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Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains

Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain.


Introduction
Infection by Mycoplasma synoviae occurs worldwide leading to respiratory diseases and synovitis in chickens and turkeys, and eggshell apex abnormality in chickens, and as a consequence to high economic losses in the poultry industry [1][2][3]. The severity of clinical signs can vary from sub-clinical to severe forms and it depends on the pathogenicity of the agent and on the presence of other bacteria or viruses (e.g. Escherichia coli or infectious bronchitis virus) [1][2]4]. M. synoviae can be transmitted horizontally, colonizing first the respiratory tract or vertically through the eggs [4][5][6]. To prevent the introduction of M. synoviae into a flock effective biosecurity programs should be applied and mycoplasma-free sources should be used for restocking. Medication can reduce the economic losses in the infected flocks and inhibit the transmission through the eggs [6]. Vaccination is another option to control the disease, especially in commercial layers [6][7]. At present, two commercial attenuated live vaccines are available against M. synoviae: the temperature sensitive (ts + ) MS-H vaccine strain (Vaxsafe1 MS, Bioproperties Pty Ltd.) which was developed by the chemical mutagenesis of an Australian field strain (86079/7NS); and the NAD independent MS1 vaccine strain (Nobilis1 MS Live, MSD Animal Health Inc.) which was obtained spontaneously during in vitro passages of the type strain (WVU 1853, ATCC 25204, NCTC 10124) [8][9]. Differentiation of vaccine and wild strains is crucial in the control programs. Several molecular techniques are available for the discrimination of the ts + MS-H strain, its tsre-isolates and field isolates, such as the sequence analysis of the vlhA gene or identification of specific point mutations in the obg gene by high resolution melting-curve (HRM) analysis or mismatch amplification mutation assays (MAMAs) [10][11][12]. The comparison of the whole genomes of the WVU 1853 parent strain and the MS1 vaccine strain revealed a single nucleotide polymorphism (SNP) in the histidine triad motif (HIT) family protein coding gene [9]. The present study provides melt-curve and agarose-gel based MAMAs specific for the SNP in the HIT family protein coding gene to discriminate MS1 vaccine and field strains. The assays were developed to be used simultaneously with the previously described MS-H1 and MS-H2 MAMAs [12], hence differentiating the MS1 vaccine, ts + MS-H vaccine, ts -MS-H vaccine re-isolate and field strains in one simple and cost-efficient step.

Methods Samples
The  (Table 1). For the validation of the assays, clinical samples including trachea swabs, trachea tissue samples and sample collection cards (Whatman FTA card, Whatman, Maidstone, UK) were collected from vaccinated (160 chickens) and clinically infected (seropositive) chickens and turkeys (total of 59 animals; Table 1). Ethical approval and specific permission were not required for the study as all samples and strains were collected by the authors during routine diagnostic examinations with the consent of the owners. