Synoviae Isolates Research Articles

Variable lipoprotein haemagglutinin (vlhA) gene sequence typing of mainly Dutch Mycoplasma synoviae isolates: comparison with vlhA sequences from Genbank and with amplified fragment length polymorphism analysis

Molecular typing techniques with sufficient discriminatory power are required to better understand the transmission of Mycoplasma synoviae, a poultry pathogen with increasing clinical and economic relevance. A promising molecular technique is polymerase chain reaction and subsequent sequencing based on the conserved 5′ region of the M. synoviae variable lipoprotein and haemagglutinin (vlhA) gene. This technique was used for genotyping 27 mainly Dutch M. synoviae isolates from different organs of various categories of poultry housed on different farms and collected during a period of 10 years. The obtained vlhA sequences were compared with those of M. synoviae strains from Genbank and data obtained by amplified fragment length polymorphism (AFLP). Grouping based on 100% similarity revealed nine genotypes. Some isolates had identical vlhA gene sequences although they originated from different geographical areas, different years and organs. AFLP analysis results largely confirmed the results obtained by vlhA sequence typing. Our findings raise concern regarding the discriminatory power of these techniques for its use in molecular epidemiology of Dutch M. synoviae isolates and for the differentiation between M. synoviae vaccine strains and field isolates, and indicate that molecular typing based on additional markers should be considered.

Molecular characterization of Mycoplasma synoviae isolates from commercial chickens in Iran

Detection of Mycoplasma synoviae (MS) by culture and polymerase chain reaction (PCR) has been reported from commercial chicken farms in different provinces of Iran. In some reports the phylogenetic analysis of MS isolates based on 16S rRNA and variable lipoprotein hemagglutinin (vlhA) genes have been carried out. The PCR product containing partial 16S rRNA genes of Iranain isolates was sequenced, and compared with 16S rRNA gene of MS sequences which were available in GenBank. Variations, polymorphisms, and differences between nucleotides of all isolates were observed. Phylogenetic analysis of these sequences showed that all MS isolates from Iran were most closely related to sequences of MS from Brazil. Sequence analysis of the N-terminal end of the hemagglutinin encoding gene vlhA were also used as an alternative for the detection and initial typing of field strains of MS in commercial poultry. The results showed that there was a complete concordance between all Iranian isolates nucleotide sequence and the 5-vlhA region sequence remained unchanged in all MS isolates and demonstrated differentiation between Iranian isolates and live commercial MS-H vaccine strain. More recently, the single-copy domain of the conserved region of vlhA gene in MS was sequenced, analyzed and verified to type MS field isolates in Iran and live vaccine MS-H strain. In addition, a restriction fragment length polymorphism (RFLP) method was established based on single nucleotide polymorphism that existed in all field isolates of Iran to differentiate between these field isolates and MS-H. This PCR-RFLP method allowed differentiating all MS field isolates from the vaccine strain.

Molecular typing of Iranian field isolates Mycoplasma synoviae and their differentiation from the live commercial vaccine strain MS-H using vlhA gene

1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains.2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain.3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity.4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains.5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.

Molecular Epidemiology of Mycoplasma synoviae Infection in Commercial Layers

Mycoplasma synoviae (M. synoviae) infection is a cause of great economic loss in commercial egg layer hens. The aims of this study were to investigate the prevalence of M. synoviae and to compare the characteristics of M. synoviae infected and free flocks of commercial layers. A total of 400 tracheal swabs and 400 blood serum samples were collected from 20 different layer flocks. Random Amplified Polymorfic DNA (RAPD) analysis was used for the molecular typing of M. synoviae isolates to determine the source of infection. M. synoviae was isolated from 89 tracheal swabs collected from 5 out of 20 flocks. The genetic similarity between field strains ranged from 53% to 100% as determined by RAPD analysis. All M. synoviae strains separated into 2 main-clusters in UPGMA dendogram. These results revealed that the 5 infected flocks were contaminated from 2 different sources. The egg production of positive flocks was statistically lower (P<0.05) than that of the pathogen free flocks. Infection was more frequent in multi-age farms and on sites with several houses. The mortality of infected flocks was higher than uninfected flocks, but this difference was not statistically significant. The mean weight of eggs and the average live weight of hens were similar in free and infected flocks. In conclusion, M. synoviae infection significantly decreased egg production in layer hens and was more frequent in multiage farms.

