Genomic diversity among Danish field strains of Mycoplasma hyosynoviae assessed by amplified fragment length polymorphism analysis

Genomic diversity among strains of Mycoplasma hyosynoviae isolated in Denmark was assessed by using amplified fragment length polymorphism (AFLP) analysis. Ninety-six strains, obtained from different specimens and geographical locations during 30 years and the type strain of M. hyosynoviae S16(T) were concurrently examined for variance in BglII-MfeI and EcoRI-Csp6I-A AFLP markers. A total of 56 different genomic fingerprints having an overall similarity between 77 and 96% were detected. No correlation between AFLP variability and period of isolation or anatomical site of isolation could be demonstrated. An examination of the clonal appearance of M. hyosynoviae isolates recovered from seven herds affected with arthritis revealed presence of several genotypically distinct variants of the organism in a single herd, indicating that different strains may simultaneously be involved in an outbreak of the disease.     

Differentiation of mycoplasma gallisepticum strains using molecular methods

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for the differentiation of MG strains. The MG strains MK-7, MS-16, S6, FS-9 and R strains and the MG live vaccine strain F were compared by random amplification of polymorphic DNA (RAPD) in this study. Using RAPD, different patterns were found among the MG strains. In addition to this, we examined the differentiating potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) primers targeted at the crmA, crmB, crmC, gapA, mgc2 and pvpA genes encoding cytadherence-related surface proteins. These proteins may take part in the pathogenesis of MG-induced disease. Differentiation of strain F is based on the identification of restriction enzyme sites in the PCR amplicons. Using HphI enzyme, crmC PCR amplicons produced different RFLP patterns. Digestion of amplicons of gapA-specific PCR with MboI enzyme also produced distinct patterns. Differences were observed among strains R and F by digestion of mgc2 PCR amplicons with HaelIl and VspI enzymes and digestion of pvpA PCR amplicons with AccI, PvulI and ScrFI endonucleases. This method can be used for the rapid differentiation of vaccine strain from wild strains. Differentiation of MG strains is a great advantage for diagnosticians or practitioners and it is useful for epidemiological studies.     

Characterization of Mycoplasma hyosynoviae strains by amplified fragment length polymorphism analysis,pulsed-field gel electrophoresis and 16S ribosomal DNA sequencing

Mycoplasma hyospnoviae strains from Denmark, Germany, Japan, Sweden, the Netherlands and the UK were examined for variations in the genomic DNA and within the 16S ribosomal RNA (rRNA) gene. Variations in the chromosomal DNA among 57 isolates recovered from the respiratory tract and joints of pigs, were investigated by analysis of amplified fragment length polymorphisms of the Bg/II and MfeI restriction sites and by pulsed-field gel electrophoresis of a BssHII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory potential for the differentiation of M. hyosynoviae isolates. Concordant results obtained with the two whole-genome fingerprinting techniques evidence the considerable intraspecies genetic heterogeneity of M. hyosynoviae. Sixteen field strains of M. hyosynoviae and the type strain S16(T) were further examined for variation within the 16S rRNA gene. Ten field strains possessed the 16S rDNA sequences identical to the type strain, while the remaining six strains had sequences that differed by one to two nucleotides from that obtained from the type strain.     

Differentiation of the vaccine F-strain from other strains of Mycoplasma gallisepticum by restriction endonuclease analysis

The electrophoretic patterns of the nucleic acids (DNA) of Mycoplasma gallisepticum strains digested with the restriction enzymes Bam HI, Eco RI and Hind III were useful for differentiating the vaccine F-strain from other strains of M. gallisepticum. The procedure was more sensitive than the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. The vaccine F-strain, represented by cultures designated F-K810 and F-F2F10 was clearly differentiated from other strains of M. gallisepticum. This procedure may be useful in field studies to determine if the vaccine strain will replace wild-type M. gallisepticum in commercial layers.     

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.     

Differentiation of Mycoplasma gallisepticum vaccine strains ts-11 and 6/85 from commonly used Mycoplasma gallisepticum challenge strains by PCR

Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses within the poultry industry. In an effort to develop tools to aid in MG research and diagnostics, we have compared sequences of the attenuated MG vaccine strain ts-11 to those of commonly used pathogenic challenge strains in search of a simple means of differentiation. Via gapA sequence alignments and comparisons, we have identified and designed primers facilitating strain differentiation. When applied to conventional polymerase chain reaction (PCR) assay at low annealing temperature, the primer sets allow for the differentiation of MG attenuated vaccine strains ts-11 as well as the attenuated MG vaccine strain 6/85 from the commonly utilized MG challenge strains R(low), R, and S6. Conventional PCR differentiation is based on the visualization of sole products with the attenuated MG strains ts-11 and 6/85 and the lack of the corresponding products from MG strains R(low), R, and S6. When applied to MG strain F, product visualization varies with the applied primer set. The differentiation of MG strains ts-11 and 6/85 from the pathogenic challenge strains was also accomplished via real-time analyses, however, the primer sets were not able to differentiate MG strains ts-11 and 6/85 from selected MG field isolates.     

