Distribution of capsular serotypes and virulence markers of Streptococcus suis isolated from pigs with polyserositis in Korea

The objective of this study was to determine the capsular serotypes and potential virulence factors of Streptococcus suis isolated from pigs with polyserositis. Among the 24 isolates evaluated, serotype 3 [7 (29%) of the isolates] and serotype 4 [5 (21%)] were the most common. The isolates were also studied for the presence of the genes mrp, epf, and sly, which encode muramidase-released protein (MRP), extracellular factor (EF), and suilysin (SLY), respectively. Of the 24 isolates, 8 carried mrp: 4 of serotype 3, 2 of serotype 2, and 2 of serotype 4. One mrp(+) isolate (serotype 2) also carried the epf gene. All 24 isolates carried the sly gene. The serotype and genotype distribution greatly differed from that reported for isolates from pigs with other clinical manifestations of S. suis infection in other countries.     

屠宰场猪链球菌分离菌株分型及其致病性和耐药性分析

为了解吉林省猪链球菌的流行情况,从屠宰场采集的猪咽拭子和鼻拭子样品中分离鉴定猪链球菌,并进行菌株血清型、基因型和毒力表型的鉴定,以及致病性和耐药性的分析。结果表明,从100份样品中共分离鉴定猪链球菌104株,其中29株鉴定为血清2型、9型和1型等几种常见的致病性血清型,其他75株不属于常见的致病性血清型。血清2型的菌株中,2株经鉴定为ST1基因型和mrp+epf+sly+毒力型,并经动物试验鉴定为强毒菌株;其他菌株均为ST28基因型和mrp+epf-sly-毒力型,具有中等毒力。血清9型的菌株,均为mrp-epf-sly-毒力型,动物试验鉴定均为强毒菌株。根据Kirby-Bauer纸片扩散法的药敏试验结果,98%的猪链球菌分离株对四环素耐药;对大环内酯类、克林霉素和链霉素的耐药率都在50%以上;对β-内酰胺类、氯霉素和喹诺酮类抗生素的耐药率小于20%。总体分析,从屠宰场分离的猪链球菌强毒菌株所占的比例并不高,但是菌株多重耐药的情况非常严重。     

Distribution and characterization of Streptococcus suis serotypes isolated from January 2015 to June 2020 from diseased pigs in Québec,Canada

Streptococcus suis is one of the most important swine bacterial pathogens causing economic losses. This report presents the serotype distribution of S. suis recovered from diseased pigs in Québec from January 2015 to June 2020. Serotypes 1/2 and 2 predominated, followed by serotypes 7, 3, 5, 4, 9, 1, and 14. Compared to previously reported data, very few changes could be observed concerning the serotype distribution, indicating a relative stability. Half of the untypable isolates did not belong to the species S. suis sensu stricto, as determined by recN polymerase chain reaction. Less than 10% of “real S. suis” isolates were untypable. The genetic diversity of S. suis serotypes 1, 2, and 14, as analyzed by multilocus sequence typing, was mainly represented by sequence type (ST)1, ST28, ST25, and ST94. All ST1 isolates (considered highly virulent) belonged to either serotype 1 or 14.     

The presence of muramidase released protein and extracellular factor protein in various serotypes of Streptococcus suis isolated from diseased and healthy pigs in Spain

A total of 142 strains from different serotypes of Streptococcus suis isolated in Spain from diseased pigs (88 strains) and healthy carrier pigs (54 strains) were studied for the presence of a muramidase released protein (MRP) and an extracellular factor (EF). The following five phenotypes: MRP+EF+, MRP+EF-, MRP-EF+, MRP+EF* and MRP*EF- were detected. A high percentage of S. suis serotype 2 strains isolated from diseased pigs (84 per cent) belonged to phenotype MRP+EF+, but this phenotype has also been noticed in other serotypes (serotypes 1, 1/2 and 14). Both proteins were detected in S. suis serotype 2 strains (26%) isolated from healthy carrier pigs and one of both proteins in serotypes 1 and 14 (phenotype MRP+EF*). The isolation of S. suis strains from healthy pigs which have shown both proteins may support the epidemiological significance of these carriers in the maintenance, transmission and distribution of virulent strains within and between swine farms.     

