CN110710493A - 一种猪链球菌9型感染豚鼠动物模型的构建及其应用 - Google Patents
一种猪链球菌9型感染豚鼠动物模型的构建及其应用 Download PDFInfo
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Abstract
本发明公开了一种猪链球菌9型感染豚鼠动物模型的构建及其应用,具体涉及疫苗和药物开发及评估研究领域,其中构建的步骤:接种动物准备、菌液制备、动物接种、临床观察和解剖鉴定。本发明豚鼠模型饲养方便,易于操作,能够较强的表现出SS9感染的典型临床症状及病理变化,与仔猪自然感染情况相似,重复性好;构建了猪链球菌9型感染豚鼠动物模型,可用于开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作。
Description
技术领域
本发明涉及疫苗和药物开发及评估研究技术领域,更具体地说,本发明涉及一种猪链球菌9型感染豚鼠动物模型的构建及其应用。
背景技术
猪链球菌(Streptococcus suis,SS)是一种重要的人畜共患病病原,是链球菌属。猪链球菌的定植部位为猪的上呼吸道,尤其是扁桃体和鼻腔。部分血清型的猪链球菌具有致病性,可引起猪的急性败血症、脑膜炎、关节炎、心内膜炎、肺炎等疾病。部分菌株可引起人类感染,造成细菌性脑炎(bacterial meningitis)或引起中毒样休克综合征(toxicshock-like syndrome)。SS呈世界性流行,对养猪行业和公共卫生安全造成重大的威胁。按荚膜抗原的特性,据其荚膜抗原(CPS)的不同,猪链球菌被分为35个血清型(1-34型和1/2型),其中1型、2型(1/2型)、7型、9型的致病力最强。近几年来,SS2多次引起猪链球菌病的暴发,并可感染人致死,成为当前重要的人畜共患病之一。血清型2为最常见和毒力最强的血清型。致病因子有荚膜、溶菌酶释放蛋白(MRP)、细胞外因子(EF)、溶血素。荚膜可以保护细菌,抵抗吞噬。溶菌酶释放蛋白、细胞外因子的存在提高了菌株的致病力。
猪链球菌2型流行最广、毒力最强,是我国主要致病血清型,近几年,猪链球菌9型(SS9)检出率和分离率呈上升趋势,急需开展猪链球菌9型流行病学、致病性、感染机制及疫苗开发的研究工作。虽然国内外有利用小鼠、兔子、豚鼠和斑马鱼来研究SS2感染的动物模型,但是却缺少研究SS9感染的动物模型,无法开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作。
发明内容
为了克服现有技术的上述缺陷,本发明的实施例提供一种猪链球菌9型感染豚鼠动物模型的构建,通过构建的猪链球菌9型感染豚鼠动物模型,用于开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作。
为实现上述目的,本发明提供如下技术方案:一种猪链球菌9型感染豚鼠动物模型的构建,其中构建的步骤:
S1、接种动物准备:选取4-6周龄豚鼠,分为正常对照组(n=6)、PBS对照组(n=6)与6个实验组(n=6×6),在相同环境下按照分组隔离饲养5-7d;
S2、菌液制备:使用稀释液对猪链球菌9型菌进行稀释,分别配置六组不同浓度的猪链球菌9型菌液;
S3、动物接种:将S1步骤中的正常对照组不做处理,PBS对照组通过腹腔注射1mL的PBS缓冲液,6个实验组分别通过腹腔注射S2步骤中制备的不同梯度浓度的猪链球菌9型菌液1mL;
S4、临床动物观察:对步骤S3中各组豚鼠在相同环境进行隔离饲养,期间进行正常的水、食喂养,观察1周豚鼠的发病及死亡率情况;
S5、对死亡豚鼠进行解剖,取脑、心、肝、脾和肺组织进行细菌分离鉴定和各个器官中细菌分布及载量测定;
