高原环境下两种变异L型细菌的分离培养及鉴定

Isolated Culture and Identification of Two Variants of L-type Bacteria in the Altitude Environment

研究高原环境下L型变异细菌的变异量和抗原特征,以期为高原家畜L型细菌引发相关性疾病的临床诊治及有效预防奠定基础。选用藏系绵羊消化道食糜、藏系绵羊鼻液、西藏黄牛粪便为分离培养对象,采用特殊培养基分离培养、染色镜检、生化鉴定、血清型鉴定及PCR检测方法进行相关性研究。结果表明:L型大肠杆菌在高渗XLD培养基上呈现3种菌落形态,西藏黄牛粪便中变异菌比例为(40.69±56.67)%的菌落呈“油煎蛋样”型(L型),(52.73±36.68)%的菌落为颗粒型(G型),(6.57±6.65)%的菌落为菌丝型(F型);藏系绵羊消化道食糜变异菌比例中(64.41±85.66)%的菌落为L型,(28.81±14.34)%的菌落为G型,(6.78±0.00)%的菌落为F型。L型巴氏杆菌在高渗TSLN培养基上生长出2种菌落形态,占藏系绵羊鼻液中变异菌的(97.67±89.50)%为G型,(2.65±10.50)%的菌落为L型;藏系绵羊消化道食糜中(82.28±88.92)%的菌落为G型,(17.72±11.08)%的菌落为L型。挑取纯化后的正常菌进行革兰染色,观察到正常大肠杆菌两端呈粉红色、钝圆,革兰阴性短杆菌;正常巴氏杆菌两端钝圆,中央微凸起,单个或成对存在的革兰阴性短杆菌;挑取L型细菌“油煎蛋样”菌落,镜检可见L型大肠杆菌呈卵圆形、长杆状的革兰阴性菌;L型巴氏杆菌呈棒状、杆状的革兰阳性菌;用鞣酸-龙胆紫染色法染色,在高倍镜下观察,L型大肠杆菌、L型巴氏杆菌全菌染成蓝紫色,菌体长是正常菌的2~3倍,菌体呈椭圆、棒状、杆状等多种形状。L型细菌因细胞壁的缺失,生化鉴定与正常菌不同。正常大肠杆菌及正常巴氏杆菌生化鉴定符合《伯杰氏系统细菌学手册》中描述的生化特性,L型大肠杆菌除与正常大肠杆菌苯丙氨酸差异最大,其余生化鉴定差异不明显;L型巴氏杆菌与正常巴氏杆菌生化鉴定相比,表现为氧化酶试纸条呈蓝色,半固体琼脂穿刺、V-P、鸟氨酸脱羧酶肉汤、赖氨酸脱羧酶肉汤、山梨醇、蜜二糖及棉子糖生化鉴定为阳性;色氨酸脱羧酶肉汤生化鉴定为阴性。L型大肠杆菌血清凝集强度低于正常大肠杆菌,最强为2+,正常大肠杆菌最强为4+。L型大肠杆菌条带亮度从高到低依次为O15-R、O91-2R、O8-F,正常大肠杆菌电泳条带亮度从高到低依次O91-2R、O15-R、O21-F;L型巴氏杆菌条带亮度从高到低依次为A-F、B-F、F-F,正常巴氏杆菌电泳条带A-F、B-F、E-F。综上,L型细菌的抗原性发生改变。不同细菌的基因型适应高原环境的能力、在强紫外线下发生变异的概率、细胞壁中抗原性等发生变异的概率也均不同。

The purpose was to research estimator of mutant bacteria and antigen characteristics of L-type mutant bacteria in plateau environment.This study could provide foundation for clinical dignosis,therapy and effective prevention this bacteria.The material from digestive tract of Tibetan sheep,nasal fluid of Tibetan sheep,dung of Tibetan cattle were researched by the method of isolation and culture on special medium,dyeing microscopic examination,biochemical identification,serotype identification and PCR test.The results showed that isolation and culture of L-type bacteria,L-type Escherichia coli were grown three colony morphologies on hypertonic XLD mediums,the percentage of mutant bacteria which its colony showed"fried egg"shape(L shape)was at(40.69±56.67)% in the feces of Tibetan Yellow cattle.Some coenobium with granular(G shape)was up to(52.73±36.68)%,some coenobium was mycelium type(F type)at(6.57±6.65)%.The percentage of mutant bacteria in the digestive tract chyme of Tibetan sheep reached(64.41±85.66)% for L shape.G shape was at(28.81±14.34)%.And F type was only(6.78±0.00)%. L-type Pasteurella had grown two colony morphologies on hypertonic TSLN medium,the proportion of mutant bacteria was up to(97.67±89.50)% in the nasal discharge of Tibetan sheep with G shape,and L type was only at(2.65±10.50)%.G shape in the digestive tract chyme of Tibetan sheep reached at(82.28±88.92)%,L type was only at(17.72±11.08)%.Staning to L-type bacteria and microscopic examination,normal purified bacteria were selected for Gram staning.Normal Escherichia coli was with pink,blunt and Gram-negative brevibacillus by microscpic examination.Pasteurella showed Gram-negative bacilli brevis with obtuse in opposite ends,and micro bulge in the central,single individual or in pairs existence.Bacterial colonies with"Fried eggs"shape were picked up for Gram stain from L-type bacteria,it showed Gram-negative bacteria with ovoid,bars and rod-shaped for L-type Escherichia coli under microscope,L-type Pasteurella showed Gram-positive bacteria with sticks and bar shape. In addition to that,Tannin-gentian violet staining dyed L-type Escherichia coli and L-type Pasteurella,that the whole bacterium exposed blue and purple,bacterial length was about 2-3 times than normal with various forms such as ellipse,stick and rod shape.Biochemical identification on L-type bacteria:L-type bacteria and normal bacteria were different in biochemical identification because of lack cell wall.Biochemical chatacters of normal Escherichia coli and Pasteurella was conformable to Berger’s Manual of Systematic Bacterriology.Phenylalanine of L-type Escherichia coli was the biggest difference with normal Escherichia coli.Others were not evident.Biochemical identification of L-type Pasteurella compared to Pasteurella showed that Oxidase strip was blue,and other biochemical tests of semi-solid AGAR puncture(dynamic tests),V-P,ornithine decarboxylase broth,lysine decarboxylase broth,sorbitol,melibiose and melitriose had positive reactions.Trytophan decarboxylase broth were negative.Serotype identification on L-type Escherichia coli,Agglutination intensity of serum of L-type Escherichia coli was less than normal Escherichia was up to 2+.Moreover,normal Escherichia coli was the strongest to 4+.The serum agglutination intensity of L-type Escherichia coli was lower than of Escherichia coli,the highest was 2+,and that of normal Escherichia coli was 4+.L-type Escherichia coli bands were O15-R,O91-2 R and O8-F from high to low,while normal Escherichia coli bands were O91-2 R,O15-R,O21-F from high to low.L-type Pasteurella bands were A-F,B-F and F-F from high to low,and normal Pasteurella electrophoresis bands were A-F,B-F and E-F.The results indicated that antigenicity of L-type bacteria had changed,the ability to adapt to high altitude environment,the probability of mutation under strong ultraviolet light,and the probability of mutation in cell wall antigenicity were also different among different bacterial genotypes.