【作者】 马增军；

【导师】 杨汉春； 房海；

【作者基本信息】 中国农业大学 ， 预防兽医， 2013， 博士

【摘要】 猪链球菌(Streptococcus suis)可引起猪败血症、脑膜炎、心内膜炎和关节炎,也是一种重要的人畜共患病病原,具有重要的公共生学意义。因此,开展猪链球菌病病原学调查与监测,对于该病的防治具有重要的理论和实践意义。本论文研究工作涉及河北省不同地区猪链球菌的血清型监测与分析,致病性猪链球菌的耐药性和毒力基因分析,利用优势致病菌株研制猪链球菌病价灭活疫苗,旨在了解河北省猪链球菌病的流行情况,为猪链球菌病的防治提供科学依据。从河北省石家庄、邢台、张家口、沧州、唐山、秦皇岛等地采集临床健康猪鼻拭子600份,经增菌分离培养、革兰染色镜检、PCR鉴定猪链球菌种及分离菌株血清型鉴定。结果表明,从600份鼻拭子样品中共检出148株猪链球菌,检出率为24.67%,其中27株为猪链球菌7型,24株为猪链球菌2型,20株为猪链球菌9型,2株为猪链球菌1型,75株未定血清型。由此表明,河北省正常猪群中猪携带的猪链球菌血清型呈现复杂多样的特点。对2005-2012年从河北省各地区临床病死猪分离获得的96株猪源链球菌进行了生物学特性鉴定及血清分群。结果表明,分离的链球菌以兰氏D群为主,占34.4%,其次为C群(18.8%)。利用PCR扩增猪链球菌的种特异性gdh基因以及荚膜多糖cpslI、cps2J、cps7H和cps9H基因,对96株链球菌分离株进行猪链球菌种的鉴定和分型。结果显示,属于猪链球菌的为47株,其中猪链球菌2型(SS2)为65.96%(31/47),SS7为2.13%(1/47),SS9为4.25%(2/47),其它血清型为27.66%(13/47)。采用微量稀释法测定了96株猪源链球菌对27种药物的体外最小抑菌浓度(MIC)。结果显示,94.8%的菌株呈现多重耐药,耐药谱型达14种；对磺胺类药物、氨基糖苷类药物、四环素类药物、林可胺类药物、大环内酯类药物的耐药性最为严重,对青霉素类、头孢菌素类、喹诺酮类、酰胺醇类耐药性次之；对头孢菌素类药物——头孢噻呋、头孢唑啉、头孢噻吩的敏感率分别为87.5%、85.4%和83.3%。由此表明,猪链球菌临床分离株对常用药物均产生了一定的耐药性。应用PCR对血清型明确的34株猪链球菌的6个毒力因子(gdh、cps、ef、sly和orf2)进行了检测。结果显示,31株猪链球菌2型分离株表现为9种毒力因子基因型,以cps2+/gdh+/sly+/ef+/mrp+/orf2+和cps2+/gdh+/sly-/ef+/mrp+/orf2+为主,分别占32.3%和35.5%,提示河北省SS2的主要流行菌株为同时具有6种毒力因子的高致病性菌株和sly基因缺失株；1株SS7的基因型为cps7+/gdh+/sly+/ef-/mrp+/orf2+;2株SS9的基因型分别为cps9+/gdh+/sly+/ef-/mrp+/orf2+和cps9+/gdh+/sly-/ef-/mrp+/orf2+。致病性试验结果发现,具有cps2+/gdh+/sly+/ef+/mrp+/orf2+和cps2+/gdh+/sly-/ef+/mrp+/orf2+基因型的菌株,对猪均具有较强的致病性和致死性,死亡率达100%。利用毒力强、分别属于C、D两个主要流行血清群的的菌株LN-2和BBD-1,研制出猪链球菌病氢氧化铝二价灭活疫苗。免疫攻毒试验结果表明,疫苗对断奶仔猪、育肥猪均具有较好的安全性,用LN-2和BBD-1菌株攻毒,免疫组可获得完全保护。更多还原

