广东地区猪链球菌感染的流行病学调查及分离株的特性分析
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摘要
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。按荚膜抗原的特性,目前分为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。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。SS可以致猪败血症、脑膜炎、关节炎等症状,SS2还致人败血症、脑膜炎、肺炎等。对于猪链球菌的致病机理研究的还不是很清楚,发现与许多毒力因子有关系。根据致病的强弱,SS可分为强毒株、弱毒株和无毒株,目前对ss毒性强弱的判定还没有标准,动物实验还是行之有效的方法。本实验对广东省地区的流行株进行了研究,具体如下。
1.广东地区猪群中猪链球菌的分离鉴定与分析
2007年9月-2008年9月,从广东省各地40多个猪场和2个屠宰场采集猪的肺、肝、心血、脾、淋巴结、扁桃体等器官,在发病猪群中采集187头份病猪的591份病料,屠宰猪群中采集193份扁桃体,202份鼻拭子样品。细菌分离培养后,用PCR方法鉴定SS,对分离到的菌株做1型(或14型)、2型(或1／2型)、7型、9型的血清型做PCR鉴定。发病猪群中72份(38.2%)样品检出SS阳性,其中61份(32.6%)分离得到SS 111株；屠宰猪群中,扁桃体样品检测到28份(14.51%),分离菌株18株(9.3%),鼻拭子样品仅检测到1株阳性,未分离到细菌。血清型鉴定发现发病猪群中39株(36.0%)为SS2型,2株为SS9型(1.8%)未定型为70株(63.2%);扁桃体样品中,6株(33.3%)检测为SS2型,1株为SS7型(5.6%),11株(61.1%)为未定型。分析表明：SS在广东地区比较流行,其中以2型为主,但有63.2%无法定型,且有血清型混合感染或者同其他疾病混合发生的现象,表明广东地区SS流行复杂,防控需要采取多种措施,才可能有效预防。
2.猪链球菌分离株主要毒力因子分布特征
依据本实验室建立方法,对猪链球菌7种主要毒力因子进行检测,包括溶菌酶释放因子(mrp)、胞外因子(efp)、溶血素(sly)、纤连蛋白/血纤蛋白原结合蛋白(fbps)、三磷酸甘油醛脱氢酶(gapdh)、谷氨酸脱氢酶(gdh)和毒力相关序列orf2,并合成4种新发现的潜在毒力因子引物,包括38KD-Protein、Sao蛋白、精氨酸脱亚氨酸酶(ads)、次黄嘌呤核苷酸脱氢酶(impdh)对分离菌株检测。检测结果表明,SS2含有的毒力因子最多,有10种及以上毒力因子的菌株是37株,占82.2%,以cps2/gdh+/sly+/ef+/mrp+/fbps+/gadph+/orf2+/38kD-pr+/impdh+/ads+/sao+基因型为主。2株SS9缺失sly、矿和mrp等主要的毒力因子。1株SS7基因型为cps7/gdh+/sly-/ef-/mrp+/fbps+/gadph+/orf2+/38kD-pr+/impdh-/ads-/sao+。未定型菌株的毒力因子缺失严重,主要含有gdh、gapdh、orf2、38kD-pr、ads毒力因子。
3.猪链球菌分离株的生化特性与药物敏感性
用微量发酵管法分析18株屠宰猪群分离株和60株发病猪群分离株的生化特性,实验结果发现,猪链球菌易发酵葡萄糖、蔗糖、海藻糖等,对无机盐类等物质不发生发酵反应。结果显示不同地区的菌株的生化特性略有不同。用药敏纸片法检测45株扁桃体分离株和56株发病猪群分离株分析对药物的敏感情况。实验结果表明,80.4%菌株对头孢唑啉和阿莫西林等药敏感,44.4%菌株对乙酰螺旋霉素、氟本尼考、氧氟沙星等中度敏感。62.5%以上的菌株对大环内酯类和四环素类等不敏感。不同地区的菌株对不同药物的敏感度有较大差异,而相同地区的菌株对药物的敏感性差异不大。
4.猪链球菌2型免疫抑制小鼠感染模型的初步建立
选用18-20g,雄性昆明小鼠,按80mg/kg剂量腹腔注射环磷酰胺,连续注射3d,建立免疫抑制模型。检测江苏分离株HA9801对免疫抑制小鼠的半数致死量(LD50)将小鼠分为7组(A、B、C、D、E、免疫对照组和空白对照组),每组6只小鼠,A-E组,分别注射5×107、1.6×107、5×106、1.6×106、5×105cfu,用Reed-Muench法计算小鼠的LD50,为4.9×106cfu。选择3株流行病学背景不同的菌株A070、A107、ZQ-3,A070和A107为临床发病猪分离株,ZQ-3为屠宰猪群分离株,以HA9801株对免疫抑制小鼠的LD50的剂量,检测其对免疫抑制小鼠的致病性。攻毒之后的小鼠,表现出精神萎靡,厌食,被毛粗乱。剖检病变明显,大脑充血、出血,肝、肺、心等充血、淤血,有的肠道充血、鼓气。在肺、肝、心、脑等多个器官分离到病原。试验结果显示A070对小鼠的致死率为50%,A107对小鼠的致死率为30%,ZQ-3对小鼠的致死率为25%,试验结果表明了免疫抑制小鼠对猪链球菌致病性敏感,能成功复制出病变,较好的区分出菌株毒性的强弱。
Streptococcus suis (SS) is an important zoonotic agent. A characteristic feature of this pathogen is diversity of serotype based on capsular polysaccharides. To date,35 serotypes (1-34 and 1/2) have been identified. Baesd on epidemiological date, serotypes 1,2,1/2,7 and 9 are considered to be more invasive than others. SS can cause serious enzootic infections worldwide. SS can cause septicemia, meningitis and arthritis in pigs and also can cause septicemia, meningitis and pneumonia in humans. Up to now, we still know little about pathopoiesis mechanism of SS, but we found that the relation with virulence factor in some way. According to the degree of virulence, SS is divided to three types: highly-virulent, weakly-virulent and non-virulent.