Prevalence, capsular type and antimicrobial susceptibility of Streptococcus suis isolated from slaughter pigs in Korea.

This study was undertaken to determine the prevalence, capsular serotype, and antimicrobial susceptibility of Streptococcus suis isolated from slaughter pigs. Capsular serotype and antimicrobial susceptibility were determined by coagglutination test and agar dilution minimum inhibitory concentration, respectively. Streptococcus suis was isolated from 55 of the 406 palatine tonsillar samples tested (13.8%) and 14 of the 29 sampled herds (48.3%). Of the 55 isolates recovered from slaughter pigs, 26 (47.3%) were untypeable. Of the remaining 29 isolates, capsular serotypes 9 (9 isolates) and 16 (4 isolates) were the most common, followed by capsular serotypes 4 (3 isolates) and 7 (3 isolates). Every capsulated isolate was typeable and no palatine tonsillar sample yielded more than one serotype. Most of isolates were susceptible to low concentrations (MIC90) of amoxicillin (2 microg/mL), ceftiofur (1 microg/mL), and penicillin (1 microg/mL). No correlation was found between antimicrobial susceptibility and capsular serotype.     

猪链球菌扁桃体分离株的毒力因子分布特征与致病性

本研究设计并合成7对引物,用PCR方法对猪链球菌7种主要毒力因子,包括谷氨酸脱氢酶(gdh)、溶血素(sly)、胞外蛋白因子(ef)、溶茵酶释放蛋白(mrp)、纤连蛋白/血纤蛋白原结合蛋白(fpbs)、三磷酸甘油醛脱氢酶(gadph)和毒力相关序列orf2进行检测,分析了29株猪链球菌扁桃体分离株的毒力因子分布特征.在被检的25株2型菌株中,共检测出7个基因型.其中10株(40%)的基因型为cps2/gdh+/sly+/ef+/mrp+/fbps+/gadph+/orf2+,7株(28%)表现为cps2/gdh+/sly-/ef-/mrp+/fbps+/gadph+/orf2+,4株(16%)表现为eps2/gdh+/sly-/ef+/mrp+/fbps+/gadph+/orl2+,基因型表现为eps2/gdh+/sly+/ef+/mrp+/fbps+/gadph+/orf2-、cps2/gdh+/sly-/ef-/mrp+/fbps+/gadph+/orf2-、eps2/gdh+/sly-/ef-/mrp*/fbps+/gadph+/orf2+、eps2/gdh+/sly-/ef-/mrp-/fbps-/gadph+/orf2-各1株;1株猪链球菌7型(SS7)分离株,基因型表现为cps7/gdh+/sly+/ef-/mrp-/fbps+/gadph+/orf2+;3株猪链球茵9型(SS9)扁桃体分离株,均表现为cps9/gdh+/sly-/ef-/mrp-/fbps+/gadph+/orf2+.可见我国SS分离株的毒力基因分布较为复杂,而且SS2的优势流行菌株是同时具有多种毒力因子的高致病茵株.通过对不同毒力基因型毒株对西藏小型猪的感染试验发现:毒力基因型为cps2/gdh+/sly-/ef+/mrp+/fbps+/gadph+/orf2+的健康扁桃体分离株SH06-21D对西藏小型猪有较强的致病性,仅次于标准株HA9801,而基因型为sly-/ef-/mrp-/fbps-/gadph+/orf2-的健康扁桃体分离株GZ06-122B对西藏小型猪不表现明显的致病性.     

