Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+) MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates

Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+) MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.


Introduction
Mycoplasma synoviae can cause respiratory disease, infectious synovitis and eggshell apex abnormality in chickens and turkeys, thus the bacterium has economic importance in poultry industry. M. synoviae infection may occur from sub-clinical to severe forms, and the clinical signs change markedly when Mycoplasma infection is associated with other pathogens [1][2][3]. M. synoviae has a worldwide distribution and its occurrence is increasing. Less attention paid to control programs, biosecurity lapses at farms and large concentration of poultry in small geographic areas could accelerate the spread of M. synoviae infection [4]. M. synoviae may be transmitted either vertically through the eggs, or laterally by direct contact or indirect contact via the environment [1,5]. There are three general aspects in the control of M. synoviae infection: eradication and maintaining pathogen-free status by prevention, medication or vaccination [4].
In situations where maintaining flocks free of M. synoviae is not feasible (e.g. at a multi-age commercial layer farm) vaccination is a practical option for infection control. The commercially available live attenuated vaccine contains the temperature sensitive (ts + ) MS-H strain (Vaxsafe MS-H, Bioproperties Pty. Ltd. Australia), and it is used in many countries [6,7]. To perform control and eradication programs, molecular typing techniques have to be able to differentiate the ts + MS-H vaccine strain from field isolates. It is also important to examine whether the vaccine strain has successfully colonized the respiratory mucosa and thus produced an efficient immune response against wild-type strains. The sequence analysis of the vlhA gene was widely used to differentiate the MS-H vaccine strain from clinical isolates [8][9][10][11]. Unfortunately, it turned out that the vlhA gene sequence profile of the MS-H vaccine strain is not unique and several Australian and European field strains share the same vlhA gene sequence [9,12].
Shahid and co-workers [13] discovered two single nucleotide polymorphisms (SNP) on the obg gene sequence, namely the A-G substitution at nt367 and C-T substitution at nt629 which are able to differentiate the ts + MS-H vaccine strain, its rare non-temperature sensitive (ts -) MS-H re-isolates and wild-type M. synoviae isolates. They developed four PCRs followed by high-resolution melting (HRM)-curve analysis for the differentiation of strains based on these SNPs [7]. Although these PCR-HRM assays work well, we think they can be simplified and new assays can be designed for efficient use on basic laboratory equipment. Therefore in our study we provide melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to distinguish the ts + MS-H vaccine strain, ts -MS-H re-isolates and wild-type M. synoviae isolates based on the substitutions at nt367 and nt629 of the obg gene.

Methods
MAMAs are used for SNP typing in many different pathogens. The detailed description of the method is presented elsewhere [14]. In brief, MAMAs are based on allele-specific primers that are SNP specific at the 3'end. A single base mismatch at the ante-penultimate (-3) position of each allele-specific primer enhances the SNP discrimination capacity of these assays. One allele-specific primer possesses an additional 15-20bp GC-clamp at the 5'end with a sequence of CGGGG and the other allele primer has no additional sequence. The GC-clamp increases the melting temperature (T m ) of the resulting PCR product by 3-5°C, a shift that is detectable by fluorescent dye on a real-time PCR platform (Melt-MAMA) and it increases the size of the PCR product, resulting in a size difference which can also be detected by 3% agarose gel electrophoresis (Agarose-MAMA).
In the present study MAMAs were designed and tested to detect the nt367 and nt629 SNPs in the obg gene of M. synoviae. The genome locations, primer sequences, annealing and melting temperatures for these assays can be found in Tables 1 and 2. Melt-MAMA PCR reactions were performed in 10 μl total volume, containing 1μl target DNA diluted in 2 μl 5X Color-less GoTaq Flexi Buffer (Promega Inc., Madison, WI), 1 μl MgCl 2 (25mM), 0.3 μl dNTP (10 mM, Qiagen Inc., Valencia, CA), 0.5 μl EvaGreen (Biotium Inc., Hayward, CA), primers (10 pmol/ μl) according to Table 1 and 0.08 μl GoTaq DNA polymerase (5 U/μl; Promega). Melt-MAMAs were performed on an Applied Biosystems Step-One Plus real-time PCR system with StepOne Softwarev2.2.2. Thermocycling parameters were 95°C for 10 min, followed by 39 cycles of 95°C for 15 sec and 58°C for 1 min. Endpoint PCR products were subjected to melt analysis using a dissociation protocol comprising 95°C for 15 sec, followed by incremental temperature ramping (0.2°C) from 58°C to 95°C. EvaGreen fluorescent intensity was measured at 525 nm at each ramp interval and plotted against temperature.
In order to test the sensitivity of the assays, 10 fold dilutions of gBlocks (Integrated DNA Technologies Inc., Coralville, IA) containing 200 ng of a 330 bp long fragment of the obg gene (from nt342 to nt672) were used (Fig 1). The specificity of the assays was tested by including the following avian

