猪源链球菌的分离鉴定及其生物学特性研究

发布时间：2018-04-17 03:27

本文选题：猪源链球菌 + 流行病学　；参考：《河南科技大学》2017年硕士论文

【摘要】：近年来,猪链球菌病在我国广泛流行并致病,已成为严重危害养猪业发展的主要病原菌之一。本研究对猪源链球菌的病原流行病学、毒力特性、生物学特性等进行了系统研究,得到了以下研究结果:1.通过对猪源链球菌的分离鉴定(2014.09~2015.12),并对前期实验室分离鉴定结果进行统计分析(2011.01~2014.08),结果表明,2011~2015年间,从河南及其周边地区规模养殖场和部分散养农户2 521份发病猪的肺脏等病料组织中分离鉴定出772株链球菌(Streptococcus),总分离率为30.6%。通过基于gdh基因的特异性PCR方法对其中310株猪源链球菌进行了PCR鉴定,共鉴定出135株猪链球菌(S.suis),占43.5%。通过基于cps基因的特异性PCR方法对135株猪链球菌进行了血清型鉴定,共鉴定出72株猪链球菌2型(占53.3%),14株7型(占10.4%),14株9型(占10.4%),2株1型(占1.5%),33株其他型(占24.4%)。这表明在我国河南及其周边省份的猪群中,链球菌为分离率最高的细菌性病原,其中猪链球菌仍占重要地位(占43.5%),在猪链球菌中,血清2型占主导地位(占53.3%)。2.2011~2015年间,猪源链球菌的分离率介于23.5~45.5%之间,其中2011年和2014年显著偏高(p0.01)。另外发现其分离率具有明显的季节性,其中2~5月和8月份显著偏高(p0.01)。在分离出链球菌的772份病料中,有274份仅分离出链球菌,没有发现其他常见病原菌,占35.5%;有498份同时分离出各种不同的共感染菌,占64.5%。链球菌最常见的共感染菌是副猪嗜血杆菌占所有共感染病料的47.4%;(236/498),其次是大肠埃希菌(35.3%;176/498)、多杀性巴氏杆菌20.9%;(104/498)、支气管败血波氏杆菌(15.3%;76/498)、葡萄球菌(6.2%;31/498)、沙门氏菌(6.1%;30/498)和胸膜肺炎放线杆菌(1.0%;5/498)。这表明在猪链球菌病的发生具有明显的季节性,且与其他病原菌的共感染情况非常严重。3.对分离鉴定的49株猪链球菌,包括2型26株、7型14株、9型9株,和实验室前期分离鉴定的2株C群马链球菌兽疫亚种强毒菌株进行了小鼠毒力试验,结果表明C群马链球菌兽疫亚种的菌株毒力表现最强,其次是猪链球菌2型、7型和9型菌株。分别选择小鼠毒力试验中表现最强的猪链球菌2型3株、7型3株、9型3株和2株C群马链球菌兽疫亚种菌株(共11株链球菌),进一步进行仔猪毒力试验,结果表明2株C群马链球菌兽疫亚种菌株和3株猪链球菌2型菌株的攻毒仔猪均能出现典型猪链球菌病的临床症状和病理变化,3株猪链球菌9型菌株中有1株(SS9-6)能导致攻毒仔猪出现典型临床症状和病理变化;但是3株猪链球菌7型菌株均不能。这表明在我国猪链球菌病的病原中,C群马链球菌兽疫亚种和猪链球菌2型菌株的致病力较强,而猪链球菌7型和9型较弱。另外,各血清型猪链球菌的小鼠毒力试验结果和仔猪毒力试验结果并不完全一致。4.以C群马链球菌兽疫亚种M1株和猪链球菌2型SS2-2株为代表菌株进行了培养基筛选试验。在含10%新生牛血清的TSA培养基、牛血清琼脂培养、鲜血琼脂培养基基、麦康凯琼脂培养基和巧克力琼脂培养基共5种固体培养基中,2株菌株均在含10%新生牛血清的TSA中生长最快。在含10%新生牛血清的TSB培养基、马丁肉汤培养基、硫乙醇酸盐培养基(TM)、THB和BHI共5种液体培养基中,2株菌株均在含10%新生牛血清的TSB中生长活菌含量最高。对上述的11株链球菌进一步研究了其在TSA固体培养基上的培养试验,结果表明,猪链球菌2型、7型、9型菌株之间的生长速度无明显差异,其24h培养菌落的直径介于1.62~1.88 mm之间,但C群菌株的生长速度显著偏快(p0.01),其平均直径为3.24 mm。在TSB液体培养基中的生长曲线测定结果表明,11株链球菌的最高活菌含量介于8.4×108~1.2×109 CFU/mL之间,均能高密度发酵培养。21种生化反应的试验比较结果表明,11株链球菌的生化反应均不一致,即使同一血清型的不同菌株之间也具有较大差别。药敏试验表明,11个强毒菌株对青霉素G、氨苄西林、阿莫西林、头孢噻肟、头孢拉定、头孢克洛、阿奇霉素等均高度敏感。这些试验筛选出了适合猪源链球菌培养的最佳培养基,同时比较了11株强毒菌株的生化特征、生长特性、药物敏感性等生物学特性,为后期灭活疫苗研发奠定基础。
[Abstract]:In recent years, Streptococcus suis are widely popular in China and has become one of the main pathogens, pathogenic bacteria and serious harm to the development of pig industry. The epidemiology of swine streptococcus, the virulence characteristics, biological characteristics were studied, the main results are as follows: 1. through the isolation and identification of Streptococcus suis (2014.09~2015.12), and the identification results of the laboratory separation for statistical analysis (2011.01~2014.08), the results showed that during 2011~2015, identified 772 strains isolated from Henan and the surrounding area the size of farms and backyard farmers 2521 incidence of swine lung diseases such as material organization (Streptococcus), the total separation rate of 30.6%. by specific the PCR method based on GDH gene of the 310 strains of Streptococcus suis were identified using PCR method, we identified 135 strains of Streptococcus suis (S.suis), accounting for 43.5%. based on CPS gene The specific PCR method was used to identify the serotype of 135 strains of Streptococcus suis were identified in 72 strains of Streptococcus suis type 2 (53.3%), 14 strains of type 7 (10.4%), 14 strains of type 9 (10.4%), 2 strains of type 1 (1.5%), 33 strains of other type (24.4%). Show in Henan and its surrounding provinces in herds, streptococcus was the highest isolation rate of bacterial pathogens, including Streptococcus suis still occupies an important position (43.5%), in Streptococcus suis serotype 2, the dominant (53.3%).2.2011~2015 years, between the separation rate between 23.5~45.5% swine streptococcus, which in 2011 and in 2014 was significantly higher (P0.01). Also found that the separation rate has obvious seasonal, which 2~5 in April and August were significantly higher (P0.01). In the separation of 772 cases of Streptococcus, 274 only isolated Streptococcus, not found in other common pathogens, accounted for 35.5%; 498 copies and isolated different Co infection of bacteria, accounted