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1.
A. A. HASSAN
. AKINEDEN C. LMMLER R. HUBER‐SCHLENSTEDT 《Zoonoses and public health》2002,49(5):257-259
In the present study three phenotypically CAMP‐negative Streptococcus agalactiae, isolated from three cows with mastitis, were characterized by molecular analysis. An identification of the S. agalactiae was performed by conventional methods and by PCR amplification of species specific parts of the 16S rRNA gene and the 16S‐23S rDNA intergenic spacer region. In addition all three phenotypically CAMP‐negative isolates harboured a normal sized CAMP‐factor encoding cfb gene indicating a reduced expression of CAMP‐factor or a gene defect elsewhere along the pathway of expression. The clonal identity of the three isolates could be demonstrated by macrorestriction analysis of their chromosomal DNA. 相似文献
2.
Intraspecific variation in the 16S rRNA genes of 17 Mycoplasma agalactiae and eight Mycoplasma bovis isolates was investigated to determine the degree of sequence variation in these two species and to determine whether the polymorphisms in the 16S rRNA genes could be used for the construction of an evolutionary tree and as epidemiological markers. A high degree of variation was found within isolates (between operons) and between isolates of both species. In contrast to M. capripneumoniae no distinct evolutionary pattern could be seen, probably because there are functional systems for gene conversion in M. agalactiae and M. bovis. However, the non-European isolates of M. agalactiae shared three characteristic nucleotides and European isolates from the same or neighbouring countries were very similar. Differences within isolates included both polymorphic positions and sequence length differences between operons. The amount of variation within isolates of the respective species ranged from zero to seven polymorphisms for M. agalactiae and from zero to four polymorphisms for M. bovis. The high degree of variation suggests the potential for misdiagnosis of species in diagnostic PCR assays based on the 16S rRNA gene sequences. All isolates of both species had a thymidine in position 912 (E. coli numbering) that causes streptomycin resistance in several bacterial species and which is characteristic for the members of the hominis group. As expected, when five M. agalactiae and three M. bovis isolates were tested for streptomycin susceptibility, they all demonstrated streptomycin resistance. M. agalactiae and M. bovis were found to have high intraspecific variation in their 16S rRNA gene and the polymorphisms patterns indicate that gene conversion takes place. 相似文献
3.
Estuningsih S Soedarmanto I Fink K Lämmler C Wibawan IW 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2002,49(4):185-187
All 83 bacterial strains isolated from seven farms in three areas of the island of Java in Indonesia investigated in the present study could be identified as Streptococcus agalactiae. Identification was performed by cultural, biochemical and serological properties and by polymerase chain reaction amplification of species-specific parts of the gene encoding the 16S rRNA, the 16S-23S rDNA intergenic spacer region and the CAMP factor (cfb) gene. All isolates were unpigmented. almost all of the isolates had the serotype pattern II/X. Despite these similarities a macrorestriction analysis of the chromosomal DNA of the bacteria revealed no significant homologies of the DNA-fingerprints of the S. agalactiae from the various areas. This last finding might possibly indicate that a single ancestral unpigmented serotype II/X S. agalactiae clone was responsible for the mastitis situation on Java and had evolved separately in the various farms and regions. 相似文献
4.
Seven group B streptococcal cultures isolated from three horses reacted with group B-specific antiserum, were CAMP positive, pigmented and showed the typical biochemical properties of Streptococcus agalactiae. The identification could be confirmed by PCR amplification of the 16S rRNA gene and a subsequent RsaI restriction pattern typical for S. agalactiae. In addition, the isolates were identified by amplification of species specific parts of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer region and by amplification of the CAMP-factor (cfb) gene. Six isolates could be classified as serotype III/Rib, one isolate as serotype Ia/cbeta. The occurrence of the protein antigens Rib and cbeta could be confirmed by PCR amplification of the respective genes. The six isolates of serotype III/Rib were hyaluronidase negative, had a hylB gene with a size of 4.6 kb and an insertion element IS1548 of 0.98 kb. The isolate of serotype Ia/cbeta was hyaluronidase positive, had a hylB gene with a size of 3.3 kb and no insertion element IS1548. In addition, all seven isolates had the insertion element ISSag2 and the gene lmb encoding the laminin binding surface protein Lmb and the gene scpB encoding C5a peptidase. According to the present results the group B streptococci isolated from horses showed characteristics of human isolates of this species. 相似文献
5.
Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens. 相似文献
6.
Specific detection by PCR of Streptococcus agalactiae in milk. 总被引：3，自引：0，他引：3
The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk. 相似文献
7.
试验旨在研究无乳链球菌的生物学特性,为防治无乳链球菌引起的奶牛乳房炎提供理论依据。根据细菌分子生物学分离鉴定无乳链球菌,参考GenBank登录的牛源无乳链球菌16S rRNA、菌属特异性cfb （CAMP因子）、毒力基因和耐药基因序列,运用Oligo 6.0和Primer Premier 5.0软件设计14对引物,建立PCR快速检测方法,并进行20种常见抗生药物的耐药试验。结果显示,试验成功鉴定出17株牛源无乳链球菌,毒力基因与NCBI上已报道的无乳链球菌相应序列具有高度同源性,均≥99%;共检测到6种耐药基因;分离菌株对青霉素、红霉素、林可霉素、克林霉素、万古霉素、氨苄西林、新生霉素、磺胺异噁唑的耐药率均较高,耐药率依次为100.0%、94.1%、94.1%、94.1%、94.1%、82.3%、82.3%和47.1%,对青霉素严重耐药;而对氨基糖苷类、四环素类、头孢菌素类、喹诺酮类耐药率均较低,耐药率分别为15.7%、14.7%、7.7%和3.9%。本研究结果表明,建立的PCR快速检测方法灵敏可靠,云南地区无乳链球菌已对部分β-内酰胺类、大环内酯类、磺胺类等抗生素出现多重耐药性。 相似文献
8.
Fitzmaurice J Sewell M King CM McDougall S McDonald WL O'Keefe JS 《New Zealand veterinary journal》2008,56(5):233-236
AIM: To develop a real-time PCR for the detection of Mycoplasma agalactiae, using PCR primers targeting the ma-mp81 gene. METHODS: A group of 15 M. agalactiae isolates, 21 other Mycoplasma spp. isolates and 21 other bacterial isolates was used in evaluation of the assay. RESULTS: All M. agalactiae isolates were detected by the assay and none of the non-target isolates was amplified. The analytical detection limit of the assay was 10 fg of purified genomic DNA and 104 cfu/ml milk inoculated with M. agalactiae. When applied to goat-milk samples collected from three herds free of M. agalactiae infection, the assay had a specificity of 100%. CONCLUSIONS: The assay would be useful in a diagnostic laboratory, providing specific, sensitive and rapid detection of M. agalactiae. 相似文献
9.
Hugh Cai Marie Archambault John F Prescott 《Journal of veterinary diagnostic investigation》2003,15(5):465-469
This study evaluated 16S rRNA gene sequence analysis methods as tools for identification of 22 phenotypically difficult to identify veterinary clinical bacterial isolates in a veterinary diagnostic laboratory. The study compared 16S rRNA gene sequencing and conventional phenotypic identification methods. Using 16S rRNA full-gene sequencing, 95% (21/22) of the isolates were identified to the genus level and 86% (19/22) to the species level. The conventional or commercially available manual identification phenotypic characterization methods presumptively identified 91% (20/22) of the isolates to the genus level and 1 isolate to the species level. However, only 55% (12/22) or 4.5% (1/22) of the phenotypic identifications were correct at the genus or species level when they were compared with the 16S rRNA full-gene sequencing. This study also compared 16S rRNA full-gene and partial-gene sequencing. The results demonstrated that the best 16S rRNA gene-sequencing approach is full-gene sequencing because it gives the most precise species identification. Sequencing of the variable regions 1, 2, and 3 of the 16S rRNA gene could be used for tentative identification because the ability of this sequencing to identify bacteria to the genus level is similar to that of the 16S rRNA full-gene sequencing. This method identified only 14% (3/22) isolates differently to the species level compared with the 16S rRNA full gene sequence. Sequencing of the variable regions 7, 8, and 9 is not recommended because it gives more ambiguous identifications. The cost of a 16S RNA full-gene-sequencing analysis was Can 160 dollars and Can 60 dollars for a partial 16S rRNA gene sequence, i.e., sequencing of variable regions 1, 2, and 3 or variable regions 7, 8 and 9. 相似文献
10.