Genetic diversity of Streptococcus suis serotypes 2 and 1/2 isolates recovered from carrier pigs in closed herds

The aim of this study was to compare, by randomly amplified polymorphic DNA (RAPD), the diversity of Streptococcus suis serotypes 1/2 and 2 isolates recovered at slaughter houses from the tonsils of clinically healthy pigs. The pigs belonged to herds with or without clinical signs of S. suis disease.

Overall, a low diversity was observed among isolates of serotype 1/2. A representative isolate recovered from a diseased animal presented a relatively high similarity (85%), with most isolates recovered from carrier pigs, from herds either with or without clinical signs of S. suis disease. For serotype 2 isolates, a relatively high degree of heterogeneity was observed in the whole population. Two subpopulations were observed for serotype 2 isolates, which arose from herds with clinical signs. Interestingly, the representative isolate coming from the diseased pig was included in a small closed cluster, with 2 isolates recovered from carrier pigs belonging to the same herd. On the other hand, most of the S. suis serotype 2 isolates originating from herds with no history of S. suis disease, were closely related (90% similarity). Furthermore, they presented different RAPD patterns from those originating from animals from the herd presenting S. suis clinical signs due to this serotype. Results suggest that, in the herds studied, clinical manifestations due to serotype 2 are probably related to the virulence of a specific isolate. Conversely, for the herd affected with serotype 1/2, clinical manifestations of the disease were more likely to be the result of inherent herd factors than the virulence of the specific isolate.

     

Prevalence of the suilysin gene in Streptococcus suis strains isolated from diseased and healthy carrier pigs

Knowledge of virulence factors of Streptococcus suis is limited. Several virulence factor candidates have been proposed, among them suilysin, which is responsible for a toxic effect on epithelial cells. The aim of this study was to detect the suilysin gene sequence in Streptococcus suis strains of various origin. In total 63 Streptococcus suis isolates were investigated. Forty four of them originated from tissues of streptococcosis affected animals. The remaining 19 strains were isolated from tonsils of healthy carrier pigs. Suilysin gene specific sequence was detected in 79% of the strains tested. In isolates obtained from pigs with signs of streptococcosis this gene sequence was recorded in 85% of cases. In Streptococcus suis strains isolated from healthy carrier pigs the suilysin gene was detected in 63% of the isolates. It seems that suilysin toxic activity is only one of the many steps involved in the pathogenesis of Streptococcus suis infection and that strain's virulence cannot be stated only on the basis of suilysin gene sequence presence.     

牦牛肠道与粪便乳酸菌的分离鉴定及PCR-16SrDNA鉴定

以取自四川省不同地区的牦牛粪便、肠道内空物为材料，用MRS琼脂双层培养基进行厌氧培养，分离到50株乳酸菌，经生化鉴定为嗜热链球菌（2株）、乳酸乳球菌（1株）、保加利亚乳杆菌（5株）、嗜粪乳杆菌（10株）、嗜酸乳杆菌（8株）、乳酸乳杆菌（9株）、肠乳杆菌（10株）、弯曲乳杆菌（5株）。采用乳酸菌16 SrDNA通用引物，对分离的8种菌的16S rDNA-段可变区序列进行扩增，均得到大小约470bp的产物；扩增产物经纯化、测序后与GenBank中标准菌株的核甘酸序列比较，同源性均大于97．5％，同源性分析与生化试验的结果是一致的。证实，牦牛肠道和粪便的乳酸菌较为丰富，且乳杆菌的数量较多，这可能与牦牛复杂的生长环境有关。     