1. Isolation and identification of Streptococcus suis in Guangdong province
Organs were collected from more than 40 nurseries and 2 abattoirs between September 2007-September 2008, such as lung, liver, heart-blood, spleen, lymph node and tonsilla. We collected 591 samples from 187 diseased pigs,193 portion of tonsil and 202 portions of nose swab samples from abataged pigs. The serotypes of the isolated strains were determined after fractional cultivation by PCR.72 samples were determined to SS, the ratio was 38.2% and 111 strains were isolated from the disease pigs. In the abataged pigs,28 samples from tonsilla were determined to be SS, the ratio was 14.51% and 18 strains were isolated, the ratio was 9.3%. Only one sample from nose swab was detected positive, but no bacterium was isolated. On the basis of PCR,39 samples from diseased pigs were determined to be SS2,2 strains were SS9, serotypes of 70 strains could not be identified and the ratio was 35.1%,1.8% and 63.2% for each serotype. As to nose swab samples,6 strains were SS2,1 strain was SS7,11 strains were unidentified serotypes and the ratio was 33.3%,5.5% and 61.1%. It is concluded that SS was prevalent among Guangdong province, especially SS2, but 63.2% of strains were unidentified serotypes. This showed that the prevalence of swine streptocosis was complicated, so we have to adopt more measures in order to control it more effectively.
2. Distribution of main virulence factor in the Streptococcus suis isolates
According to the approach established in our lab, seven common virulence factors were detected, such as muramidase-released protein(MRP), extracellular factor (EF), Suilysin(SLY), fibronectin-binding protein(FBPS), glyceraldehyde-3-phosphate dehydro-Genase (GAPDH), glutamate dehydrogenase (GDH) and virulence-related rank orf2, and four new found larvaceous virulence factors, such as 38-KD protein, Sao protein, arginine deiminase (ADS), Inosine-5-monop-hosphate dehydrogenase (IMPDH). The results showed that the virulence factors in SS2 strain were most common, and 37 strains contained 10 virulence factors or more, the ratio was 82.2%. cps2/gdh+/sly+/ef+/mrp+/fbps+/gadph+/ orf2+/38kD-pr+/impdh+/ads+/sao+ was the most common among these strains.2 strains of SS9 was absent of sly, ef and mrp. The genotype of 1 strain of SS7 was cps7/gdh+/sly-/ef-/mrp+/fbps+/gadph+/orf2+/38kD-pr+/impdh-/ads-/sao+. The strains of unidentified serotype contained less virulence factors, such as gdh, gapdh, orf2, 38kD-protein and ads.