The present study was performed to investigate streptococci of serological group B obtained from various sources and group B streptococcal reference strains for serotype, hyaluronate lyase enzyme activity, the occurrence of the hylB gene and the insertion sequence IS1548. All group B streptococci were identified by cultural, biochemical, and serological properties and by polymerase chain reaction amplification of species-specific parts of the 16S-23S rDNA intergenic spacer region, the 16S rRNA gene and the CAMP-factor (cfb) gene. Of the 73 group B streptococci investigated, 59 strains displayed hyaluronate lyase enzyme activity. All hyaluronate-lyase-positive strains and three phenotypically hyaluronate-lyase-negative strains had a hylB gene with an amplicon size of 3.3kb. Eleven of the 14 phenotypically hyaluronate-lyase-negative strains generated a hylB gene PCR product with a size of 4.6kb, and 10 of these strains displayed a IS1548 amplicon with a size of 0.98kb. The hyaluronate-lyase-negative isolates were mainly observed among group B streptococci of serotype III/Rib. All strains harbouring IS1548 had an additional copy of IS1548 located downstream of the C5a peptidase (scpB) gene. 相似文献
11.
分析河南、陕西分离的14株鸡杆菌(Gallibacterium)之间的进化关系,及gyrB基因序列比较在该菌进化分析中的作用。PCR扩增鸡杆菌分离株的gyrB、16SrRNA和rpoB3个看家基因,PCR产物纯化后直接测序。将鸡杆菌分离株、国外参考株的3个看家基因序列进行比较分析,用Phylip 3.67软件构建进化树。结果表明,14株鸡杆菌与鸭源鸡杆菌(Gallibacterium anatis)模式株间的相似性为96.3%～98.0%(gyrB)、97.7%～99.6%(16SrRNA)和97.7%～99.0%(rpoB);14株鸡杆菌与鸡杆菌复合群1(Gallibacterium genomosp.1)参考株间的相似性为88.8%～89.9%(gyrB)、96.2%～97.5%(16SrRNA)和92.6%～93.6%(rpoB)。基于3个看家基因序列的进化分析,均显示14株鸡杆菌和鸭源鸡杆菌模式株形成单独的一个群。14株鸡杆菌分离株均属于鸭源鸡杆菌种;在3个看家基因位点,鸡杆菌河南株与陕西株之间、鸡杆菌输卵管炎病鸡分离株与健康鸡分离株之间均无明显遗传上的差异;gyrB基因序列分析可用于鸡杆菌分离株的种类鉴定,且对14株鸡杆菌与复合群1参考株的区别能力优于另外2个看家基因。 相似文献
12.
Daignault D Guévremont E Guillemette JM Messier S Gottschalk M Higgins R 《The Canadian veterinary journal. La revue veterinaire canadienne》2003,44(3):217-220
Streptococcus agalactiae remains an important pathogen of dairy herds in Québec, but data about antigenic characteristics of this microorganism are sparse. This study was conducted to determine the variety of S. agalactiae serotypes in dairy herds in Québec. Two hundred and ninety-five isolates cultured from the milk of individual cows from 7 regions of Québec were serotyped. Sixty-two percent of the isolates were untypable. Among the 38% of typeable isolates, serotype III was found most frequently. In conclusion, the heterogeneity found among antigenic determinants of isolates from bovine milk suggests that an immunological method for the detection of S. agalactiae performed directly on bovine milk would not be a practical approach. 相似文献
13.
