猪链球菌防治新技术研究
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摘要
本研究对16起疑似猪链球菌感染病料进行分菌鉴定后,确认14起为猪链球菌感染,其中2型3起,7型5起,6起是1、2、7和9型之外的血清型,一起弓形虫感染,一起未查明病因。对甘、青、宁三省的猪链球菌2型的血清学调查结果表明,临床健康猪群的猪链球菌2型抗体阳性率为0～16.7%;连续5年从甘肃武威健康猪只采取扁桃体分菌,猪链球菌的分离率为27.2%,其中2型5.5%,9型13.2%, 7型11.4%,1型3.7%,66%是1、2、7和9型之外的血清型。药敏检测表明多重耐药性菌株占分离菌株的84%以上。
通过基因筛选、不相合性检测及不同基因序列组合分型,发现由dnaK-EFTu-potA-eno-uvrA-guaB的基因合并序列所构建的系统发生树在各节点处的自举检验值均≥85%,该系统发育树从根本上提高了链球菌分型结果的可靠性。据此,链球菌分为3群,第一群为mitis群链球菌、猪链球菌和热链球菌,猪链球菌与mitis群链球菌的遗传关系极为密切;第二群为pyogenic群链球菌;第三群为变异链球菌和牛链球菌。该系统发育树从根本上提高了链球菌分型的准确性。potA基因上一段碱基序列,能够在种的水平上对各种链球菌分类。
通过灭活猪链球菌2型强毒株基因组上的D-丙氨酸消旋酶基因,筛选到依赖外源D-丙氨酸才能复制的变异菌株24株。接着通过生长试验筛选出5株生长良好的变异菌株。为了选择制苗菌株,将5个变异菌株分别接种于10只22-24g SPF昆明小鼠。4周后,根据小鼠不同组织器官中变异菌株的存留情况以及免疫效力,筛选出一株制苗候选菌株,命名为S.suis serotype 2 11△alr 2/24。为测定该菌株实用免疫剂量,分别以5×10~8,1×10~9,2×10~9,3×10~9,4×10~9,5×10~9CFU剂量接种3-4周龄猪链球菌2型抗体阴性小猪5只,同时用5只小猪作为空白对照。免疫4周后,用1.7×10~7剂量的猪链球菌强毒株通过耳静脉攻毒。结果对照组小猪在1周内全部死亡(5/5),5×10~8组和1x10~9组歌存活3头, 2×10~9组以及3×10~9组各存活3头,4×10~9和5×10~9组全部存活。从而确定4×10~9CFU为试制疫苗的实用剂量。用该剂量试制疫苗2批,在单次超剂量免疫仔猪的安全性试验和单剂量多次免疫仔猪的安全性试验中,从免疫仔猪的肝、心、脾、肺、脑和扁桃体均未检测到1△alr 2/24菌株存在,说明该疫苗是安全可靠的。试制疫苗2批,按实用免疫剂量接种小猪各5只。4周后,用约2.1×10~7剂量的猪链球菌强毒株通过耳静脉攻毒,结果未免疫小猪全部死亡,免疫组全部存活。本研究的结果表明该营养缺陷细菌基因工程疫苗具有良好的安全性和免疫效力,为研制新一代安全高效的猪链球菌外源营养依赖型疫苗奠定了基础。
In this research, the identification of pathogens from samples originating from 16 farms that were suspected to be affected with Streptococcus suis (S. suis ) revealed: 14 infections caused by S. suis, one caused by Toxoplasma.gondii, and for one case the pathogen was not identified. Of the S. suis samples that were isolated, three were S. suis serotype 2, five were S. suis serotype 7 and six were not identified. Serological detection of S. suis serotype 2 from the sera collected from different regions from Gansu, Qinghai, and Ningxia provinces revealed the positive rates for S. suis serotype 2 ranged from 0 to 16.7%. In the 2007-2011 period, S. suis strains were isolated from tonsils of pigs from Wuwei: 272 S. suis isolates were collected and the positive sample rate was 27.2%, of which, 5.5% were S. suis serotype 2, 13.2 % were serotype 9, 11.4% were serotype 9, and 3.7% were serotype 1. The drug resistance ability test revealed that 84% of the isolated S. suis strains had multidrug resistance.
