Examination of the 16S-23S rRNA intergenic spacer sequences of 'Candidatus Mycoplasma haemobos' and Mycoplasma haemofelis

The intergenic spacer region between the 16S and 23S rRNA genes of mycoplasmas has been used for a genetic marker for identification of the species. Here we show the intergenic spacer regions of two hemotropic mycoplasmas, Mycoplasma haemofelis and 'Candidatus Mycoplasma haemobos (synonym: 'C. M. haemobovis')' are also useful for classification of this particular group of mycoplasms. The spacer region of M. haemofelis and `C. M. haemobos' consisted of 209 and 210 base pairs, respectively, and both lacked the spacer tRNA genes. Phylogenetic analysis suggested a monophyletic relationship among hemoplasmas and M. fastidiosum. A hypothetical secondary structure predicted in the spacer regions tentatively assigned the boxA and boxB motifs peculiar to the members of the genus Mycoplasma. M. haemofelis and 'C. M. haemobos' possessed a stem-loop structure in common, despite the presence of a palindromic nucleotide substitution in the stem region.     

Molecular identification of Arcanobacterium bialowiezense and Arcanobacterium bonasi based on 16S-23S rRNA intergenic spacer region sequences

In the present study, the 16S-23S rDNA intergenic spacer region (ISR) of Arcanobacterium (A.) bialowiezense DSM 17162, A. bonasi DSM 17163, A. bernardiae DSM 9152, A. haemolyticum DSM 20595, A. hippocoleae DSM 15539, A. phocae DSM 10002, A. pluranimalium DSM 13483 and A. pyogenes DSM 20630 was amplified, sequenced and compared with the corresponding 16S rRNA gene sequences yielding comparable phylogenetic relationships. The ISR sequence of A. bialowiezense and A. bonasi allowed the design of species-specific oligonucleotide primers which could successfully be used for PCR-mediated identification of previously characterized A. bialowiezense and A. bonasi isolated from infections of the European bison. The presented molecular identification might help to improve a future diagnosis of both newly described bacterial pathogens.     

Characterization of the 16S-23S rRNA intergenic spacer regions among strains of the Fusobacterium necrophorum cluster

The 16S-23S rRNA intergenic spacer regions (ISRs) of Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme were characterized. Products of two sizes, about 360 bp (small) and 530 bp (large), were generated by PCR amplification from the 16S-23S rRNA ISR of all the strains tested. The large and small 16S-23S rRNA ISRs of F. necrophorum exhibited a level of sequence similarity of 93.9% to 99.7% and 94.2% to 98.6% homologies within the species, respectively. Only the large spacer regions in these bacteria contained one or two tRNA genes. F. necrophorum subsp. necrophorum contains the isoleucine and alanine tRNA gene, whereas F. necrophorum subsp. funduliforme contains the isoleucine tRNA gene.     

Polymerase chain reaction with a primer pair in the 16S-23S rRNA spacer region for detection of Mycoplasma pulmonis in clinical isolates

To develop a diagnostic tool to identify Mycoplasma pulmonis (M. pulmonis) in clinical isolates, we developed a polymerase chain reaction (PCR) assay using primers specific for the 16S-23S rRNA intergenic spacer region (SR) of M. pulmonis. One pair of PCR primers reacted specifically with two reference strains of M. pulmonis tested and seven samples isolated from naturally infected rats. The primer pair did not produce PCR products of the correct size from any other rodent or human mycoplasmas or cellular DNA from rodent lungs. Specificity of the PCR assay was confirmed by Southern blotting with probe specific for the SR of M. pulmonis. The PCR assay for detection of M. pulmonis established in this study is suitable for diagnosis of M. pulmonis infection in clinical cases.     

Comparison of the 16S-23S rRNA intergenic spacer regions between Fusobacterium varium and "Fusobacterium pseudonecrophorum" strains

The 16S-23S rRNA intergenic spacer regions (ISRs) of five strains of "Fusobacterium pseudonecrophorum" which had been proposed as a new species, were compared with those of F. varium ATCC 8501T. All the strains of "F. pseudonecrophorum" exhibited of sequence similarities of 97.7% to 100% to the strain of F. varium in their 16S-23S rRNA ISR sequences. This indicates that the strains of "F. pseudonecrophorum" and the type strain of F. varium are identical at the species level.     