Use of mgc2-polymerase chain reaction-restriction fragment length polymorphism for rapid differentiation between field isolates and vaccine strains of Mycoplasma gallisepticum in Israel

Increasing use of Mycoplasma gallisepticum (MG) live vaccines has led to a need for a rapid test for differentiation of MG field strains from the live vaccine strains ts-11 and 6/85. We examined the differentiating potential of diagnostic polymerase chain reaction (PCR) primers targeted to the gene mgc2, encoding a cytadherence-related surface protein uniquely present in MG. The mgc2-PCR diagnostic primers are specific for MG in tests of all avian mycoplasmas or bacteria present in the chicken trachea and are sensitive enough to readily detect MG in tracheal swabs from field outbreaks. Differentiation of vaccine strain ts-11 was based on identification of restriction enzyme sites in the 300-base-pair (bp) mgc2-PCR amplicon present in ts-11 and missing in MG isolates from field outbreaks in Israel. Restriction sites for the enzymes HaeII and SfaN1 were identified in the amplified region in strain ts-11 and were not found in 28 field isolates of MG, comprising a representative cross section of all the MG isolates from the period 1997-2003. In practice, differential diagnosis of MG is achieved within 1 day of submission of tracheal swab samples by mgc2-PCR amplification and restriction of the amplicon with HaeII, giving a 270-bp fragment for ts-11 or no restriction for other MG strains tested. Application of the mgc2-PCR-restriction fragment length polymorphism (mgc2-PCR-RFLP) assay enabled differential diagnosis of both components of a mixture of ts-11 and non-ts-11 DNA, detecting the field strain in the presence of a large excess of ts-11. The test was successfully applied in vivo for monitoring vaccinates in a ts-11 vaccine trial. In principle, the test may also be used to identify the 6/85 vaccine strain, which yields a 237-bp product, readily differentiated from the approximately 300-bp PCR product of all other strains tested. Further testing of field isolates will be necessary to determine the applicability of this test in the United States and other countries.     

猪链球菌荧光DNA扩增片段长度多态性检测方法的建立

利用荧光标记技术,采用2对引物初步建立检测猪链球菌荧光DNA扩增片段长度多态性方法,结果表明12株猪链球菌扩增的多态性位点数从51～98条不等,该方法能够检测猪链球菌的多态性,区分不同血清型以及同一血清型不同特性的菌株,可用于菌株鉴定及流行病学研究中细菌源的追踪。     

Staphylococcus aureus: study of genomic similarity of strains isolated in veterinary pathology using amplified fragment length polymorphism (AFLP)

Staphylococcus aureus is a widespread pathogen causing infections in different animal species. The extensive use of antibiotics, particularly methicillin, causes the rise of antibiotic-resistant strains (MRSA). In order to verify the epidemiology and genetic relatedness among MRSA and sensible strains (MSSA), an accurate fingerprinting technique, the amplified fragment length polymorphism (AFLP), was carried out. The isolates were cultured, subdivided on MRSA and MSSA and submitted for the genomic DNA extraction that was utilized for AFLP. The data were analysed for genetic similarity using the Dice coefficient. The results of genomic analysis among MRSA and MSSA and within them revealed that the major component of variation was due to variation within strains (82.12%), while variance among strains was lower (17.88%). The low level of genomic similarity found among S. aureus strains implies high level of genetic diversity. Different similarity was found as well in all strains independently of the source.     

Molecular variability of house finch Mycoplasma gallisepticum isolates as revealed by sequencing and restriction fragment length polymorphism analysis of the pvpA gene

Mycoplasma gallisepticum, a major pathogen of chickens and turkeys, has caused significant declines in house finch (Carpodacus mexicanus) populations in the eastern United States since it was first observed in this species in 1994. There is evidence that M. gallisepticum infection is now endemic among eastern house finches, although disease prevalence has declined, suggesting an evolving host-parasite relationship. Studies based on randomly amplified polymorphic DNA (RAPD) have documented the presence of a single, unique RAPD profile in house finch M. gallisepticum isolates, suggesting a single point source of origin, which agrees with the known epidemiologic observations. In the present study, we evaluated the molecular variability of 55 house finch isolates as well as 11 chicken and turkey isolates including reference strains of M. gallisepticum. Molecular variability was evaluated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis and nucleotide sequencing of the pvpA gene, which encodes for the putative cytadhesin protein PvpA. Three different RFLP groups and 16 genotypes were evident from the 55 house finch isolates evaluated. Sequence analysis of pvpA gene PCR products showed that although most house finch M. gallisepticum isolates clustered more closely to each other, others clustered more closely to either turkey or chicken field isolates. These findings suggest that house finch isolates are more polymorphic than previously recognized by RAPD studies. This feature may allow us to learn more about the molecular evolution and epidemiology of this emerging disease host-parasite relationship.     