Traditional microbiological methods for identification of Streptococcus spp. causing bovine mastitis have been demonstrated to be less than highly reliable. PCR-RFLP analysis of 16S-23S ribosomal DNA was used to characterise seven reference strains of streptococcal mastitis pathogens as well as four reference strains of other gram-positive, catalase-negative cocci of bovine origin to allow comparative identification of field isolates. RFLP analysis of PCR products, using a combination of two restriction endonucleases in single reactions (HaeIII and AluI, HaeIII and RsaI or AluI and RsaI) generated unique patterns for species of Streptococcus, Enterococcus and Lactococcus. One hundred field isolates of Streptococcus spp. collected from cows with clinical or subclinical mastitis were tested. Fifty-seven isolates, classified by conventional tests as S. uberis, were identified as 47 S. uberis and six S. parauberis by their unique RFLP patterns. The remaining four isolates had RFLP patterns distinct from the reference strains and two of these were identified as closely related to S. iniae and two to Aerococcus viridans by 16S rRNA sequencing. Conventional identification of 17 S. agalactiae and 26 S. dysgalactiae subsp. dysgalactiae was confirmed by RFLP. Use of a combination of restriction enzymes in a single tube enabled the rapid, accurate, cost effective and easily performed identification of all major streptococcal mastitis pathogens. 相似文献
14.
应用生物信息学手段和查阅文献资料设计了金黄色葡萄球菌、大肠埃希菌、无乳链球菌、绿脓杆菌、停乳链球菌、乳房链球菌6种奶牛乳房炎主要致病菌的通用引物和金黄色葡萄球菌、大肠埃希菌、绿脓杆菌的寡核苷酸探针及无乳链球菌、停乳链球菌、乳房链球菌的特异引物,并用这3种特异引物扩增片段的纯化产物作为这3种链球菌的检测探针。在引物对样品中细菌的相应基因片段扩增的同时进行靶基因的生物素标记,扩增的产物与硝酸纤维素膜上的探针进行杂交,酶联、显色后根据芯片扫描仪的判读结果来确定奶牛乳房炎致病菌感染的种类。结果表明,建立的以16S rDNA为对象的基因芯片技术可以快速的检测出以上6种细菌,整个检测过程需要6h~7h,灵敏度高,特异性好,能快速的对奶牛乳房炎的主要致病菌做出诊断。 相似文献
15.
多重PCR快速检测奶牛乳房炎3种主要病原体 总被引：10，自引：0，他引：10
奶牛乳房炎是引起奶牛业经济损失的一种重要疫病,目前还没有快速、特异检测奶牛乳房炎主要致病原的方法。本试验根据金黄色葡萄球菌、无乳链球菌、大肠杆菌各自保守的16S或23S rRNA基因序列,合成了3对特异性引物,建立了三重PCR检测方法。特异性试验表明,该方法对所有参与测试的金黄色葡萄球菌、无乳链球菌和大肠杆菌都能扩增出各自的阳性条带,而对所有参与测试的对照菌株则不能扩增出任何条带。敏感性试验表明该方法能检测到4个菌的金黄色葡萄球菌、无乳链球菌和2个菌的大肠杆菌。对送检的乳房炎奶样36份直接进行PCR检测,金黄色葡萄球菌阳性7份,无乳链球菌阳性2份,大肠杆菌阳性6份。 相似文献
16.