To obtain the best possible phylogenetic analysis among genus Streptococcus, the Multilocus sequence typing (MLST) method was used and the concatenated sequence of partial sequences from dnaK-EFTu-potA-eno-uvrA-guaB genes was found to be most suitable for analysis. The bootstrap values in every node for the 14 Streptococcus species described in this research were≥86. From the phylogenetic analysis, it was found that the species used in this research could be divided into three major clusters. Cluster one includes the mitis group-related S. suis and S. thermophilus which belonged to the Salivarious group. The second cluster includes the pyogenic group and the third cluster includes S. mutans and S. gallolyticus which belong to the equines group. Based on this research, a partial 638 bp sequence from the gene coding for ABC-type spermidine/putrescine transport system was found to be usable for genotyping among genus Streptococcus at the species level. To make a safe and highly efficient vaccine, we produced, using the pSET4s vector, twenty four D-alanine authophic S. suis serotype 2 strain 06TS08 by gene knockout of the alanine racemase gene that is responsible for the supply of D-alanine. One D-alanine authophic S. suis serotype 2 strain that was named 11△alr 2/24 was selected for S. suis serotype 2 live vaccine research. To assess the immune dosage of the authophic S. suis serotype 2 strain as a vaccine, 30 piglets that were S. suis serotype 2 negative were distributed into 6 groups of 5 piglets, each of which were administrated by intramuscular injection with 5×10~8, 1×10~9,2×10~9,3×10~9,4×10~9,5×10~9 CFU of 11△alr 2/24. Simultaneously, five piglets were used as control which were injected with PBS. Four weeks later, all the piglets were challenged with 1.7×10~7 CFU S. suis serotype 2 by auricular vein injection. The non-immunized piglets all died within one week. For immunized pigs: in the 5×10~8 group, three survived;in the 1×10~9 group three survived; 2×109 group four survived; in the 3×10~9 group four survived; in the 4×10~9 group five survived and in the 5×10~9 group five survived. Based on these results, 4×10~9 CFU was considered to be a suitable dosage. Two batches of vaccines were prepared based on this dosage and the safety test of the vaccine revealed that S. suis serotype 2 11△alr 2/24 was not found in liver, heart, spleen, lung and tonsils of immunized piglets 28 days post injection. At the same time, for every vaccine batch, 10 piglets were distributed into two groups: 5 piglets were administrated by intramuscular injection with 1ml of the prepared vaccine and 5 piglets were used as the control group which injected PBS. Four weeks later, all the piglets were challenged with 2.1×10~7 CFU Serotype 2 by auricular vein injection. The non-immunized piglets were all dead within one week while all the immunized piglets survived. These results revealed that the mutant S. suis serotype 2 strain 11△alr 2/24was safe and effective to make vaccines.
通过基因筛选、不相合性检测及不同基因序列组合分型,发现由dnaK-EFTu-potA-eno-uvrA-guaB的基因合并序列所构建的系统发生树在各节点处的自举检验值均≥85%,该系统发育树从根本上提高了链球菌分型结果的可靠性。据此,链球菌分为3群,第一群为mitis群链球菌、猪链球菌和热链球菌,猪链球菌与mitis群链球菌的遗传关系极为密切;第二群为pyogenic群链球菌;第三群为变异链球菌和牛链球菌。该系统发育树从根本上提高了链球菌分型的准确性。potA基因上一段碱基序列,能够在种的水平上对各种链球菌分类。
通过灭活猪链球菌2型强毒株基因组上的D-丙氨酸消旋酶基因,筛选到依赖外源D-丙氨酸才能复制的变异菌株24株。接着通过生长试验筛选出5株生长良好的变异菌株。为了选择制苗菌株,将5个变异菌株分别接种于10只22-24g SPF昆明小鼠。4周后,根据小鼠不同组织器官中变异菌株的存留情况以及免疫效力,筛选出一株制苗候选菌株,命名为S.suis serotype 2 11△alr 2/24。为测定该菌株实用免疫剂量,分别以5×10~8,1×10~9,2×10~9,3×10~9,4×10~9,5×10~9CFU剂量接种3-4周龄猪链球菌2型抗体阴性小猪5只,同时用5只小猪作为空白对照。免疫4周后,用1.7×10~7剂量的猪链球菌强毒株通过耳静脉攻毒。结果对照组小猪在1周内全部死亡(5/5),5×10~8组和1x10~9组歌存活3头, 2×10~9组以及3×10~9组各存活3头,4×10~9和5×10~9组全部存活。从而确定4×10~9CFU为试制疫苗的实用剂量。用该剂量试制疫苗2批,在单次超剂量免疫仔猪的安全性试验和单剂量多次免疫仔猪的安全性试验中,从免疫仔猪的肝、心、脾、肺、脑和扁桃体均未检测到1△alr 2/24菌株存在,说明该疫苗是安全可靠的。试制疫苗2批,按实用免疫剂量接种小猪各5只。4周后,用约2.1×10~7剂量的猪链球菌强毒株通过耳静脉攻毒,结果未免疫小猪全部死亡,免疫组全部存活。本研究的结果表明该营养缺陷细菌基因工程疫苗具有良好的安全性和免疫效力,为研制新一代安全高效的猪链球菌外源营养依赖型疫苗奠定了基础。