全自动微生物分析系统和16S rDNA序列分析技术对奶牛乳腺炎病原菌的鉴定

从四川成都、绵阳和眉山地区部分奶牛场共分离奶牛乳腺炎病原菌120株,采用VITEK全自动微生物分析系统和16S rDNA序列分析技术对分离菌株进行鉴定。研究发现引起四川成都、绵阳和眉山奶牛乳腺炎以细菌混合感染为主,主要致病菌有葡萄球菌、大肠杆菌、单胞菌属、鲁氏不动杆菌、吉氏库特氏菌和芽孢杆菌属。其中,葡萄球菌的感染最为普遍,检出率最高(33株,27.50%);其次为大肠杆菌(17株,14.17%)、单胞菌属(12株,10%)、鲁氏不动杆菌(11株,9.17%)、吉氏库特氏菌(9株,7.50%)和芽孢杆菌属(9株,7.50%);再次为巨型球菌(6株,5%)、阴沟肠杆菌(6株,5.00%)、产吲哚金黄杆菌(6株,5.00%)和链球菌属(4株,3.33%);最低的是棒杆菌属(3株,2.50%)、粪肠球菌(2株,1.67%)、弗氏志贺菌(1株,0.83%)和普通变形杆菌(1株,0.83%)。本研究为防治奶牛乳腺炎奠定了基础。     

湖南猪源粪肠球菌的分离鉴定及16S rDNA系统进化分析

从湖南各地送检至实验室的临床样本中分离到42株革兰氏染色阳性及过氧化氢酶阴性的球菌,其中78.6%来自于肺脏,菌落形态及染色镜检均与粪肠球菌参考株ATCC 29212相似,兰氏分群鉴定97.6%（41/42）为D群,生化特性符合粪肠球菌特征。16SrDNA测序鉴定显示：42个分离株与同步测序的ATCC 29212同源性在99.2%到100%之间,与所选择粪肠球菌参考序列的同源性在98.7%到100%之间,NCBI在线BLAST分析发现42株均与GeneBank收录的粪肠球菌序列同源性最高。湖南分离株16SrDNA序列与E.faecalis参考序列进化关系与地域分布无关,来源于不同宿主的菌株的16SrDNA变异并不大,而与E.faecium、E.canis、E.avium、E.hirae等4种肠球菌则有多处变异,这些变异区域在这4种肠球菌中却是保守的。     

血清2型猪链球菌河南分离株毒力基因的鉴定及其cps2j全基因序列分析

为了解血清2型猪链球菌(S.suis2)河南分离株的毒力基因型及cps2j全基因突变情况,本研究扩增S.suis2毒力基因gdh、cps2j、mrp、ef 、sly、orf2和fbps,并对s.suis2的cps2j进行全基因序列测定和同源性分析.结果表明,gdh、cps2j、mrp、ef、sly、orf2和fbps基因在8个S.suis2分离株中的检出率分别为100％(8/8)、100％(8/8)、62.5％(5/8)、75％(6/8)、87.5％(7/8)、100％(8/8)和100％(8/8);其中cps2j全基因核苷酸及推导的氨基酸序列与来源不同的S.suis2分离株同源性分别达98％和97％以上,以该基因构建的系统发育树表明8株S.suis2河南分离株与国内外不同参考株同源性高,亲缘关系密切.     

Phenotypic characterisation and 16S rRNA sequence analysis of veterinary isolates of Streptococcus pluranimalium

Forty-two isolates of Streptococcus pluranimalium were identified from cattle (n=38), sheep (n=2), an alpaca (n=1) and a pheasant (n=1) in the United Kingdom. The isolates were confirmed as S. pluranimalium by 16S rRNA sequence analysis but could not be differentiated reliably from Streptococcus acidominimus by phenotypic characterisation using commercial kits routinely used in veterinary laboratories. The alanyl-phenylalanyl-proline arylamidase reaction could be used to differentiate S. pluranimalium (positive) from Aerococcus urinae (negative).     