Ⅶ型分泌系统(T7SS)是近年来发现的分泌系统,分泌两种胞外蛋白,EsxA基因编码的ESAT6蛋白和EsxB基因编码的CFP-10蛋白。分泌蛋白具有良好的免疫原性,促进细菌从巨噬细胞吞噬体逃逸、影响巨噬细胞凋亡及裂解细胞等生物学功能,与致病性密切相关。以罗非鱼源无乳链球菌为模板,克隆到294bp的EsxA基因,将目的序列克隆到pMD18-T载体,经测序,目的序列与无乳链球菌EsxA基因同源率在99%以上。EsxA基因克隆到pET32a载体中,重组载体在28℃、0.5mmol/L IPTG诱导条件下表达量最大,可溶性表达。将纯化的ESAT6蛋白免疫Balb/c鼠,成功制备ESAT6蛋白鼠多克隆抗体,经Western blot,ESAT6蛋白具有良好的反应原性。研究结果为进一步进行无乳链球菌ESAT6蛋白的免疫学功能研究奠定了基础。 相似文献
17.
Joo Youn Park Lawrence K. Fox Keun Seok Seo Mark A. McGuire Yong Ho Park Fred R. Rurangirwa William M. Sischo Gregory A. Bohach 《Veterinary microbiology》2011,147(1-2):142-148
Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens from cows with intramammary infection (IMI). Although API STAPH ID 20, a commercially available identification system, and PCR-restriction fragment length polymorphism (PCR-RFLP) of the gap gene (gap PCR-RFLP) have been successfully applied for the identification of CNS isolates from human specimens, their accuracy in the identification of veterinary isolates has not been fully established. In this study, we identified 263 CNS isolates from bovine IMI at species level by partial 16S rRNA gene sequence analysis as the definitive test. Species identification obtained using partial 16S rRNA gene sequence analysis was compared to results from the API STAPH ID 20 and gap PCR-RFLP analysis. Eleven different CNS species were identified by partial 16S rRNA gene sequence analysis. Only 76.0% (200/263) of the species identification results obtained by API STAPH ID 20 matched those obtained by partial 16S rRNA gene sequence analysis, whereas 97.0% (255/263) of the species identification results obtained by the gap PCR-RFLP analysis matched those obtained by partial 16S rRNA gene sequence analysis. The gap PCR-RFLP analysis could be a useful and reliable alternative method for the species identification of CNS isolates from bovine IMI and appears to be a more accurate method of species identification than the API STAPH ID 20 system. 相似文献
18.
19.
The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects. 相似文献
20.
Ingestion and killing of Streptococcus agalactiae by bovine granulocytes in the presence of natural opsonins 总被引：3，自引：0，他引：3
An enzyme-linked immunosorbent assay (ELISA) demonstrated the presence of naturally acquired antibodies against Streptococcus agalactiae in normal bovine serum (NBS). In milk wheys, ELISA values were much lower than in sera. Pre-colostral calf serum (PCS) was shown to lack antibodies to type II and III S. agalactiae. The opsonic requirements of 10 human and 10 bovine strains were investigated by evaluating the phagocytosis-induced reduction of the incorporation of radiolabeled thymidine by streptococci. Antibodies present in NBS were required for the efficient ingestion of both human and bovine isolates type II by bovine granulocytes. Three out of five type III bovine isolates were opsonized in the absence of specific antibodies (opsonization by PCS) and type II and III bovine isolates did not require complement opsonization. By contrast, inactivation of complement reduced phagocytosis of human isolates and only one type III strain of human origin was opsonized by PCS. These findings suggest that human isolates had higher opsonic requirements. The phagocytic killing of 6 type III strains (5 mastitis isolates and the reference typing strain) was investigated. Opsonization by normal serum enabled bovine blood granulocytes to ingest and kill S. agalactiae. Nevertheless, greater than or equal to 35% of bacteria remained viable at the end of the phagocytosis incubation in 10% NBS. Heat treatment of serum decreased the efficacy of killing for only 3 of the 6 tested strains. An IgG2 fraction of normal adult bovine serum promoted active ingestion, which was still increased in the presence of PCS. Normal wheys displayed large variations in their ability to promote ingestion of S. agalactiae by blood granulocytes. The promoting effect was systematically less than that of serum from the same cow, and this can be related to the lower ELISA values found in wheys. 相似文献