S6、对分离到的细菌通过猪链球菌谷氨酸脱氢酶基因和荚膜多糖基因PCR方法进行SS9鉴定并测序;
S7、对各组豚鼠进行解剖,对其的脑、心、肝、脾和肺进行组织病理切片鉴定。
在一个优选地实施方式中,所述S1步骤中各组豚鼠长度和体重应选取大致相当,并模拟生态环境进行饲养。
在一个优选地实施方式中,所述S2步骤中的稀释液设置为PBS缓冲液。
在一个优选地实施方式中,所述S2步骤中六组猪链球菌9型菌液的浓度分别设置为5.0×108CFU/mL、1.0×109CFU/mL、2.0×109CFU/mL、3.0×109CFU/mL、4.0×109CFU/mL和5.0×109CFU/mL。
在一个优选地实施方式中,所述S7步骤中的组织病理切片鉴定步骤为:取0.5cm*0.5cm*1cm的组织块→使用100mL/L浓度的福尔马林固定→经酒精脱水→石蜡包埋→切片→贴片→HE染色→镜检。
在一个优选地实施方式中,所构建的模型可用于SS9感染的典型临床症状及病理变化,该模型还可用于开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作。
本发明的技术效果和优点:
本发明通过构建的猪链球菌9型感染豚鼠动物模型,用于开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作,且豚鼠模型饲养方便,易于操作,能够较强的表现出SS9感染的典型临床症状及病理变化,与仔猪自然感染情况相似,重复性好。
附图说明
图1为本发明的荚膜多糖基因(cps)扩增产物测序图。
图2为本发明的主要器官病理学变化图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例:
本发明提供了一种猪链球菌9型感染豚鼠动物模型的构建,其中构建的步骤:
S1、接种动物准备:选取4-6周龄豚鼠,分为正常对照组(n=6)、PBS对照组(n=6)与6个实验组(n=6×6),在相同环境下按照分组隔离饲养5-7d,采用豚鼠作为仔猪的替代感染物,体型较小,方便饲养,各组豚鼠长度和体重应选取大致相当,并模拟生态环境进行饲养,一方面可保证其在相同的生活环境内,避免外界因素对实验数据的干扰,另一方面采用模拟生态的方式对各组豚鼠进行统一的饲养,可避免环境及进食的不同对实验数据的干扰,保证数据的真实、有效性;
S2、菌液制备:使用PBS缓冲液对猪链球菌9型菌进行稀释,分别配置6组不同浓度的猪链球菌9型菌液,6组猪链球菌9型菌液的浓度分别设置为5.0×108CFU/mL、1.0×109CFU/mL、2.0×109CFU/mL、3.0×109CFU/mL、4.0×109CFU/mL和5.0×109CFU/mL;
S3、动物接种:将S1步骤中的正常对照组不做处理,PBS对照组通过腹腔注射1mL的PBS缓冲液,6个实验组分别通过腹腔注射S2步骤中制备的不同梯度浓度的猪链球菌9型菌液1mL,向PBS对照组的豚鼠注射PBS缓冲液,用于对比配置的猪链球菌9型菌液中的PBS缓冲液对豚鼠产生的影响,正常对照组不做任何处理,可模拟正常状态下的豚鼠的生命体质状态,分别对8组接种动物分隔进行饲养;
S4、临床动物观察:对步骤S3中各组豚鼠在相同环境进行隔离饲养,期间进行正常的水、食喂养,观察1周豚鼠的发病及死亡率情况;
按S3项方法接种后的豚鼠在S4项进行观察后的死亡情况,所得结果见表1:
表1不同组豚鼠接种后的死亡情况统计(单位:只)
S5、对死亡豚鼠进行解剖,取脑、心、肝、脾和肺组织进行细菌分离鉴定和各个器官中细菌分布及载量测定,所得结果见表2;
表2部分死亡豚鼠及对照组豚鼠组织器官细菌分布及载量
S6、对分离到的细菌通过猪链球菌谷氨酸脱氢酶(gdh)基因和荚膜多糖基因(cps)PCR方法进行SS9鉴定并测序,所得结果见图1;