【Abstract】 Streptococcus suis (SS) is the causative agent which can cause septicemia, meningitis, endocarditis and arthritis in pigs. It is also a zoonostic pathogen with potential significance to public health. Therefore, the etiological investigation on swine streptococusis possesses remarkable theoretical and practical values. This study carried out a serotype survey of SS prevalent in different areas of Hebei Province, and analyzed the drug resistances and virulence genes of SS, and developed bivalent inactivated vaccine of swine streptococcosis using dominant pathogenic strains of SS prevalent in the areas of Hebei province in order to provide scientific basis for the prevention of swine streptococcosis.An enrichment culture, Gram’s staining and microscopy, and PCR was implemented from600samples of nosal swabs collected from clinical healthy pigs in Shijiazhuang, Xingtai, Zhangjiakou, Cangzhou, Tangshan and Qinghuangdao of Hebei province. The results showed that148strains (24.67%) of Streptococcus suis were isolated from the samples, and of them,27strains (18.24%) belonged to serotype7,24strains (16.22%) were serotype2,20strains (13.51%) were serotype9, and2strains belonged to serotype1, and the serotypes of remaining75strains (50.68%) were undetermined, suggesting that there are various complex serotypes of SS in clinical healthy pigs.Ninety-six isolates of SS from sick or dead pigs during2005to2012were identified by biological characteristics and serum grouping. Among these isolates,34.4%mainly belonged to Group D, and18.8%belonged to Group C. The species and genotypes of96isolates were further identified by PCR amplifying species-specific gdh gene and genes (cpsl I, cps2J, cps7H and cps9H) of capsular polysaccharide of SS. The results indicated that47of96isolates were SS in which65.96%(31/47) belonged to serotype2(SS2),2.13%(1/47) was SS7,4.25%(2/47) was SS9and27.66%(13/47) belonged to other serotypes.The drug resistance of96isolates to27antibiotics was analyzed in vitro by microdilution. The results revealed that most of isolates exhibited obvious drug resistance to14antimicrobial drugs and94.8%of isolates displayed multi-drug resistance in which resistance to sulfonamides, aminoglycosides, tetracyclines, lincosamides and macrolides were most frequent, and then to penicillins, cephalosporins, quinolones and amphenicols.87.5%,85.4%and83.3%of isolates were susceptible to ceftiofur, cefazolin and cephalothin, respectively, showing that SS isolated from clinical sick or dead pigs are resistant to common antimicrobial drugs to some degree.Six virulence genes(gdh, cps, mrp, ef, sly and orf2) for34isolates of SS were detected by PCR. It was indicated that31out of34SS had9genotypes in which32.3%(10/31) was positive in cps2+lgdh+lepf+/mrp+/sly+lorf2+and35.5%(11/31) in cps2+gdh+sly-ef+mrp+orf2+, showing that the dominant strains of SS in Hebei province are highly pathogenic strains with six virulence genes and those of sly gene deletion; out of34,1(SS7) was positive in cps7+/gdh+/sly+/ef-/mrp+/orf2+, and2(SS9) were positive to cps9+/gdh+/sly+/ef-/mrp+/orf2+and cps9+/gdh+/sly-/ef-/mrp+/orf2+, respectively. Furthermore, animal trials showed that the strains with genotypes of cps2+/gdh+/sly+/ef+/mrp+/orf2+and cps2+/gdh+/sly-/ef+/mrp+/orf2+had stronger pathogenicity and fatality (100%mortality) for pigs.Bivalent inactivated vaccine with aluminium hydroxide adjuvant of swine streptococcosis was developed with two highly virulent strains of LN-2and BBD-1belonging to serogroup C and D. Challenge trials for the vaccinated animals indicated that the vaccine was safe for weaning pigs and finishing pigs which could provide complete protection against LN-2and BBD-1.更多还原