Characterization of Arcanobacterium abortisuis by phenotypic properties and by sequencing the 16S-23S rDNA intergenic spacer region

The present study was designed to characterize phenotypically and genotypically nine Arcanobacterium abortisuis strains collected from specimen of pigs in a period of nine years. All nine A. abortisuis strains and A. abortisuis reference strain DSM 19515 displayed a synergistic hemolytic reaction with Staphylococcus aureus β-hemolysin, Rhodococcus equi, and Arcanobacterium haemolyticum indicator strains and showed the typical biochemical properties of this species. The species identity could be confirmed by identification and sequencing of the 16S-23S rDNA intergenic spacer region (ISR), which appeared to be a useful target for genotypic characterization of this bacterial species. The A. abortisuis strains of the present study were isolated from specimen of pigs together with various other bacterial species indicating that the pathogenic importance of this newly described species remains to be elucidated.     

Development and evaluation of a diagnostic PCR for Mycoplasma synoviae using primers located in the intergenic spacer region and the 23S rRNA gene

Mycoplasma synoviae (Ms) is an important pathogen of poultry, causing economic losses to this industry. Early and reliable diagnosis is a key to controlling the spread of this organism. In this study, a polymerase chain reaction with one primer based on the intergenic spacer region (ISR) was validated for detection of Ms. The ISR primer was paired with a general primer from within the 23S rRNA gene. The PCR primers were tested with the 22 other recognised avian Mycoplasma species to check the specificity and with 21 field isolates of Ms from various hosts and countries, and with several swab samples. The PCR appeared to be specific and sensitive. Four different sample preparation methods were compared for use in this PCR, and the amplification protocol was compared with three others, confirming the comparative sensitivity of the new PCR.     

16S-23S rRNA基因间隔序列PCR及RFLP对奇异变形杆菌分离株的鉴别与系统发育分析

根据临床常见致病菌16S-23S rRNA基因间隔序列(ISR)两端的16S及23S rRNA保守序列设计PCR扩增的通用引物,对9株奇异变形杆菌和6株相近菌株应用通用引物PCR扩增16S-23S rRNA ISR序列.通过PCR长度多态性比较、RFLP分析以及部分序列测序比较,分析鉴别奇异变形杆菌.结果显示,PCR长度多态性可以将奇异变形杆菌同其余菌种进行区分;RFLP分析可以将所有试验菌种进行区分;部分序列测序可以对奇异变形杆菌进行分型.由此表明,16S 23S rRNA ISR序列PCR及RFLP分析可以简单、快速、准确的鉴定奇异变形杆菌.     

Identification of Bartonella strains isolated from wild and domestic ruminants by a single-step PCR analysis of the 16S-23S intergenic spacer region

Of the 20 species or subspecies of Bartonella currently known, 7 cause various diseases in humans with many being zoonotic. However, some Bartonella species appear only to cause asymptomatic bacteraemia in their hosts. In ruminants, three Bartonella species (B. bovis, B. capreoli and B. schoenbuchensis) have recently been described. However, limited or no information has yet been published concerning their mode of transmission and their possible pathogenicity for domestic cattle. The phylogenetic relationship of these species with other bacteria of the Bartonella genus has only been recently investigated. It is therefore necessary to develop appropriate tools that will easily allow identification of these ruminant strains for epidemiological and clinical studies. A single-step PCR assay, based on the amplification of a fragment of the 16S-23S rRNA intergenic spacer (ITS), was evaluated for identification of Bartonella isolated from domestic cattle and from free-ranging or captive cervids. For each Bartonella species tested, the PCR assay led to a product that was unique either for its length or its sequence. All ruminant isolates tested could be easily differentiated among themselves and from the other Bartonella species. Furthermore, sequence analysis of the PCR products revealed a close relationship between all ruminant Bartonella strains. Therefore, ITS PCR testing appears to be a convenient tool for a quick diagnosis of ruminant Bartonella species.     

