枯草芽孢桿菌對豬皰疹病毒1型體內外拮抗作用研究
發(fā)布時間：2018-07-25 21:20
【摘要】：豬皰疹病毒1型是多種哺乳動物共患的偽狂犬病的病原。主要引起母豬生殖障礙、公豬不育、仔豬大量死亡、呼吸困難、瘙癢及神經癥狀,給畜牧業(yè)帶來極大的威脅,造成了較大的經濟損失。近些年來,變異毒株的出現(xiàn),使得偽狂犬病的防治變得越發(fā)困難。枯草芽孢桿菌作為一種微生態(tài)制劑在自然界中普遍存在且安全,可以抑制多種細菌和真菌,但對于枯草芽孢桿菌抗病毒作用的報道較少。因此,本文進行了此方面的研究,希望為枯草芽孢桿菌更廣泛地應用于畜牧業(yè)生產提供依據(jù)。研究經16S rDNA和氣相色譜鑒定了一株枯草芽孢桿菌CCBS-1,可以抑制金黃色葡萄球菌及大腸桿菌,從其發(fā)酵液中經硫酸銨沉淀法提取蛋白粗提物,甲醇抽提法提取脂肽粗提物,并測定其濃度。將發(fā)酵液和兩種粗提物用于體外抑制豬皰疹病毒1型試驗,經安全濃度測定、阻斷、抑制和殺滅等試驗,發(fā)現(xiàn)發(fā)酵液和蛋白粗提物分別在0.042mg/mL和0.167mg/mL濃度下對豬皰疹病毒1型有抑制作用,0.111mg/mL濃度下的脂肽對豬皰疹病毒1型具有殺滅作用。以昆明鼠為試驗動物進行枯草芽孢桿菌體內抗病毒試驗。選取18g~22g昆明鼠34只,10只用于安全試驗測定,其余隨機分為3組,設置PBS對照組(PBS組)、枯草芽孢桿菌預防組(BS組)和豬皰疹病毒1型攻毒組(PRV組);預飼喂7d后BS組1.2×108CFU/只/d劑量灌胃枯草芽孢桿菌,PBS組與PRV組灌胃相同劑量的PBS,連續(xù)飼喂14天;每隔1d稱重1次,試驗第7d時分別對PBS組和BS組昆明鼠尾靜脈采血,試驗14d后PBS組和BS組隨機選取4只鼠眼球取血后無菌取脾臟及胸腺進行免疫器官指數(shù)的測定,然后取脾細胞依次進行淋巴細胞轉化率檢測、CD3+CD4+及CD3+CD8+T細胞亞群細胞數(shù)量檢測、IgG非特異性抗體檢測、IL-4、IFN-γ分泌水平檢測和NK細胞活性檢測,用收集到7d和14d的血清分別進行抗體及IL-4和IFN-γ水平測定,最后對剩余PRV組及BS組鼠進行106.70 TCID50豬皰疹病毒1型腦部攻毒試驗,昆明鼠死后及時取腦組織制作病理切片。結果表明:(1)飼喂枯草芽孢桿菌對昆明鼠體重的影響表明BS組體重明顯升高;(2)對于昆明鼠胸腺指數(shù)和脾臟指數(shù)而言,BS組的胸腺指數(shù)極顯著高于PBS組,但其脾臟指數(shù)與PBS組差異不顯著;(3)兩組非特異性抗體水平都呈上升趨勢,14d較7d BS組非特異性抗體水平有所增長,且極顯著高于PBS組;(4)BS組在非特異性刺激劑作用下刺激指數(shù)高于PBS組;(5)BS組CD3+CD4+、CD3+CD8+T淋巴細胞數(shù)量與PBS相比差異顯著;(6)BS組NK細胞活性顯著高于PBS組,NK細胞活性提高25%左右;(7)BS組血清和細胞上清中IL-4和IFN-γ含量都顯著高于PBS組;(8)BS組攻毒保護率提高25%;(9)病理切片中攻毒組具有明顯的水腫、出血及炎性浸潤等現(xiàn)象。從以上研究結果可知,枯草芽孢桿菌CCBS-1株的發(fā)酵液成分對豬皰疹病毒1型具有一定的抑制作用,該菌可提高實驗鼠的體液免疫和細胞免疫能力,對偽狂犬病具有一定的預防作用。
[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- 緯 and NK cell activity were detected. The antibodies and the levels of IL-4 and IFN- 緯 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- 緯 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.
【學位授予單位】：吉林農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：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- 緯 and NK cell activity were detected. The antibodies and the levels of IL-4 and IFN- 緯 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- 緯 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.
【學位授予單位】：吉林農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S852.65
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相關期刊論文 前10條
1 馮志華;邵麗瑋;趙國先;龔建剛;張一為;;枯草芽孢桿菌-刺五加多糖合生元對蛋雛雞免疫器官指數(shù)及盲腸微生物的影響[J];飼料工業(yè);2016年09期
2 鄒清青;歐陽琛璨;堀川貴生;柳澤惠^，
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