猪链球菌2型安徽分离株多位点序列分型与毒力特征研究

为了解安徽地区猪链球菌2型(SS2)临床分离株毒力基因分布、分子分型特征及其与致病性的相关性,本研究收集了58株源自临床患病猪的SS2,应用PCR技术检测其毒力基因:胞外蛋白因子基因(epf)、溶菌酶释放蛋白基因(mrp)和溶血素基因(sly),应用多位点序列分型方法(MLST)对其进行基因分型,并分析其与动物致病性之间的相关性。结果显示,58株分离菌,epf^+/mrp^+/sly^+型为44.83%(26/58),epf^+/mrp^-/sly^+型为25.86%(15/58),epf^-/mrp^+/sly^+型为18.97%(11/58),epf^-/mrp^-/sly^+和epf^-/mrp^-/sly^-型均为5.17%(3/58)。共分出10种ST型,其中ST1 26株(44.83%),ST7 18株(31.03%),ST28 6株(10.34%),ST117、ST156和ST308均为1株,各占1.72%,另有4种新发现的ST型,即ST958 2株(3.45%),ST957、ST959、ST970各1株(1.72%);分为4种克隆复合物(ST1 complex、ST27 complex、ST87 complex、ST188 complex)。ST1以epf^+/mrp^+/sly^+型为主(69.23%),其中23株(88.46%)分离自全身感染病猪;ST7以epf^+/mrp^-/sly^+型为主(55.56%),其次为epf^+/mrp^+/sly^+型(38.89%),其中13株(72.22%)分离自全身感染病猪。通过对斑马鱼、昆明鼠感染试验和LD50的测定,筛选出的6株强毒菌株分为ST1型(2株)和ST7型(4株)。结果表明,epf^+/mrp^+/sly^+是安徽地区SS2的优势毒力基因型,其次为epf^+/mrp^-/sly^+型。获得10种ST型,其中4种为首次发现,并且ST1和ST7为优势型。具有ST1/epf^+/mrp^+/sly^+遗传特征的菌株在临床上均倾向于导致全身感染。本研究结果揭示了安徽地区SS2毒力基因型、MLST遗传特征与毒力、动物临床症状之间的相关性,为开展SS2流行病学研究,区分不同菌株间毒力差异的研究提供参考依据。     

7种鸭源细菌的分离与16S rDNA测序鉴定

从病死鸭的脏器中无菌分离细菌.根据培养特性、菌落形态、革兰染色和生化特性,鉴定出7种细菌分离株.以分离株的基因组DNA为模板,用16S rDNA试剂盒扩增其DNA片段,测序后NCBI网站进行BLAST搜索比对,鉴定出细菌种类,分别为鼠伤寒沙门菌、大肠杆菌、鸭疫里默氏杆菌、铜绿假单胞杆菌、施氏假单胞菌、浅绿气球菌和麦氏棒杆菌.前3种为鸭的常见分离菌,其余的较为少见.因此在鸭病控制过程中应注意可能存在一些不常见细菌的混合感染.     

Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.     

3种兔球虫18S rDNA部分序列测定与系统发育分析

采用单卵囊分离法从河北某兔场分离大型艾美耳球虫、黄艾美耳球虫及肠艾美耳球虫,接种无球虫兔后获得大量纯种卵囊,CTAB法提取孢子化卵囊基因组DNA。利用艾美耳属球虫18S rDNA保守引物,PCR扩增3种兔球虫18S rDNA片段,产物纯化后测序。将3种球虫18S rDNA测序结果与GenBank中发布的兔球虫18S rDNA序列用DNAStar软件进行比对。使用MEGA4.0软件对兔球虫18S rDNA进行同源性比较,并绘制遗传进化树。结果表明,大型艾美耳球虫扩增出大小为1 521bp的18S rDNA片段;黄艾美耳球虫及肠艾美耳球虫均扩增出大小为1 520bp的18S rDNA片段。序列比对结果显示,3种河北株兔球虫与GenBank中相应的3种兔球虫18S rD-NA(EF694016、EF694011、EF694012)相似性分别为99.6%、99.6%和100%。3种河北株兔球虫序列和GenBank中兔球虫18S rDNA序列(EF694007-EF694017)位于一个单系集群。