪大量死亡、呼吸困难、瘙痒及神经症状,给畜牧业带来极大的威胁,造成了较大的经济损失。近些年来,变异毒株的出现,使得伪狂犬病的防治变得越发困难。枯草芽孢杆菌作为一种微生态制剂在自然界中普遍存在且安全,可以抑制多种细菌和真菌,但对于枯草芽孢杆菌抗病毒作用的报道较少。因此,本文进行了此方面的研究,希望为枯草芽孢杆菌更广泛地应用于畜牧业生产提供依据。研究经16S rDNA和气相色谱鉴定了一株枯草芽孢杆菌CCBS-1,可以抑制金黄色葡萄球菌及大肠杆菌,从其发酵液中经硫酸铵沉淀法提取蛋白粗提物,甲醇抽提法提取脂肽粗提物,并测定其浓度。将发酵液和两种粗提物用于体外抑制猪疱疹病毒1型试验,经安全浓度测定、阻断、抑制和杀灭等试验,发现发酵液和蛋白粗提物分别在0.042mg/mL和0.167mg/mL浓度下对猪疱疹病毒1型有抑制作用,0.111mg/mL浓度下的脂肽对猪疱疹病毒1型具有杀灭作用。以昆明鼠为试验动物进行枯草芽孢杆菌体内抗病毒试验。选取18g~22g昆明鼠34只,10只用于安全试验测定,其余随机分为3组,设置PBS对照组(PBS组)、枯草芽孢杆菌预防组(BS组)和猪疱疹病毒1型攻毒组(PRV组);预饲喂7d后BS组1.2×108CFU/只/d剂量灌胃枯草芽孢杆菌,PBS组与PRV组灌胃相同剂量的PBS,连续饲喂14天;每隔1d称重1次,试验第7d时分别对PBS组和BS组昆明鼠尾静脉采血,试验14d后PBS组和BS组随机选取4只鼠眼球取血后无菌取脾脏及胸腺进行免疫器官指数的测定,然后取脾细胞依次进行淋巴细胞转化率检测、CD3+CD4+及CD3+CD8+T细胞亚群细胞数量检测、IgG非特异性抗体检测、IL-4、IFN-γ分泌水平检测和NK细胞活性检测,用收集到7d和14d的血清分别进行抗体及IL-4和IFN-γ水平测定,最后对剩余PRV组及BS组鼠进行106.70 TCID50猪疱疹病毒1型脑部攻毒试验,昆明鼠死后及时取脑组织制作病理切片。结果表明:(1)饲喂枯草芽孢杆菌对昆明鼠体重的影响表明BS组体重明显升高;(2)对于昆明鼠胸腺指数和脾脏指数而言,BS组的胸腺指数极显著高于PBS组,但其脾脏指数与PBS组差异不显著;(3)两组非特异性抗体水平都呈上升趋势,14d较7d BS组非特异性抗体水平有所增长,且极显著高于PBS组;(4)BS组在非特异性刺激剂作用下刺激指数高于PBS组;(5)BS组CD3+CD4+、CD3+CD8+T淋巴细胞数量与PBS相比差异显著;(6)BS组NK细胞活性显著高于PBS组,NK细胞活性提高25%左右;(7)BS组血清和细胞上清中IL-4和IFN-γ含量都显著高于PBS组;(8)BS组攻毒保护率提高25%;(9)病理切片中攻毒组具有明显的水肿、出血及炎性浸润等现象。从以上研究结果可知,枯草芽孢杆菌CCBS-1株的发酵液成分对猪疱疹病毒1型具有一定的抑制作用,该菌可提高实验鼠的体液免疫和细胞免疫能力,对伪狂犬病具有一定的预防作用。
[Abstract]:Porcine herpesvirus 1 is a common pathogen of pseudorabies in many mammals. It mainly causes reproductive disorders in sows, male infertility, death of piglets, dyspnea, itching and neurological symptoms, which poses a great threat to animal husbandry and results in great economic losses. In recent years, the emergence of variant strains makes the prevention and treatment of pseudorabies more difficult. Bacillus subtilis (Bacillus subtilis), as a microecological agent, is widespread and safe in nature. It can inhibit many kinds of bacteria and fungi, but there are few reports on the antiviral effect of Bacillus subtilis. Therefore, this paper has carried on the research in this aspect, hoped to provide the basis for the Bacillus subtilis to be more widely used in animal husbandry production. A strain of Bacillus subtilis CCBS-1 was identified by 16s rDNA and gas chromatography, which can inhibit Staphylococcus aureus and Escherichia coli. The crude protein extract was extracted by ammonium sulfate precipitation method, and the crude lipopeptide extract was extracted by methanol extraction. Its concentration was determined. Fermentation broth and two kinds of crude extracts were used to inhibit porcine herpesvirus type 1 in vitro. It was found that fermentation broth and protein extract could inhibit porcine herpesvirus type 1 at the concentration of 0.042mg/mL and 0.167mg/mL respectively. The lipopeptide at the concentration of 0.111 mg / mL could kill porcine herpesvirus type 1. The antiviral test of Bacillus subtilis in vivo was carried out in Kunming mice. 34 18g~22g Kunming mice were selected for safety test and the rest were randomly divided into 3 groups. PBS control group (PBS group), Bacillus subtilis prevention group (BS group) and porcine herpesvirus type 1 attack group (PRV group) were given intragastrically the same dose of PBS as PRV group for 14 days. On the 7th day, blood was collected from the tail veins of Kunming rats in PBS group and BS group. After 14 days, 4 mice in PBS group and BS group were randomly selected to measure