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1.
Ribosomal RNA gene probes to detect intraspecies heterogeneity in Mycoplasma gallisepticum and M. synoviae 总被引：3，自引：0，他引：3
Intraspecies genotypic heterogeneity among strains of Mycoplasma gallisepticum and M. synoviae was tested using genomic fingerprints with a ribosomal RNA (rRNA) gene probe. The organism's DNA was digested by a restriction endonuclease, electrophoresed, transferred to a nitrocellulose sheet, and hybridized with 32P-labeled pMC5 plasmid carrying the highly conserved rRNA genes of M. capricolum. The resulting hybridization patterns indicated a degree of genotypic heterogeneity among M. gallisepticum strains more pronounced than among the M. synoviae strains tested. Most importantly, the live vaccine F strain of M. gallisepticum could be distinguished from virulent field isolates of this species, enabling the detection and identification of the F strain in areas in which vaccination with this strain has taken place. Genomic fingerprints with an rRNA gene probe can thus be added to the battery of tools useful in taxonomy at the intraspecies level and in epidemiology of mycoplasmosis in poultry. 相似文献
2.
Marques LM Buzinhani M Guimaraes AM Marques RC Farias ST Neto RL Yamaguti M Oliveira RC Timenetsky J 《Veterinary microbiology》2011,152(1-2):205-211
Ureaplasma diversum infection in bulls may result in seminal vesiculitis, balanoposthitis and alterations in spermatozoids. In cows, it can cause placentitis, fetal alveolitis, abortion and the birth of weak calves. U. diversum ATCC 49782 (serogroups A), ATCC 49783 (serogroup C) and 34 field isolates were used for this study. These microorganisms were submitted to Polymerase Chain Reaction for 16S gene sequence determination using Taq High Fidelity and the products were purified and bi-directionally sequenced. Using the sequence obtained, a fragment containing four hypervariable regions was selected and nucleotide polymorphisms were identified based on their position within the 16S rRNA gene. Forty-four single nucleotide polymorphisms (SNP) were detected. The genotypic variability of the 16S rRNA gene of U. diversum isolates shows that the taxonomy classification of these organisms is likely much more complex than previously described and that 16S rRNA gene sequencing may be used to suggest an epidemiologic pattern of different origin strains. 相似文献
3.
Hong Y García M Leiting V Bencina D Dufour-Zavala L Zavala G Kleven SH 《Avian diseases》2004,48(3):606-616
Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene-targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 x 10(2) color changing units/ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene-targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples. 相似文献
4.
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
5.
Siarkou VI Mylonakis ME Bourtzi-Hatzopoulou E Koutinas AF 《Veterinary microbiology》2007,125(3-4):304-312
The purpose of this study was to characterize, at the molecular level, the Ehrlichia canis strains involved in naturally occurring canine monocytic ehrlichiosis (CME) in Greece, and to investigate if any sequence diversity exists between the 16S rRNA genes of those involved in the mild non-myelosuppressive or the severe myelosuppressive form of CME. To this end, amplification of the ehrlichial 16S rRNA gene was attempted by nested polymerase chain reaction (PCR) assays in bone marrow (BM) aspirates from 20 dogs tentatively diagnosed as having non-myelosuppressive (n=10, group A) or myelosuppressive (n=10, group B) CME. PCR assay using E. canis-specific primers revealed that 15 BM samples, including all group A and 5 group B dogs, were positive. Using universal PCR primers, a nearly full-length 16S rRNA gene could be amplified from 13 BM samples, including 9 group A and 4 group B dogs. The 16S rDNA analysis based on secondary structure revealed that all sequences of the Greek strains were identical to each other and indicated 100% identity among some American (Venezuelan and Brazilian), European (Greek), Middle Eastern (Turkish) and Asiatic (Thailand) strains. The results of this study suggest that the E. canis strains involved in the non-myelosuppressive and myelosuppressive forms of CME in Greece share an identical 16S rRNA genotype. 相似文献
6.
