Isolation of M. synoviae field isolates
M. synoviae field isolates were obtained from registered commercial chicken farms, including breeder flocks, broiler flocks and layer flocks, in Thailand during 2020. Chickens were individually swabbed at articular joint or respiratory tract; choanal cleft, trachea and airsac; by using a sterilized cotton swab. Each swab samples were identified and inoculated into 2 ml of FMS broth, Frey’s broth medium supplemented with 15% swine serum 23 . Broth samples were then submitted to determine by PCR assays based on the 16S rRNA geneand the vlhA gene. The use of experimental animals was approved by Chulalongkorn University Animal Care and Use Committee (IACUC), protocol No.1931051. All methods were performed in accordance with the relevant guidelines and regulations. Good practice principles were respected to minimize the discomfort and provide the well-being to chickens.
M. synoviae vaccine, reference and field strains
Live M. synoviae vaccine; Vaxsafe MS® (Bioproperties, Australia); is MS-H strain, the temperature-sensitive strain developed in Australia since 1996 and introduced in Thailand since 2012. The reference strains of M. synoviae and M. gallisepticum, using as positive and negative controls in PCR assays, were M. synoviae WVU 1853 strain and M. gallisepticum S6 strain, respectively. Thai M. synoviae field isolates, identified as types C, E, and L 17 , were also included in this study. M. synoviae MS-H vaccine, M. gallisepticum S6, M. synoviae WVU 1853, and Thai M. synoviae field strains were cultured in 2 ml of FMS broths and subsequently analyzed by PCR assays. An appropriate risk assessment was approved by Institution Biosafety Committee (CU-VET-BC), protocol No. IBC1931023.
Culture method
FMS broth samples were first incubated at 37oC in a humidified chamber for 5-7 days until the broth color changed from pink-red to orange-yellow. Then, broth samples were divided into 2 portions. First portion was subjected to extract the DNA for M. synoviae specific PCR assay, 16S rRNA gene-based PCR assay. The remaining portion was immediately diluted for culture on FMS agar and incubated at 37oC in humidified condition before sampling a single colony of M. synoviae isolate. Consequently, five selected single M. synoviae colonies were passaged into fresh FMS broth and incubated at 37oC in humidified condition until the broth color changed from pink to orange-yellow. The FMS cultured broth, showing mycoplasma growth, was then equally divided into 3 portions. First portion was extracted the DNA for the 16S rRNA gene and the vlhA -based PCR assays. Second and third portion were stored at -80oC as frozen stock of each pure M. synoviae isolate for further study 15 .
DNA templates preparation
DNA was extracted from FMS broth samples using the modified rapid boiling DNA extraction 24 . Broth samples were centrifuged at 16,000x g for 6 min, washed two times with sterile phosphate-buffered saline (PBS), and resuspended in 50 µl of sterile PBS. Pack cell was boiled at 100oC for 10 min, placed on ice for 10 min, and centrifuged at 16,000x g for 6 min. Supernatant containing DNA template was collected and stored at -20oC until used. Concentration of DNA template was determined by using a NanoDrop™ Spectrophotometer.
In addition to DNA templates of M. gallisepticum S6 strain, M. synoviae WVU 1853 strain, M. synoviae MS-H vaccine isolate and Thai M. synoviae field isolates identified as types C, E, and L, the DNA template mixtures of Thai M. synoviae field strains and M. synoviae MS-H vaccine strain were also prepared for further molecular approaches.
M. synoviae-specific PCR assay
DNA templates were examined by the Lauerman 16S rRNA gene-based PCR assay 16 . PCR mixture 50 µl contained 5 µl of 5x Green GoTaq® Flexi Buffer (Promega, Madison, WI, USA), 2.5 µl of 1.25 mM MgCl2, 1 µl of 10 mM dNTP (Fermentas, Leon-Rot, Germany), 0.5 µl of each 10 µM primer MSL-1 (5'-GAA GCA AAA TAG TGA TAT CA-3') and primer MSL-2 (5'-GTC GTC TCC GAA GTT AAC AA-3') (Qiagen®, Valencia, CA, USA), 0.5 µl of 5 U/µl GoTaq® Flexi DNA Polymerase (Promega, Medison, WI, USA) and DNA template 5 µl. M. gallisepticum S6 strain and M. synoviae WVU 1853 strain were used as negative and positive controls, respectively. The PCR mixtures were amplified in a DNA thermal cycler (Life express, BIOER®, ROC) starting with 94oC for 5 min and 40 cycles of 94oC for 1 min, 55oC for 1 min and 72oC for 2 min and then followed by 72oC for 5 min at the final extension. The PCR products were analyzed using gel electrophoresis.
PCR amplification of partial vlhA gene
The vlhA gene fragment of M. synoviae positive samples were amplified by using the revised Hammond vlhA gene-targeted PCR assay 18 . The 50 µl PCR mixture contained 5 µl of 5x Colorless GoTaq® Flexi Buffer (Promega, Madison, WI, USA), 2.5 µl of 1.25 mM MgCl2, 1 µl of 10 mM dNTP (Fermentas, Leon-Rot, Germany) 0.5 µl of each 10 µM primer MSRH-1 (5'- GGC CAT TGC TCC TRC TGT TAT -3') and primer MSRH-2 (5'- AGT AAC CGA TCC GCT TAA TGC -3') (Qiagen®, Valencia, CA, USA), 0.5 µl of 5 U/µl GoTaq® Flexi DNA Polymerase (Promega, Madison, WI, USA) and DNA template 5 µl. M. gallisepticum S6 strain and M. synoviae WVU 1853 strain were used as negative and positive controls, respectively.