the immune organ index of spleen and thymus after eyeball blood extraction. The lymphocyte transformation rate of CD3 CD4 and the number of CD3 CD8 T cell subsets were measured, and the levels of IL-4, IFN- 纬 and NK cell activity were detected. The antibodies and the levels of IL-4 and IFN- 纬 in serum collected for 7 days and 14 days were determined respectively. Finally, the 106.70 TCID50 porcine herpesvirus type 1 brain attack test was carried out in the remaining PRV group and BS group. The brain tissue of Kunming rats was taken out in time after death to make pathological sections. The results showed that: (1) the effect of Bacillus subtilis on the body weight of Kunming mice showed that the body weight of BS group was significantly higher than that of PBS group, and (2) the thymus index and spleen index of BS group were significantly higher than that of PBS group. However, there was no significant difference between the spleen index and PBS group. (3) the level of non-specific antibody in both groups showed an upward trend of 14 days compared with that in BS group on 7 days, and the level of non-specific antibody increased in both groups. The number of CD3 CD4 CD3 CD8 T lymphocytes in BS group was significantly higher than that in PBS group, (6) the NK cell activity in BS group was significantly higher than that in PBS group, (4) the stimulation index in BS group was higher than that in PBS group, (5) the number of CD3 CD4 CD3 CD8 T lymphocytes in BS group was significantly higher than that in PBS group, and (6) the NK cell activity in BS group was significantly higher than that in PBS group. (7) the contents of IL-4 and IFN- 纬 in serum and supernatant of BS group were significantly higher than those in PBS group; (8) the protective rate of drug attack in BS group was increased by 25%; Bleeding and inflammatory infiltration. The results show that the fermentation broth of Bacillus subtilis CCBS-1 strain has a certain inhibitory effect on porcine herpesvirus type 1, which can improve the humoral and cellular immunity of the experimental mice. It has certain preventive effect on pseudorabies.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
[Abstract]:Porcine herpesvirus 1 is a common pathogen of pseudorabies in many mammals. It mainly causes reproductive disorders in sows, male infertility, death of piglets, dyspnea, itching and neurological symptoms, which poses a great threat to animal husbandry and results in great economic losses. In recent years, the emergence of variant strains makes the prevention and treatment of pseudorabies more difficult. Bacillus subtilis (Bacillus subtilis), as a microecological agent, is widespread and safe in nature. It can inhibit many kinds of bacteria and fungi, but there are few reports on the antiviral effect of Bacillus subtilis. Therefore, this paper has carried on the research in this aspect, hoped to provide the basis for the Bacillus subtilis to be more widely used in animal husbandry production. A strain of Bacillus subtilis CCBS-1 was identified by 16s rDNA and gas chromatography, which can inhibit Staphylococcus aureus and Escherichia coli. The crude protein extract was extracted by ammonium sulfate precipitation method, and the crude lipopeptide extract was extracted by methanol extraction. Its concentration was determined. Fermentation