Polymerase chain reaction amplification of 16S-23S spacer region for rapid identification of Salmonella serovars

Polymerase chain reaction (PCR) was used to amplify the spacer regions between the 16S and 23S genes of rRNA genetic loci of Salmonella serovars for their rapid identification. These genetic loci revealed a significant level of polymorphism in length across the species/serovar lines. When the 16S-23S spacer region amplification products were subjected to agarose electrophoresis, the patterns observed could be used to distinguish all the serovars of Salmonella tested. Unique elements obtained in amplification products were mostly clustered at serovar level, although certain genus-specific patterns were also observed. On the basis of the results obtained, the amplification of 16S-23S ribosomal spacer region could suitably be used in a PCR-based identification method for Salmonella serovars.     

Rapid and direct detection of clostridium chauvoei by PCR of the 16S-23S rDNA spacer region and partial 23S rDNA sequences

Clostridium chauvoei causes blackleg, which is difficult to distinguish from the causative clostridia of malignant edema. Therefore, a single-step PCR system was developed for specific detection of C. chauvoei DNA using primers derived from the 16S-23S rDNA spacer region and partial 23S rDNA sequences. The specificity of the single-step PCR system was demonstrated by testing 37 strains of clostridia and 3 strains of other genera. A 509 bp PCR product, which is a C. choauvoei-specific PCR product, could be amplified from all of the C. chauvoei strains tested, but not from the other strains. Moreover, this single-step PCR system specifically detected C. chauvoei DNA in samples of muscle from mice 24 hr after inoculation with 100 spores of C. chauvoei, and in clinical materials from a cow affected with blackleg. These results suggest that our single-step PCR system may be useful for direct detection of C. chauvoei in culture and in clinical materials from animals affected with blackleg.     

Identification and differentiation of canine Mycoplasma isolates by 16S-23S rDNA PCR-RFLP

Conventional serological methods for the identification of canine mycoplasma isolates depend on the availability of a panel of species-specific diagnostic antisera and are not always reliable in terms of specificity. To enable simultaneous identification of field isolates, PCR-RFLP analysis of the 16S-23S rRNA intergenic spacer region was used to characterize the type strains of the 12 currently described canine mycoplasmas of the Genus Mycoplasma which represent the "classic" non-hemotropic species. The use of 16S-23S rDNA PCR in the first step of this analysis revealed specific size differences of amplicons which allowed to classify these 12 canine Mycoplasma species into three groups. Depending on the length of the amplicon, subsequent RFLP analysis of PCR products using two restriction endonucleases in a single digest (ApoI/DdeI or TaqI/VspI) generated unique banding patterns. For further evaluation of the 16S-23S rDNA PCR-RFLP assay system as identification and differentiation tool, a total of 262 field isolates collected from the canine genital tract were tested. PCR-RFLP results for 251 field isolates correlated with traditional serological test results. The remaining 11 isolates had an RFLP pattern distinct from the type strains included in this study and were identified by 16S rDNA sequencing as closely related to M. sp. HRC689. The PCR-RFLP assay established in this study enabled a rapid, accurate and easily performed identification and differentiation of all 12 currently described non-hemotropic canine Mycoplasma species.     

Streptococcus agalactiae and Streptococcus difficile 16S-23S intergenic rDNA: genetic homogeneity and species-specific PCR

Streptococcus difficile is a non-hemolytic Gram-positive bacterial coccus that causes septicemia and meningoencephalitis in farmed tilapia (Oreochromis sp.) and rainbow trout (Oncorhynchus mykiss). Recent studies have demonstrated S. difficile to be a group B, type Ib streptococcus with a whole cell protein electrophoretic profile indistinguishable from S. agalactiae and a biochemical profile similar to that observed for other group B, type Ib streptococci isolated from fish and frogs. The aim of this study was to expand on these findings by comparative nucleic acid sequence analysis of the 16S-23S ribosomal DNA (rDNA) intergenic spacers of S. difficile and S. agalactiae. The 97.7% sequence homology identified in these studies supports the taxonomic relationship of these two organisms. The sequence data generated were also used to construct a pair of species-specific PCR primers for use in molecular detection and identification schemes.     