鸡败血支原体GapA高变区在大肠杆菌中高效表达

本研究通过对鸡败血支原体(Mycoplasma gallisepticum,MG)强毒株黏附素GapA的N端(98aa～322aa)和C端(820aa～1 115aa)2个高变区进行克隆,并进行原核表达,制备获得抗GapA多抗,结果显示,GapAN获得高效表达,制备获得的抗体对MG黏附真核细胞有一定的抑制作用,这说明GapA对MG的黏附有着非常重要的影响.这一抗原成分的成功表达和抗体的制备为研制特异性单抗和筛选GapA缺失型突变株奠定了坚实的基础;并且利用不同毒株在GapA N端功能区的基因序列的明显差异,使基于该抗体建立新型快速的免疫学方法以鉴别MG强弱毒株亦将成为可能.     

Glycoconjugate heterogeneity among five strains of Mycoplasma gallisepticum

Five strains of Mycoplasma gallisepticum (MG) were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis for the presence of carbohydrate-containing components. Staining with periodic acid-Schiff (PAS) demonstrated carbohydrate components in three of the five strains studied. The PAS-reactive bands counterstained for protein, indicating a possible glycoprotein nature. Western blot analysis using three biotinylated lectin probes demonstrated the presence of additional glycoconjugates in the blot profiles of each MG strain. The carbohydrate specificity of lectin binding was demonstrated by competition experiments using specific sugars. Differences in the number, electrophoretic mobility and the morphology of PAS and lectin reactive bands were reproducible among separate preparations of each MG strain. These findings indicate substantial phenotypic diversity among the five MG strains in their ability to produce or acquire glycoconjugates.     

Pathogenicity of two strains of Mycoplasma gallisepticum in broilers

Strains F and R of Mycoplasma gallisepticum (MG) were compared in two laboratory trials for their relative pathogenicity in terms of inducing airsacculitis and antibody production to MG. Chickens exposed to the R strain had significantly higher incidence of air-sac lesions (P less than 0.05) and greater severity of airsacculitis than did chicks exposed to the F strain. In both trials, chickens vaccinated simultaneously with Newcastle disease-infectious bronchitis vaccine and exposed to MG had more severe lesions than did chickens exposed to mycoplasma alone. chickens exposed to the F strain had significantly lower geometric mean hemagglutination-inhibition antibody titers to MG than did chicks exposed to the R strain. Chickens vaccinated simultaneously with Newcastle disease-infectious bronchitis vaccine and exposed to R strain had significantly lower body weights than did chickens in the other group.     

Complementary randomly amplified polymorphic DNA (RAPD) analysis patterns and primer sets to differentiate Mycoplasma gallisepticum strains.

Randomly amplified polymorphic DNA (RAPD) analysis was used to differentiate 7 strains of Mycoplasma gallisepticum. Six commercially available primers or primer combinations were screened for their ability to differentiate vaccine and type strains. Although major and minor bands were produced with each primer, many of the primers were unsuitable for strain differentiation. The use of primer 6 and combined primers 3 and 4 resulted in complementary RAPD banding patterns for each M. gallisepticum strain. Eleven different isolates representing 7 different strains were segregated into 7 different patterns, corresponding to the 7 strains.     

鸡毒支原体敏感株与耐药株超微结构的比较

对临床分离和实验室药物压力下筛选的鸡毒支原体耐药株与敏感株进行超微结构的观察和比较.结果显示鸡毒支原体敏感株呈多形性,柔软且有较大的变形性,可清晰观察到细胞膜分为外、中、内三层膜,并可观察到裂殖繁殖方式;有的支原体在繁殖时先在极端产生泡状突起,形成不均等分裂.在恩诺沙星药物压力下敏感株产生耐药性后其外膜显著增厚,导致支原体的多形性减弱或消失,呈现出较为一致的圆形;细胞膜内层周围存在排列整齐、结构紧密、类似微管样的结构,胞内电子密度明显升高.临床分离的耐药株超微结构观察结果与实验室条件下筛选的耐药株一致:凡是超微结构发生变化的,均存在耐药表型,而且高水平耐药株的超微结构变化最为突出.研究结果表明耐药性的产生可导致鸡毒支原体超微结构明显的改变,并可能引起抗原性变异.     

应用多重套式PCR检测鸡毒支原体和鸡滑液囊支原体

根据已发表的鸡毒和鸡滑液支原体血凝素基因序列pMGA和vlhA各设计两对引物,建立鉴别诊断两种支原体的多重套式PCR方法,对其进行温度条件、Ⅱ步模板浓度优化及特异性、敏感性实验。该方法在两步PCR后能特异性地扩增出MG(408 bp)和MS(688 bp)两个目的片段。应用于临床样品检测,与支原体分离、SPA检测比较结果PCR灵敏度高于病原分离。     

Differentiation of twelve Actinobacillus pleuropneumoniae serotypes by outer membrane lipoprotein gene-based restriction fragment length polymorphism

Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)-amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950-base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR-based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR-based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.