Mycoplasmas have been implicated in certain clinical syndromes in ostriches and are associated with upper respiratory tract infections. As these infections result in production losses, they are of considerable economic importance to the South African ostrich industry. Although poultry mycoplasmas have been shown to infect ostriches, the existence of unique ostrich-specific mycoplasmas has been suggested. In this study, mycoplasmas were isolated from ostriches in the Klein Karoo, Central Karoo and Garden Route areas of the Western and Northern Cape Provinces of South Africa and identified using 16S rRNA gene sequencing. These sequences indicated that ostriches in these areas carry three unique mycoplasmas and were not infected with chicken mycoplasmas. Phylogenetic analysis of the 16S rRNA sequences of the three isolated ostrich mycoplasmas showed them to be quite divergent and to fall into two distinct phylogenetic groupings. Unique sequences within the 16S rRNA gene of the ostrich mycoplasmas were subsequently used for the development of specific primers for the detection and diagnosis of mycoplasma infections in ostriches. Chickens kept in close proximity to infected ostriches were not infected with these ostrich mycoplasmas. 相似文献
7.
8.
Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus 总被引：1，自引：0，他引：1
Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected. 相似文献
9.
The aim of the study was to analyse experimental transmission of Mycoplasma synoviae, an avian pathogen. Three experiments using specific pathogen-free day-old chicks placed in isolators were conducted. In the first experiment, the birds were introduced in an isolator previously contaminated with a M. synoviae broth culture. After 34 days, these birds were eliminated and, for the second trial, the chicks were introduced in the same isolator without disinfecting. In the third assay, the chicks were placed in an isolator containing a mixture of food, feathers and dust collected less than an hour earlier from a M. synoviae infected laying hen flock. In the second and third experiments in order to exacerbate the M. synoviae infection, the birds were inoculated with infectious bronchitis (IB) virus. The presence of M. synoviae in the environment and in tracheal swabs was monitored by culture, a multiplex PCR (mPCR) detecting M. synoviae and Mycoplasma 16S rDNA and a multiplex RT-PCR (mRT-PCR) detecting the M. synoviae mRNA coding for a membrane protein and Mycoplasma 16S rRNA. In in vitro experimental conditions, M. synoviae mRNA and 16S rRNA were detected up to 20 min and 23 h respectively after mycoplasma death. In the first assay, the first infected bird was detected on the 13th day. In the second trial, culturable M. synoviae or viable M. synoviae were detected in the isolator for 3 or 4 to 5 days respectively after depopulation of the birds of the first assay whereas the first culture positive tracheal swabs were detected on the 33rd day, after IB inoculation. In the third experiment, the first infected birds were detected on the 54th day. Thus, the different assays showed that M. synoviae contaminated material (dust, feathers and food) can infect chicks, sometimes after remarkably long silent periods. 相似文献
10.
Capurro A Artursson K Waller KP Bengtsson B Ericsson-Unnerstad H Aspán A 《Veterinary microbiology》2009,134(3-4):327-333
In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections. 相似文献
11.
Aguirre E Sainz A Dunner S Amusategui I López L Rodríguez-Franco F Luaces I Cortés O Tesouro MA 《Veterinary parasitology》2004,125(3-4):365-372
This paper reports the first isolation and culture of Ehrlichia canis in Spain from a naturally infected dog using the DH82 cell line. After DNA extraction and PCR amplification, a nearly complete (1412bp) sequence of the 16S rRNA gene of the new E. canis strain was obtained. The GenBank accession number for the nucleotide sequence of this strain is AY394465. This sequence was aligned with the 16S rRNA gene sequences of other Ehrlichia strains accessible in GenBank. The 16S rRNA gene sequence of the E. canis strain reported here showed a high percentage of similarity with the 16S rRNA gene sequence of E. canis from different geographic areas including Japan, Venezuela and Israel. These data confirm the presence of E. canis in Spain. 相似文献
12.
Restriction endonuclease analysis of Mycoplasma synoviae strains 总被引：1，自引：0，他引：1
A diverse group of Mycoplasma synoviae strains from various hosts, pathological processes, and geographic areas collected over about 25 years were analyzed by restriction endonuclease analysis. The results suggest that restriction endonuclease analysis of M. synoviae strains may be a useful strain identification tool to study epidemiological problems. 相似文献
13.
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. 相似文献
14.
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
15.