Phylogenetic analysis of Mycoplasma suis isolates based on 16S rRNA gene sequence in China

To perform phylogenetic analysis of Mycoplasma suis isolates derived from China to define the nature of this pathogen, nearly complete of 16S rRNA genes from Chongqing, Sichuan, Henan and Guangdong isolates were amplified by PCR and sequenced. The four sequences from the blood samples in this study, with other 17 Hemoplasmas sequences and related 3 mycoplasma sequences available in the GenBank, were aligned using Clustal X (version 1.83) sequences alignment program. Maximum parsimony, neighbor-joining and minimum evolution (MEGA 4.0) algorithms were used to create phylogenetic trees. Phylogenetic analysis of these sequences showed that all hemoplasma species were located within a single clade and were most closely related to M. pneumoniae group. The hemoplasma species were further subdivided into two distinct groups, one containing M.wenyonii, M.suis and Candidatus M. haemominutum and the other containing M. haemofelis and M. haemocanis. Within the former clade, four M.suis isolates from Mainland China and other M.suis species formed a monophyletic group in the tree. A tendency of clear geographical grouping of the isolate was evident.     

Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region

In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii. A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia. The PCR was performed with total DNA from 26 strains which included seven different Clostridia species. These bacteria were differentiated at species level by the different PCR product patterns. To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced. The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e. tRNA gene sequences. The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene.     

鸡毒支原体红霉素和替米考星耐药体外诱导及其23S rRNA基因V域突变特征分析

鸡毒支原体（Mycoplasma gallisepticum，MG）是禽类主要病原菌之一，其感染引起鸡慢性呼吸道病（CRD）和火鸡传染性窦炎。目前预防鸡毒支原体感染仍主要依靠抗菌药物，但长期广泛应用抗菌药物造成鸡毒支原体耐药性不断发展。有调查表明，鸡毒支原体临床株对四环素类、大环内酯类及氟喹诺酮类等抗菌药物已产生了耐药性，造成药物疗效下降甚至失效。由于支原体分离培养困难，国内外对动物源支原体耐药性研究十分有限，至今未见有鸡毒支原体大环内酯类抗生素耐药机制的研究报道。本文以鸡毒支原体S6株为模式菌，研究红霉素和替米考星在体外诱导鸡毒支原体的耐药情况，并分析耐药菌株23S rRNA基因V域的耐药突变特征。     

Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes

Gallibacterium was recently established as a new genus including organisms previously reported as Pasteurella anatis, [Actinobacillus] salpingitidis and avian Pasteurella haemolytica-like organisms. The aim of the present study was to develop a PCR method allowing unambiguous identification of Gallibacterium. PCR primers positioned in the 16S rRNA (1133fgal) and 23S rRNA (114r) genes were defined and their specificity was subsequently tested on 122 strains. Twenty-five of the strains represented all of the presently available 15 phenotypic variants of Gallibacterium from different geographical locations, 22 other strains represented other poultry associated bacterial species or bacteria which could pose a differential diagnostic problem including members of the families Pasteurellaceae, Enterobacteriaceae and Flavobacteriaceae, and finally 75 Gallibacterium field strains isolated from Mexican chicken egg-layers. Specific amplicons were generated in all 100 Gallibacterium strains tested, whereas none of the non-Gallibacterium strains tested positive. Correct identification was confirmed by hybridization with the Gallibacterium specific probe GAN850. Two internal amplification control strategies were successfully incorporated into the PCR assay, one based on amplification of the house-keeping gene rpoB (sharing target DNA) and another based on addition of trout DNA (foreign target DNA) and amplification with beta-actin specific primers. In conclusion, the described PCR assay enables specific identification of Gallibacterium and will thus stand as a strong alternative to the present diagnostic methods.     

Molecular characterization of Streptococcus suis strains by 16S–23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.