PCR mixtures were amplified in a DNA thermal cycler (Life express, BIOER®, ROC) starting with 95oC for 3 min and 40 cycles of 94oC for 1 min, 56oC for 1 min and 72oC for 1 min and then followed by 72oC for 5 min at the final extension. The PCR products were analyzed using gel electrophoresis.
Sequence analysis of partial vlhA gene
The vlhA gene PCR products from the revised Hammond vlhA gene-targeted PCR assay containing vlhA DNA fragment were purified and subjected to sequencing at A T G C Co. Ltd. (Thailand Science Park, Pathum Thani, Thailand). A similarity of nucleotide sequence was analyzed by using the BLAST program (www.ncbi.nlm.nih.gov/BLAST). Sequencing alignment analyses, corresponding to the N-terminal vlhA gene of M. synoviae K1968 strain classified as type B, were performed by using the molecular evolutionary genetic analysis (MEGA 6) software (http://www.megasoftware.net). M. synoviae isolates were typed based on the description of vlhA fragment. M. synoviae types A, B, C, D, E, F, G, H, I, J, K, and L were classified based on the length of PRR fragment of 38, 45, 32, 23, 19, 36, 51, 46, 28, 20, 12 and 35 amino acids, respectively 15,17,21,22,25 .
PCR-RFLP assay
The PCR-RFLP assay was developed by using the restriction enzyme map analysis tool on the Genescript webpage (https://www.genscript.com/tools/restriction-enzyme-map-analysis) to reveal the restriction enzyme TasI (ThermoFisher Scientific, San Jose, CA, USA), cuting best at 65°C in B buffer, as the suitable restriction enzyme which could digest vlhA gene-targeted PCR products of M. synoviae positive samples were consequently digested by restriction enzyme TasI (ThermoFisher Scientific, San Jose, CA, USA). Briefly, 20 µl of vlhA gene-based PCR product containing DNA at least 0.05 µg/ µl were added to the 23 µl of TasI mixture containing 4 µl of 10x Buffer B, 1 µl of TasI (10 U/ µl; ThermoFisher Scientific, San Jose, CA, USA) and 18 µl of distilled water. M. gallisepticum S6 strain and M. synoviae WVU 1853 strain were used as negative and positive controls, respectively. After incubation at 65°C for 2 hr, all digested PCR products were separated and analyzed using gel electrophoresis.
Gel electrophoresis
The PCR products resulted from PCR and PCR-RFLP assays were analyzed in 2% agarose gel (Vivantis Technologies, Malaysia), in a 1x TBE buffer at 100 volts for 35 min, pre-stained with MaestroSafeTM dye (Maestrogen, Las Vegas, NV, USA), visualized by UV transilluminator, and photographed. Each M. synoviae-specific PCR amplicon size was compared to standard 100bp DNA ladder (New England Biolab, UK).
Determination of antimicrobial susceptible profiles
A viable count of M. synoviae isolates; 10 field strains and WVU 1853 strain; in color changing unit (CCU) was obtained using the most probable number (MPN) determined using an MPN table 26,27 . Briefly, 20 μl of each M. synoviae isolate culture broth from frozen stocks was filled into each well of 1st column of the 96-well plate containing 180 μl of FMS broth and then serially 10-fold diluted from 1st until 11th column; with the 12th column containing only 200 μl of FMS broth. Each cultured plate was incubated at 37oC in humidified condition for 14 days before the number of wells in the last 3 columns showing color change from red to yellow were counted and estimated the CCU.
The frozen stocks were also used as inocula for evaluating the MIC of antimicrobials including enrofloxacin, oxytetracycline, doxycycline, tiamulin, tylosin, tilmicosin, tylvalosin and lincomycin-spectinomycin. The eight tested antimicrobials; registered and approved by the Food and Drug Administration, Ministry of Public Health, Thailand; were formulated and diluted in FMS broth. Antimicrobial susceptible profiles were determined by final MIC values using serial broth dilution method 28 . Briefly, duplicate wells of antimicrobials were two-folded, serially diluted in a 100 µl of FMS broth in the sterile 96-well, flat-bottomed microtitration plates. The 100 µl of FMS broth containing M. synoviae organisms approximately 105 CCU/ml was added to each well at 1st until 11th column containing same amount of antimicrobial at final concentrations 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.0781, 0.0391, 0.0195 and 0.0098 µg/ml. Positive control consisting only M. synoviae cultured broth was also included at the 12th column in each plate. The MIC values were recorded daily after the positive control broth color changing and final MIC values were assessed at 14 days after incubation. The lowest concentration of each antimicrobial that completely prevented the broth change of color from pink to orange-yellow was considered as MIC.
Statistical analysis
Comparison of MIC values between Thai M. synoviae field isolates, which were classified as types E and L or originated from choanal cleft and joint, was analyzed by using independent samples T-test. Statistical analyses were performed by using IBM SPSS Statistics 22 software for windows and differences were considered as significant at P < 0.05.