3. Biochemical characteristics and drug sensitivity of the Streptococcus suis isolates
The biochemistry characteristics of 18 SS isolates from slaughter pigs and 60 strains from diseased pigs were analyzed by gleam fermentation tube. The results showed that the SS isolates could fement glucose, cane sugar and trehalose easily, but inorganic salt could not be used in any way. At the same time we found that the biochemistry characteristics differed among different areas. The drug sensitivity of 45 SS isolates from tonsils and 56 SS strains from invased pigs were detected. The strains were sensitive to amoxicillin, inter-sensitive to acetylspiramycin, florfenicol, ofloxacin and resistant to macrolides and tetracyclines. There was no significant difference in the sensitivity for the isolate from a same area.
4. Establishment of immunosuppression mice model of SS2
The murine model of SS was established with 18-20g maleness mice by intraperitoneal injection with 80 mg/kg cyclophosphamide for 3 days. Six mice for each group, and administrated 5×107,1.6×107,5×106,1.6×106 and 5×105 cfu, respectively, were used to determine the LD50 of HA9801 isolated from Jiangsu province. The LD50 was calculated to be 4.9 x 106cfu by means of Reed-Muench. Virulences of three strains A070, A107 and ZQ-3 were evaluated in the immunosuppression mice based on the LD50 of HA9801, and the pathogenicities of these strains were deteermined. In clinical signs, the infected mice was depressed, anorexy, clothing hair in disorder. After the mice were dissected, we found that congestion occured in cerebrum, liver, lung, heart and intestinal tract. Furthermore, the bacterial strain used for infection was isolated from some organs, such as lung, liver, heart and brain. It suggested the immunosuppression mice were sensitive to SS and can be used as the model of Streptococcus suis type 2 infection
1.广东地区猪群中猪链球菌的分离鉴定与分析
2007年9月-2008年9月,从广东省各地40多个猪场和2个屠宰场采集猪的肺、肝、心血、脾、淋巴结、扁桃体等器官,在发病猪群中采集187头份病猪的591份病料,屠宰猪群中采集193份扁桃体,202份鼻拭子样品。细菌分离培养后,用PCR方法鉴定SS,对分离到的菌株做1型(或14型)、2型(或1／2型)、7型、9型的血清型做PCR鉴定。发病猪群中72份(38.2%)样品检出SS阳性,其中61份(32.6%)分离得到SS 111株；屠宰猪群中,扁桃体样品检测到28份(14.51%),分离菌株18株(9.3%),鼻拭子样品仅检测到1株阳性,未分离到细菌。血清型鉴定发现发病猪群中39株(36.0%)为SS2型,2株为SS9型(1.8%)未定型为70株(63.2%);扁桃体样品中,6株(33.3%)检测为SS2型,1株为SS7型(5.6%),11株(61.1%)为未定型。分析表明：SS在广东地区比较流行,其中以2型为主,但有63.2%无法定型,且有血清型混合感染或者同其他疾病混合发生的现象,表明广东地区SS流行复杂,防控需要采取多种措施,才可能有效预防。