In this research, the identification of pathogens from samples originating from 16 farms that were suspected to be affected with Streptococcus suis (S. suis ) revealed: 14 infections caused by S. suis, one caused by Toxoplasma.gondii, and for one case the pathogen was not identified. Of the S. suis samples that were isolated, three were S. suis serotype 2, five were S. suis serotype 7 and six were not identified. Serological detection of S. suis serotype 2 from the sera collected from different regions from Gansu, Qinghai, and Ningxia provinces revealed the positive rates for S. suis serotype 2 ranged from 0 to 16.7%. In the 2007-2011 period, S. suis strains were isolated from tonsils of pigs from Wuwei: 272 S. suis isolates were collected and the positive sample rate was 27.2%, of which, 5.5% were S. suis serotype 2, 13.2 % were serotype 9, 11.4% were serotype 9, and 3.7% were serotype 1. The drug resistance ability test revealed that 84% of the isolated S. suis strains had multidrug resistance.
To obtain the best possible phylogenetic analysis among genus Streptococcus, the Multilocus sequence typing (MLST) method was used and the concatenated sequence of partial sequences from dnaK-EFTu-potA-eno-uvrA-guaB genes was found to be most suitable for analysis. The bootstrap values in every node for the 14 Streptococcus species described in this research were≥86. From the phylogenetic analysis, it was found that the species used in this research could be divided into three major clusters. Cluster one includes the mitis group-related S. suis and S. thermophilus which belonged to the Salivarious group. The second cluster includes the pyogenic group and the third cluster includes S. mutans and S. gallolyticus which belong to the equines group. Based on this research, a partial 638 bp sequence from the gene coding for ABC-type spermidine/putrescine transport system was found to be usable for genotyping among genus Streptococcus at the species level. To make a safe and highly efficient vaccine, we produced, using the pSET4s vector, twenty four D-alanine authophic S. suis serotype 2 strain 06TS08 by gene knockout of the alanine racemase gene that is responsible for the supply of D-alanine. One D-alanine authophic S. suis serotype 2 strain that was named 11△alr 2/24 was selected for S. suis serotype 2 live vaccine research. To assess the immune dosage of the authophic S. suis serotype 2 strain as a vaccine, 30 piglets that were S. suis serotype 2 negative were distributed into 6 groups of 5 piglets, each of which were administrated by intramuscular injection with 5×10~8, 1×10~9,2×10~9,3×10~9,4×10~9,5×10~9 CFU of 11△alr 2/24. Simultaneously, five piglets were used as control which were injected with PBS. Four weeks later, all the piglets were challenged with 1.7×10~7 CFU S. suis serotype 2 by auricular vein injection. The non-immunized piglets all died within one week. For immunized pigs: in the 5×10~8 group, three survived;in the 1×10~9 group three survived; 2×109 group four survived; in the 3×10~9 group four survived; in the 4×10~9 group five survived and in the 5×10~9 group five survived. Based on these results, 4×10~9 CFU was considered to be a suitable dosage. Two batches of vaccines were prepared based on this dosage and the safety test of the vaccine revealed that S. suis serotype 2 11△alr 2/24 was not found in liver, heart, spleen, lung and tonsils of immunized piglets 28 days post injection. At the same time, for every vaccine batch, 10 piglets were distributed into two groups: 5 piglets were administrated by intramuscular injection with 1ml of the prepared vaccine and 5 piglets were used as the control group which injected PBS. Four weeks later, all the piglets were challenged with 2.1×10~7 CFU Serotype 2 by auricular vein injection. The non-immunized piglets were all dead within one week while all the immunized piglets survived. These results revealed that the mutant S. suis serotype 2 strain 11△alr 2/24was safe and effective to make vaccines.
引文
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