broth and two kinds of crude extracts were used to inhibit porcine herpesvirus type 1 in vitro. It was found that fermentation broth and protein extract could inhibit porcine herpesvirus type 1 at the concentration of 0.042mg/mL and 0.167mg/mL respectively. The lipopeptide at the concentration of 0.111 mg / mL could kill porcine herpesvirus type 1. The antiviral test of Bacillus subtilis in vivo was carried out in Kunming mice. 34 18g~22g Kunming mice were selected for safety test and the rest were randomly divided into 3 groups. PBS control group (PBS group), Bacillus subtilis prevention group (BS group) and porcine herpesvirus type 1 attack group (PRV group) were given intragastrically the same dose of PBS as PRV group for 14 days. On the 7th day, blood was collected from the tail veins of Kunming rats in PBS group and BS group. After 14 days, 4 mice in PBS group and BS group were randomly selected to measure the immune organ index of spleen and thymus after eyeball blood extraction. The lymphocyte transformation rate of CD3 CD4 and the number of CD3 CD8 T cell subsets were measured, and the levels of IL-4, IFN- 纬 and NK cell activity were detected. The antibodies and the levels of IL-4 and IFN- 纬 in serum collected for 7 days and 14 days were determined respectively. Finally, the 106.70 TCID50 porcine herpesvirus type 1 brain attack test was carried out in the remaining PRV group and BS group. The brain tissue of Kunming rats was taken out in time after death to make pathological sections. The results showed that: (1) the effect of Bacillus subtilis on the body weight of Kunming mice showed that the body weight of BS group was significantly higher than that of PBS group, and (2) the thymus index and spleen index of BS group were significantly higher than that of PBS group. However, there was no significant difference between the spleen index and PBS group. (3) the level of non-specific antibody in both groups showed an upward trend of 14 days compared with that in BS group on 7 days, and the level of non-specific antibody increased in both groups. The number of CD3 CD4 CD3 CD8 T lymphocytes in BS group was significantly higher than that in PBS group, (6) the NK cell activity in BS group was significantly higher than that in PBS group, (4) the stimulation index in BS group was higher than that in PBS group, (5) the number of CD3 CD4 CD3 CD8 T lymphocytes in BS group was significantly higher than that in PBS group, and (6) the NK cell activity in BS group was significantly higher than that in PBS group. (7) the contents of IL-4 and IFN- 纬 in serum and supernatant of BS group were significantly higher than those in PBS group; (8) the protective rate of drug attack in BS group was increased by 25%; Bleeding and inflammatory infiltration. The results show that the fermentation broth of Bacillus subtilis CCBS-1 strain has a certain inhibitory effect on porcine herpesvirus type 1, which can improve the humoral and cellular immunity of the experimental mice. It has certain preventive effect on pseudorabies.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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相关期刊论文 前10条
1 冯志华;邵丽玮;赵国先;龚建刚;张一为;;枯草芽孢杆菌-刺五加多糖合生元对蛋雏鸡免疫器官指数及盲肠微生物的影响[J];饲料工业;2016年09期
2 邹清青;欧阳琛璨;堀川贵生;柳泽惠^，
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