2.猪链球菌分离株主要毒力因子分布特征
依据本实验室建立方法,对猪链球菌7种主要毒力因子进行检测,包括溶菌酶释放因子(mrp)、胞外因子(efp)、溶血素(sly)、纤连蛋白/血纤蛋白原结合蛋白(fbps)、三磷酸甘油醛脱氢酶(gapdh)、谷氨酸脱氢酶(gdh)和毒力相关序列orf2,并合成4种新发现的潜在毒力因子引物,包括38KD-Protein、Sao蛋白、精氨酸脱亚氨酸酶(ads)、次黄嘌呤核苷酸脱氢酶(impdh)对分离菌株检测。检测结果表明,SS2含有的毒力因子最多,有10种及以上毒力因子的菌株是37株,占82.2%,以cps2/gdh+/sly+/ef+/mrp+/fbps+/gadph+/orf2+/38kD-pr+/impdh+/ads+/sao+基因型为主。2株SS9缺失sly、矿和mrp等主要的毒力因子。1株SS7基因型为cps7/gdh+/sly-/ef-/mrp+/fbps+/gadph+/orf2+/38kD-pr+/impdh-/ads-/sao+。未定型菌株的毒力因子缺失严重,主要含有gdh、gapdh、orf2、38kD-pr、ads毒力因子。
3.猪链球菌分离株的生化特性与药物敏感性
用微量发酵管法分析18株屠宰猪群分离株和60株发病猪群分离株的生化特性,实验结果发现,猪链球菌易发酵葡萄糖、蔗糖、海藻糖等,对无机盐类等物质不发生发酵反应。结果显示不同地区的菌株的生化特性略有不同。用药敏纸片法检测45株扁桃体分离株和56株发病猪群分离株分析对药物的敏感情况。实验结果表明,80.4%菌株对头孢唑啉和阿莫西林等药敏感,44.4%菌株对乙酰螺旋霉素、氟本尼考、氧氟沙星等中度敏感。62.5%以上的菌株对大环内酯类和四环素类等不敏感。不同地区的菌株对不同药物的敏感度有较大差异,而相同地区的菌株对药物的敏感性差异不大。
4.猪链球菌2型免疫抑制小鼠感染模型的初步建立
选用18-20g,雄性昆明小鼠,按80mg/kg剂量腹腔注射环磷酰胺,连续注射3d,建立免疫抑制模型。检测江苏分离株HA9801对免疫抑制小鼠的半数致死量(LD50)将小鼠分为7组(A、B、C、D、E、免疫对照组和空白对照组),每组6只小鼠,A-E组,分别注射5×107、1.6×107、5×106、1.6×106、5×105cfu,用Reed-Muench法计算小鼠的LD50,为4.9×106cfu。选择3株流行病学背景不同的菌株A070、A107、ZQ-3,A070和A107为临床发病猪分离株,ZQ-3为屠宰猪群分离株,以HA9801株对免疫抑制小鼠的LD50的剂量,检测其对免疫抑制小鼠的致病性。攻毒之后的小鼠,表现出精神萎靡,厌食,被毛粗乱。剖检病变明显,大脑充血、出血,肝、肺、心等充血、淤血,有的肠道充血、鼓气。在肺、肝、心、脑等多个器官分离到病原。试验结果显示A070对小鼠的致死率为50%,A107对小鼠的致死率为30%,ZQ-3对小鼠的致死率为25%,试验结果表明了免疫抑制小鼠对猪链球菌致病性敏感,能成功复制出病变,较好的区分出菌株毒性的强弱。
Streptococcus suis (SS) is an important zoonotic agent. A characteristic feature of this pathogen is diversity of serotype based on capsular polysaccharides. To date,35 serotypes (1-34 and 1/2) have been identified. Baesd on epidemiological date, serotypes 1,2,1/2,7 and 9 are considered to be more invasive than others. SS can cause serious enzootic infections worldwide. SS can cause septicemia, meningitis and arthritis in pigs and also can cause septicemia, meningitis and pneumonia in humans. Up to now, we still know little about pathopoiesis mechanism of SS, but we found that the relation with virulence factor in some way. According to the degree of virulence, SS is divided to three types: highly-virulent, weakly-virulent and non-virulent.