THE VLHA GENE SEQUENCING OF IRANIAN MYCOPLASMA SYNOVIAE ISOLATES

Mycopl asma synoviae expressed variable lipoprotein haemagglutinin (VlhA) is believed to play a major role in pathogenesis of the disease by mediating adherence and immune evasion. The aim of this study was sequencing Iranian M. synoviae isolates for the detection of nucleotide variation in the M. synoviae vlhA gene. Using oligonucleotide primers complementary to the single-copy conserved 5´ end of vlhA gene, amplicons of ~400 bp were generated from 10 M. synoviae isolated from commercial broiler chicken farms in Iran, afterward the conserved domain of the vlhA gene of M. synoviae was sequenced and analyzed for Iranian isolates. The results showed that, there was a complete concordance between all Iranian isolates nucleotide sequence (1-386 nt). In comparison with vaccine MS-H strain sequence, all Iranian isolates; entire vlhA sequence downstream of nucleotide 386 was different. It was also observed in all Iranian M. synoviae isolates, point mutations and frame-shift mutation. This study was demonstrated a difference between Iranian isolates and live commercial vaccine MS-H strain. Furthermore, these data indicated that changes in the vlhA gene sequence could introduce into the expressed vlhA gene amino acid codons and effective in pathogenesis rate in flocks.

Phylogenetic Analysis of Mycoplasma Synoviae Isolated from Commercial Iranian Chicken Farms Compared with Other Genebank Isolate Sequences Based on 16S rRNA Gene

Mycoplasma synoviae ( M. synoviae) is an important avian and extracellular pathogen which causes great economic losses in poultry industry. We have performed a phylogenetic analysis of M. synoviae isolates derived from commercial chicken farms in Iran based on 16S rRNA gene. Partial sequences of 16S rRNA genes obtained from 19 isolates of central region of Iran were amplified by PCR in 207bp fragment, then sequenced and next compared with the 16S rRNA gene of M. synoviae sequences that were available in GeneBank. Sequences were aligned using the ClustalW method, the UPGMA algorithm was used to create the phylogenetic tree. Phylogenetic analysis of these sequences demonstrated that all 19 M. synoviae isolated from Iran were most closely related to sequences of M. synoviae from Brazil. Variations, polymorphisms, and differences between nucleotides of all isolates were observed. The results of this study suggest that the close relationship, different molecular structure and heterogeneity among M. synoviae isolated may be explained by the transmission of mutations and variations between countries due to trading or to the stabilisation of spontaneous mutations because of regional conditions.

English

&nbsp; Mycoplasma synoviae&nbsp;(MS)&nbsp;is one of the important pathogens in chicken and turkey which cause great economic losses in poultry industry.&nbsp;M. synoviae&nbsp;has one serotype but there is heterogeneity among MS strains. The aim of this study was to analyze the DNA sequence of Mycoplasma&nbsp;synoviae&nbsp;isolates from Mazandran province poultry flocks of Iran and compare them with MS from other countries. For this purpose, 32 samples were collected from choanal cleft, trachea and air sac of flocks with respiratory symptom and cultured in specific PPLO medium. Ten out of the 32 samples were positive in culture&nbsp;and reacted with specific antisera. The 10 positive samples were subsequently used for molecular study. The PCR products containing 16s rRNA genome were generated with specific primers and then sequenced. Multiple alignment and phylogenic analysis of DNA sequences was performed with DNASTAR program. Alignment and comparison of the sequences in DANSTAR program revealed that 7 of 10 isolate are quite similar and 3 other isolate are different from group one. Phylogenic analysis of&nbsp;M. synoviae&nbsp;isolated from Mazandran province and 21&nbsp;M. synoviae&nbsp;from the entire world revealed low homology among most of them. However, some of the Mazandran isolates showed high homology with&nbsp;M. synoviaeisolated from Brasilia. Overall, these results revealed the different molecular structure and heterogeneity among&nbsp;M. synoviae&nbsp;isolated from Mazandran province in this study. &nbsp; Key words:&nbsp;Mycoplasma sinoviae, sequence, phylogenetic, poultry, Mazandran.