从河北某养鸡场80日龄左右鸡群所发生的禽霍乱病死鸡中，分离到了相应病原菌多杀巴斯德氏菌（pasteurella multocida）。对分离获得的16株菌（HPs-1至HPs-16）进行了形态特征、培养特性、理化特性等方面的鉴定，同时选取代表菌株（HPs-1）进行了16SrRNA基因的分子鉴定，测定了16SrRNA序列，构建了系统发育树。结果表明分离鉴定的16株细菌均为多杀巴斯德氏菌败血亚种（P．multocida subsp．septica），所测代表菌株的16SrRNA基因长度142bp，在GenBank登录号为AY999017，该菌株与检索出的22株巴斯德氏菌属（Pasteurella Trevisan，1887）细菌16SrRNA基因序列同源性均在98％～100％。另外，药敏试验结果显示分离株对供试的青霉素G等33种抗菌药物高敏、对克林霉素敏感、对苯唑青霉素等3种耐药。 相似文献
16.
为了探讨鸭疫里默氏杆菌(Riemerella anatipestifer, RA)云南流行株的外膜蛋白A (OmpA)的基因序列差异及其与16S rRNA序列的相关性,PCR扩增18株云南流行株鸭疫里默氏杆菌OmpA基因及16S rRNA核苷酸序列,分别构建其系统进化树,分析其系统进化关系。结果表明,18株鸭疫里默氏杆菌OmpA基因分为2个群,其同源性分别为86%~99.2%和92.6%~100%。18株鸭疫里默氏杆菌16S rRNA基因同属1个群,同源性高达96.1%~100%。 RA-1、RA-2、RA-11和RA-39 4株分离株的OmpA基因位于进化树的同一个亚群,其16S rRNA基因也位于进化树的同一亚群,两者呈现出明显的相关关系,其他14株分离株的OmpA基因系统进化树与16S rRNA基因系统进化树无明显的相关关系。 相似文献
17.
为了解宁夏及其周边不同地区蛋鸡场中的鸡滑液囊支原体的种类及其致病力,采用改良Frey氏鸡滑液囊支原体培养基,从疑似感染鸡滑液囊支原体的不同蛋鸡群中分离出病原株,设计鸡滑液囊支原体vlhA基因特异性引物,对分离到的病原进行基因序列扩增并测序,使用MEGA6.0软件中的邻接法(Neighbor-joining,NJ)构建分离株系统发育树并进行遗传进化分析,采用从临床症状最明显的鸡体分离的菌株进行动物感染试验,制作石蜡切片,HE染色,病理组织学观察。结果表明,所分离到的病原菌均为鸡滑液囊支原体,部分菌株之间极其相似;鸡滑液囊支原体不仅可以使鸡只关节肿胀、生长缓慢,也可引起鸡只的肝脏轻微肿大,肝细胞部分坏死、间质增生;脾脏结缔组织增生,出血。 相似文献
18.
Neubauer C Hess M 《Journal of veterinary medicine. B, Infectious diseases and veterinary public health》2006,53(8):376-381
The objective of this study was to develop a multiplex polymerase chain reaction (PCR) to detect and differentiate food-borne pathogens of the three genera Campylobacter, Arcobacter and Helicobacter in a single step procedure. One common reverse primer and three genus-specific forward primers were designed by hybridizing to the 16S rRNA of selected reference strains. Besides the species with significance as food-borne pathogens isolated from poultry meat--Campylobacter jejuni, Campylobacter coli, Arcobacter butzleri and Helicobacter pullorum--several other members of these genera were tested to determine the specificity of the designed multiplex PCR. In total, 20 ATCC and NCTC reference strains of Campyobacter, Arcobacter and Helicobacter were used to evaluate the PCR. Specific amplificates were obtained from all thermophilic species of Campylobacter as well as from species of Arcobacter and Helicobacter. No amplification product was obtained from the non-thermophilic Campylobacter, C. hyointestinalis and C. fetus. Furthermore, a total of 43 field strains of the three genera isolated from poultry, pigs, cattle and humans were investigated using this PCR. To confirm the classification of 10 H. pullorum strains the 16S rRNAs were sequenced. The developed PCR is a helpful diagnostic tool to detect and differentiate Campylobacter, Arcobacter and Helicobacter isolated from poultry and poultry products. 相似文献
19.