1. Isolation and identification of Streptococcus suis in Guangdong province
Organs were collected from more than 40 nurseries and 2 abattoirs between September 2007-September 2008, such as lung, liver, heart-blood, spleen, lymph node and tonsilla. We collected 591 samples from 187 diseased pigs,193 portion of tonsil and 202 portions of nose swab samples from abataged pigs. The serotypes of the isolated strains were determined after fractional cultivation by PCR.72 samples were determined to SS, the ratio was 38.2% and 111 strains were isolated from the disease pigs. In the abataged pigs,28 samples from tonsilla were determined to be SS, the ratio was 14.51% and 18 strains were isolated, the ratio was 9.3%. Only one sample from nose swab was detected positive, but no bacterium was isolated. On the basis of PCR,39 samples from diseased pigs were determined to be SS2,2 strains were SS9, serotypes of 70 strains could not be identified and the ratio was 35.1%,1.8% and 63.2% for each serotype. As to nose swab samples,6 strains were SS2,1 strain was SS7,11 strains were unidentified serotypes and the ratio was 33.3%,5.5% and 61.1%. It is concluded that SS was prevalent among Guangdong province, especially SS2, but 63.2% of strains were unidentified serotypes. This showed that the prevalence of swine streptocosis was complicated, so we have to adopt more measures in order to control it more effectively.
2. Distribution of main virulence factor in the Streptococcus suis isolates
According to the approach established in our lab, seven common virulence factors were detected, such as muramidase-released protein(MRP), extracellular factor (EF), Suilysin(SLY), fibronectin-binding protein(FBPS), glyceraldehyde-3-phosphate dehydro-Genase (GAPDH), glutamate dehydrogenase (GDH) and virulence-related rank orf2, and four new found larvaceous virulence factors, such as 38-KD protein, Sao protein, arginine deiminase (ADS), Inosine-5-monop-hosphate dehydrogenase (IMPDH). The results showed that the virulence factors in SS2 strain were most common, and 37 strains contained 10 virulence factors or more, the ratio was 82.2%. cps2/gdh+/sly+/ef+/mrp+/fbps+/gadph+/ orf2+/38kD-pr+/impdh+/ads+/sao+ was the most common among these strains.2 strains of SS9 was absent of sly, ef and mrp. The genotype of 1 strain of SS7 was cps7/gdh+/sly-/ef-/mrp+/fbps+/gadph+/orf2+/38kD-pr+/impdh-/ads-/sao+. The strains of unidentified serotype contained less virulence factors, such as gdh, gapdh, orf2, 38kD-protein and ads.
3. Biochemical characteristics and drug sensitivity of the Streptococcus suis isolates
The biochemistry characteristics of 18 SS isolates from slaughter pigs and 60 strains from diseased pigs were analyzed by gleam fermentation tube. The results showed that the SS isolates could fement glucose, cane sugar and trehalose easily, but inorganic salt could not be used in any way. At the same time we found that the biochemistry characteristics differed among different areas. The drug sensitivity of 45 SS isolates from tonsils and 56 SS strains from invased pigs were detected. The strains were sensitive to amoxicillin, inter-sensitive to acetylspiramycin, florfenicol, ofloxacin and resistant to macrolides and tetracyclines. There was no significant difference in the sensitivity for the isolate from a same area.
4. Establishment of immunosuppression mice model of SS2
The murine model of SS was established with 18-20g maleness mice by intraperitoneal injection with 80 mg/kg cyclophosphamide for 3 days. Six mice for each group, and administrated 5×107,1.6×107,5×106,1.6×106 and 5×105 cfu, respectively, were used to determine the LD50 of HA9801 isolated from Jiangsu province. The LD50 was calculated to be 4.9 x 106cfu by means of Reed-Muench. Virulences of three strains A070, A107 and ZQ-3 were evaluated in the immunosuppression mice based on the LD50 of HA9801, and the pathogenicities of these strains were deteermined. In clinical signs, the infected mice was depressed, anorexy, clothing hair in disorder. After the mice were dissected, we found that congestion occured in cerebrum, liver, lung, heart and intestinal tract. Furthermore, the bacterial strain used for infection was isolated from some organs, such as lung, liver, heart and brain. It suggested the immunosuppression mice were sensitive to SS and can be used as the model of Streptococcus suis type 2 infection
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