Analysis of the 16S to 23S rRNA intergenic spacer region of Mycoplasma synoviae field strains

Mycoplasma synoviae has been associated with economic loss in the chicken and turkey industries. The molecular characterization of M. synoviae at strain level allows the analysis of relationships between strains that may be valuable in epidemiological investigations. In the present study, the intergenic spacer region (ISR) between the 16S and 23S rRNA genes was examined to see whether useful information about strains could be derived. M. synoviae has two copies of this region, which may not be exactly the same (intercistronic heterogeneity). Sequencing of the ISRs of 21 M. synoviae isolates and the type strain revealed that 19 of them had such heterogeneity so DNA cloning was performed where necessary. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and a dendrogram was constructed. The length of the ISRs varied between 305 and 309 base pairs. Apart from having extra A/Ts in poly-A or poly-T regions and the presence of a few polymorphisms, the sequences of the M. synoviae strains were similar. Based on phylogenetic analysis, the strains were assigned to 10 groups—taking into account that within each group the DNA similarity was 100%, while the lowest similarity between groups was 95.8%. The results were compared with those obtained with the vlhA gene, resulting in very similar M. synoviae groups. Although the ISR could be a good target for strain typing, as has been shown by others for Mycoplasma gallisepticum, the method may be too cumbersome for routine use with M. synoviae because of complications with intercistronic heterogeneity. However, if the ISR sequence information was to be combined with other mutation detection techniques it could increase the discriminatory power.

In vitro antibiotic susceptibility of Dutch Mycoplasma synoviae field isolates originating from joint lesions and the respiratory tract of commercial poultry

The in vitro susceptibility of 17 Dutch Mycoplasma synoviae isolates from commercial poultry to enrofloxacin, difloxacin, doxycycline, tylosin and tilmicosin was examined. Three isolates originated from joint lesions and 14 were from the respiratory tract. The type strain M. synoviae WVU 1853 was included as a control strain. Antibiotic susceptibility was tested quantitatively using the broth microdilution test. Based on initial and final minimum inhibitory concentration values, all tested isolates were susceptible to doxycycline, tylosin and tilmicosin. Two isolates from the respiratory tract were resistant to enrofloxacin and showed intermediate resistance to difloxacin.

In vitro susceptibilities to fluoroquinolones in current and archived Mycoplasma gallisepticum and Mycoplasma synoviae isolates from meat-type turkeys

Monitoring of susceptibility to antibiotics in field isolates of pathogenic avian mycoplasmas is important for appropriate choice of treatment. Our study compared in vitro susceptibility to enrofloxacin and difloxacin in recent (2005–2006) isolates of Mycoplasma gallisepticum and Mycoplasma synoviae from meat-type turkey flocks with archived (1997–2003) isolates and reference strains. Comparison of minimal inhibitory concentration (MIC) values determined by microtest, agar dilution and commercial Etest showed good agreement, but underscored the need for standardized methods for testing. Notably, while the commercial Etest was convenient and accurate for determining MICs for enrofloxacin in the range 0.002–0.094 μg/ml, the endpoint of inhibition for M. gallisepticum and M. synoviae strains with MIC values ≥1.0 μg/ml could not be determined. A decrease in susceptibility to both fluoroquinolones was detected in archived strains but to a greater degree in recent isolates, most of which had MICs above the NCCLS susceptibility breakpoint for these antibiotics (≤0.5 μg/ml). In contrast, except for one flock, M. synoviae isolates were susceptible, although intrinsically less susceptible than M. gallisepticum. Overall for the 88 strains tested (45 M. gallisepticum, 43 M. synoviae), the MIC 50 for both enrofloxacin and difloxacin was 0.5 μg/ml. The isolation of fluoroquinolone-resistant M. gallisepticum isolates from breeder and broiler flocks as well as from meat-type turkeys suggests that these strains have become established in Israel, necessitating a reevaluation of antibiotic therapy. Periodic survey of MICs in field isolates of avian mycoplasmas to monitor for the possible appearance of resistant strains is recommended.