Results
Both melt-MAMAs clearly differentiated the ts + MS-H vaccine strain genotype, ts -MS-H reisolate genotype and wild-type M. synoviae strain genotype ( Table 4). The MS-H1 assay (nt367 SNP position) distinguished ts + MS-H vaccine strain and ts -MS-H re-isolates from wild-type  M. synoviae isolates with 80.1°C and 75.0°C melting temperatures, respectively (Fig 2). The MS-H2 assay (nt629 SNP position) distinguished ts + MS-H vaccine strain and wild-type M. synoviae strains from ts -MS-H re-isolates with 76.8°C and 70.9°C melting temperatures, respectively (Fig 3). The agarose-MAMA versions of the MS-H1 and MS-H2 assays also differentiated the ts + MS-H vaccine strain genotype, ts -MS-H re-isolate genotype and wild-type M. synoviae strain genotype based on the 19-20bp fragment size differences of the PCR products obtained from certain genotypes (Table 4 and Fig 4).

Discussion
M. synoviae causes infectious synovitis, airsacculitis and eggshell apex abnormality in chickens and turkeys, which eventually result in significant economic losses. The ts + MS-H vaccine strain is used worldwide in order to reduce economic losses and to eliminate wild-type M. synoviae strains from poultry farms. The ability to differentiate the vaccine strain from wild-type isolates is essential for the evaluation of the effect of vaccination and eradication programs.  Micro-titrating, incubating and quantifying the isolated strains at two different temperatures in order to differentiate the ts + MS-H vaccine strain from the ts -MS-H re-isolates and wild-type strains is time-consuming [6,15]. The sequence analysis of the vlhA gene with different assays is a widely used method to differentiate M. synoviae isolates. However, this is also a relatively time-consuming and expensive method, but its biggest pitfall is that it is unable to distinguish several Australian and European wild-type M. synoviae isolates and ts -MS-H reisolates from the ts + MS-H vaccine strain [7,9,12].
Shahid and co-workers [7,13] discovered two specific SNPs on the obg gene and developed HRM-curve analysis based assays to differentiate the ts + MS-H vaccine strain, ts -MS-H re-isolates and wild-type M. synoviae strains. In the current study we presented novel genotyping assays targeting these two SNPs on the obg gene which clearly differentiate the ts + MS-H vaccine strain, ts -MS-H re-isolates and wild-type M. synoviae strains. Our assays are specific and sensitive enough (10 3 −10 4 copy numbers) to detect and describe M. synoviae strains, when wild or vaccine strains are present as single specific pathogen in the sample. We also think that our assays are preferable to the assays developed earlier for the following three reasons: they are rapid, they can be performed directly on clinical samples (e.g. swabs) and the assays can be run simultaneously at the same annealing temperature. They are also simple as they can be performed on basic real-time PCR machines (without the HRM-curve analysis function) and on conventional PCR equipment coupled with agarose gel electrophoresis. They are cost effective  as they do not require the expensive culture process, PCR product sequencing or costly reagents (e.g. TaqMan probes).
Unfortunately, besides the above listed advantages, the presented methods also have their pitfalls similarly to previous assays based on the obg gene. Namely, the MS-H 4 like genotype ts -MS-H re-isolates show the same obg gene profile as wild-type isolates, and the rare 94036-2-1a genotype ts + MS-H re-isolates show the same obg gene profile as the average ts -MS-H re-isolates. These re-isolates can only be ascertained by using vlhA gene based HRM assay in combination with obg gene based assays (Table 4) [7,9,13]. Another pitfall is that the genotyping of mixed infections (e.g. wild type strain superinfected vaccine strain) is unreliable because of the characteristics of the MAMA method (e.g. competing primers).
We hope that the presented assays will be helpful for both well-equipped laboratories and those with basic facilities throughout the world to differentiate the ts + MS-H vaccine strain, ts -MS-H re-isolates and wild-type M. synoviae strains and thus facilitate M. synoviae control programs.