S7、对各组豚鼠进行解剖,对其的脑、心、肝、脾和肺按照取0.5cm*0.5cm*1cm的组织块→使用100mL/L浓度的福尔马林固定→经酒精脱水→石蜡包埋→切片→贴片→HE染色→镜检的步骤进行组织病理切片鉴定,所得结果见图2。
对正常对照组和PBS对照组的豚鼠进行剖检,眼观和显微镜下观察组织切片均为正常组织状态,无病变,随着接种链球菌浓度提高,眼观可见胸腹部出现纤维型渗出,黏连,肺脏充血肿胀,脾脏肿大,组织切片可见心肌纤维白细胞浸润,肝脏损伤程度从肝组织充血到多量肝细胞核浓染,肺脏组织损伤程度从轻微充血出血到严重充出血,肾脾组织充血出血。
通过上述结果可得实验结论:与正常对照组和PBS对照组比较,不同浓度猪链球菌9型菌液实验组出现毛发异常,行动异常,活动度逐渐减少,饮食饮水明显减少,体重减轻。不同感染浓度小鼠出现不同死亡率,猪链球菌9型菌液浓度越高,发病率及死亡率越高。通过对死亡豚鼠各个器官中细菌分布及载量测定、PCR鉴定和病理学变化结果表明,成功建立了猪链球菌9型感染豚鼠动物模型,可用于开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作。
最后:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种猪链球菌9型感染豚鼠动物模型的构建,其特征在于,其中构建的步骤:
S1、接种动物准备:选取4-6周龄豚鼠,分为正常对照组(n=6)、PBS对照组(n=6)与6个实验组(n=6×6),在相同环境下按照分组隔离饲养5-7d;
S2、菌液制备:使用稀释液对9型猪链球菌进行稀释,分别配置6组不同浓度的猪链球菌9型菌液;
S3、动物接种:将S1步骤中的正常对照组不做处理,PBS对照组通过腹腔注射1mL的PBS缓冲液,6个实验组分别通过腹腔注射S2步骤中制备的不同梯度浓度的猪链球菌9型菌液1mL;
S4、临床动物观察:对步骤S3中各组豚鼠在相同环境进行隔离饲养,期间进行正常的水、食喂养,观察1周豚鼠的发病及死亡率情况;
S5、对死亡豚鼠进行解剖,取脑、心、肝、脾和肺组织进行细菌分离鉴定和各个器官中细菌分布及载量测定;
S6、对分离到的细菌通过猪链球菌谷氨酸脱氢酶基因和荚膜多糖基因PCR方法进行SS9鉴定并测序;
S7、对各组豚鼠进行解剖,对其的脑、心、肝、脾和肺进行组织病理切片鉴定。
2.根据权利要求1所述的一种猪链球菌9型感染豚鼠动物模型的构建,其特征在于:所述S1步骤中各组豚鼠长度和体重应选取大致相当,并模拟生态环境进行饲养。
3.根据权利要求1所述的一种猪链球菌9型感染豚鼠动物模型的构建,其特征在于:所述S2步骤中的稀释液设置为PBS缓冲液。
4.根据权利要求1所述的一种猪链球菌9型感染豚鼠动物模型的构建,其特征在于:所述S2步骤中六组猪链球菌9型菌液的浓度分别设置为5.0×108CFU/mL、1.0×109CFU/mL、2.0×109CFU/mL、3.0×109CFU/mL、4.0×109CFU/mL和5.0×109CFU/mL。
5.根据权利要求1所述的一种猪链球菌9型感染豚鼠动物模型的构建,其特征在于:所述S7步骤中的组织病理切片鉴定步骤为:取0.5cm*0.5cm*1cm的组织块→使用100mL/L浓度的福尔马林固定→经酒精脱水→石蜡包埋→切片→贴片→HE染色→镜检。
6.根据权利要求1所述的一种猪链球菌9型感染豚鼠动物模型的应用,其特征在于:所构建的模型可用于SS9感染的典型临床症状及病理变化,该模型还可用于开展SS9致病性、感染机制及疫苗和药物开发及评估研究工作。
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