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

Mycoplasma synoviae is an economically important pathogen of poultry worldwide, causing respiratory infection and synovitis in chickens and turkeys. Identification of M. synoviae isolates is of critical importance, particularly in countries in which poultry flocks are vaccinated with the live attenuated M. synoviae strain MS-H. Using oligonucleotide primers complementary to the single-copy conserved 5' end of the variable lipoprotein and haemagglutinin gene (vlhA), amplicons of approximately 400 bp were generated from 35 different M. synoviae strains/isolates from chickens and subjected to mutation scanning analysis. Analysis of the amplicons by single-strand conformation polymorphism (SSCP) revealed 10 distinct profiles (A-J). Sequencing of the amplicons representing these profiles revealed that each profile related to a unique sequence, some differing from each other by only one base-pair substitution. Comparative high-resolution melting (HRM) curve analysis of the amplicons using SYTO 9 green fluorescent dye also displayed profiles which were concordant with the same 10 SSCP profiles (A-J) and their sequences. For both mutation detection methods, the Australian M. synoviae strains represented one of the A, B, C or D profiles, while the USA strains represented one of the E, F, G, H, I or J profiles. The results presented in this study show that the PCR-based SSCP or HRM curve analyses of vlhA provide high-resolution mutation detection tools for the detection and identification of M. synoviae strains. In particular, the HRM curve analysis is a rapid and effective technique which can be performed in a single test tube in less than 2 h.

Identification of major immunogenic proteins of Mycoplasma synoviae isolates

Mycoplasma synoviae isolates differ in patterns of immunogenic proteins, but most of them have not been identified yet. The main aim of this study was their identification in two closely related M. synoviae isolates, ULB 02/P4 and ULB 02/OV6, recovered recently from chickens in Slovenia. N-terminal sequencing identified 17 M. synoviae proteins. Amongst them were 14 major, highly expressed but previously unidentified proteins, including enzymes, chaperones and putative lipoproteins. ULB 02/P4 proteins with increasing molecular weight ( M w) in the region above the lipoprotein MSPB (∼40 kDa) were elongation factor EF-Tu, enolase, NADH oxidase, haemagglutinin MSPA, ATP synthase beta chain, trigger factor, pyruvate kinase and chaperone DnaK. Enolase (∼47 kDa) seemed to be immunogenic for chickens infected with M. synoviae, whereas EF-Tu, which might cross-react with antibodies to the P1 adhesin of Mycoplasma pneumoniae, was not. ULB 02/OV6 synthesized several immunogenic proteins and those with M w of approximately 70, 78, 82, 90, 110 and 160 kDa, cross-reacted with antibodies to Mycoplasma gallisepticum. They remain to be identified, because besides putative lipoproteins, protein bands of 78, 82, 85 and 110 kDa contained also dehydrogenase PdhD, elongation factor EF-G, enzyme PtsG and putative neuraminidase, respectively.

Specific Detection and Typing of Mycoplasma synoviae Strains in Poultry with PCR and DNA Sequence Analysis Targeting the Hemagglutinin Encoding GenevlhA

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.

Field studies on the association between amyloid arthropathy and Mycoplasma synoviae infection, and experimental reproduction of the condition in brown layers

Approximately 20% of outbreaks of amyloid arthropathy in The Netherlands were earlier attributed to Enterococcus faecalis but there was no explanation for the remaining cases. In a further study, material from the joints of 10 birds from each of 10 affected brown layer flocks and two broiler flocks was cultured in several bacteriological media. E. faecalis was isolated from one layer flock but Mycoplasma synoviae was recovered from purulent joints in six of the layer flocks and both broiler flocks. Brown layers were then infected experimentally with one of the M. synoviae isolates to assess its arthropathic and amyloidogenic potential. Using Congo red staining, articular amyloid deposits were seen 12 weeks after intra-articular or intravenous inoculation in almost all birds. After intra-articular inoculation of the left knee joint the contralateral hock tendon sheath tended to be affected, while hock and foot joints were mainly affected after intravenous inoculation. Amyloid deposits were seen in livers and spleens of many birds. This is the first demonstration of arthropathic strains of M. synoviae with an association with amyloid arthropathy, while for one isolate its amyloidogenic potential was shown experimentally.

Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin

Mycoplasma synoviae is a major avian pathogen that synthesizes hemagglutinin VlhA, an abundant immunodominant surface lipoprotein. In most M. synoviae strains, the VlhA protein cleaves into the N-terminal part, a lipoprotein MSPB, and a C-terminal part MSPA, which mediates binding to erythrocytes. VlhA is encoded by the vlhA gene of which the 5′-end is present in the genome as a single copy, which does not change its sequence during recombination of the vlhA gene with pseudogenes. In this study, sequence analyses of the 5′-end vlhA sequences of 30 M. synoviae isolates revealed a highly polymorphic region encoding the proline-rich repeats (PRR) in the N-terminal part of MSPB. Pathogenic strain K1968 had an insertion encoding sequence DNPQNPN in PRR, whereas strains F10-2AS, K2581, K3344 and five strains belonging to two related clusters of strains isolated recently from chickens in Slovenia lacked one PRR repeat of 19 amino acids. The predicted length variations correlated well with the lengths of the corresponding MSPB proteins detected in immunoblots with specific antibodies. Comparison of the 5′-end vlhA sequences of 30 M. synoviae strains showed 11 different types of vlhA sequences indicating that the analysis of this vlhA part is useful for strain differentiation. Distinct sequence motifs seem to be characteristic for vlhA genes of individual M. synoviae strains or clusters of strains and can be used as markers for tracing their spreading between poultry farms.

Comparison ofMycoplasma synoviaeisolates by immunoblotting

SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblot profiles using antisera from M. synoviae F10-2AS-inoculated chickens detected heterogeneity among 10 M. synoviae isolates. This variation was primarily seen in immunoblots as a difference in the molecular size of immunoreactive proteins below 50 kDa and corresponded to an area of variability seen in SDS-PAGE. No two isolates had the same immunoblot profile below 50 kDa. Hyperimmune antiserum to a M. synoviae F10-2AS protein of 41 kDa (p41) reacted with protein(s) ranging from 41 to 48 kDa in nine of 10 isolates and strongly with one of 87 kDa in one isolate. The immunoreactivity of antiserum from inoculated chickens and the hyperimmune antiserum to p41 varied from intense to weak with the 10 M. synoviae isolates. All of the highly immunogenic proteins of M. synoviae F10-2AS, including p41, partitioned into the Triton X-114 detergent phase, indicating that they are amphiphilic in nature and integral membrane proteins. The data suggest that M. synoviae is capable of varying the molecular size of a highly immunogenic integral membrane protein.

An Outbreak of Mycoplasma synoviae Infection in North Carolina Turkeys: Comparison of Isolates by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Restriction Endonuclease Analysis

Mycoplasma synoviae (MS) isolates made in 1988-89 from turkey flocks in North Carolina, Missouri, and Ontario, Canada, were compared with each other and MS reference strains (WVU-1853, F10-2AS, Neb-3S, and K1968) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell proteins and restriction endonuclease analysis (REA) of DNA. SDS-PAGE and REA indicated considerable homology among MS reference strains and recent field isolates. However, sufficient differences were resolved to identify the MS reference strains as different from each other and the field isolates, and to classify seven of nine recent field isolates as a cluster of nearly identical strains. The results suggest that flocks infected with members of the cluster were epizootiologically associated, possibly by a common or point source of infection.

Lyophilization of Mycoplasma synoviae Hemagglutination Antigen

Two hemagglutination (HA) antigens produced from Mycoplasma synoviae isolates WVU-1853m and FMT grown in Frey's mycoplasma broth were lyophilized for HA preservation. Some increase in the HA titer occurred following lyophilization.

Haemadsorption inhibition test for the identification ofMycoplasma gallisepticumandMycoplasma synoviae

Summary Colonies of the avian mycoplasma strains Mycoplasma gallisepticum S6 and Mycoplasma synoviae WVU 1853 and two Mycoplasma synoviae isolates from this laboratory were shown to be haemadsorption positive for chicken erythrocytes. Three Mycoplasma synoviae isolates from this laboratory proved to be haemadsorption and haemagglutination negative. The haemadsorption of the mycoplasma colonies mentioned above was inhibited with specific antisera of either high or low titre. No cross-inhibition was observed. It is suggested that this test could be used for a quick tentative identification of the two avian mycoplasmas on primary solid-medium cultures.

Studies on the Effect of Butylated Hydroxytoluene on Mycoplasma synoviae

Butylated hydroxytoluene (BHT) is one of the antioxidants added to human and animal feed products in concentrations ranging from 50 to 200 ppm to delay degradation of the labile lipid components. BHT is known to be an inactivator of mammalian and bacterial viruses that contain lipid.Results of in vitro studies showed that 10 ppm of BHT in Mycoplasma medium prevented growth of each of the six Mycoplasma synoviae (Ms) isolates. BHT added at 100, 200, and 400 ppm in the feed did not have any significant in vivo effect on the Ms serological responses or show any chemoprophylactic effect on birds infected with Ms.