for the most common 64.5%. co infection of streptococcus bacteria Haemophilus parasuis disease material co infection accounted for all 47.4%; (236/498), followed by Escherichia coli (35.3%; 176/498), Pasteurella multocida (20.9%; 104/498), b.bronchiseptica (15.3%; 76/498). Staphylococcus (6.2%; 31/498), Salmonella (6.1%; 30/498) and Actinobacillus pleuropneumoniae (1%; 5/498). This shows that in the highly seasonal occurrence of Streptococcus suis disease, and other pathogenic bacteria infection is very serious on the identification of.3. from 49 strains of Streptococcus suis type 2, including 26 strains, 14 strains of type 7, type 9 and 9 strains of lab isolated 2 strains of group C Streptococcus equi subsp.zooepidemicus virulent strain of mouse virulence test, the results showed that C of Streptococcus equi subspecies Gunma strains showed the highest virulence, followed by Streptococcus suis type 2, type 7 and type 9 choose small strain. In the performance of the strongest virulence test of 3 strains of Streptococcus suis type 2, type 7 and 3 strains, 3 strains of type 9 and 2 strains of group C Streptococcus equi subsp.zooepidemicus strains (11 strains), further piglets toxicity test, clinical symptoms and pathological changes showed that the 2 strains of group C Streptococcus equi subsp. Strains and 3 strains of Streptococcus suis type 2 strains of inoculated piglets were typical Streptococcus, 3 strains of Streptococcus suis type 9 strains in 1 strains (SS9-6) can cause the inoculated piglets showed typical clinical symptoms and pathological changes; but 3 strains of Streptococcus suis serotype 7 strains were not. This shows that the Streptococcus suis disease in China the pathogen, Streptococcus equi subspecies C strong horses and the pathogenicity of Streptococcus suis type 2 strains, and 7 Streptococcus suis type 9 and weak. In addition, mouse virulence test results of various serotypes of Streptococcus suis in piglets and virulence test results are not entirely consistent with the.4. C group streptococcus beast NDV M1 strain and subspecies of Streptococcus suis serotype 2 strain SS2-2 as the representative of the culture medium screening test strains. Cultured in TSA containing 10% fetal calf serum, bovine serum agar, blood agar base, Jimmy Kang Kai agar and chocolate agar 5 solid medium, 2 strains were in TSA containing 10% fetal calf serum in the culture medium. The fastest growth in TSB containing 10% fetal calf serum, Martin broth, thioglycollate medium (TM), THB and BHI 5 kinds of liquid medium, 2 strains were in TSB containing 10% fetal calf serum in growth the content of the highest living bacteria. 11 strains of Streptococcus further research the culture test in TSA solid medium showed that Streptococcus suis type 2, type 7, no significant difference in growth rate between the 9 strains, the 24h culture colony diameter ranged from 1.62~1.88 mm, but C strains of the students Long too fast speed significantly (P0.01), the average diameter of 3.24 mm. growth curve in TSB liquid medium were tested. The results show that among the 11 strains of the highest viable content ranging from 8.4 * 108~1.2 * 109 CFU/mL, were able to test high density fermentation.21 biochemical reactions and the comparison results showed that the biochemical Reaction 11 strains are inconsistent, even between different strains of the same serotype has great difference. The drug sensitivity test showed that 11 virulent strain of penicillin G, amoxicillin, ampicillin, cefotaxime, Cefradine, cefaclor, spectinomycin, were highly sensitive. These tests selected the best medium for cultivation of swine streptococcus, and compare the biochemical characteristics of 11 strains of virulent strains of growth characteristics, biological characteristics of drug sensitivity, laying the foundation for the later development of the inactivated vaccine.

【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.611

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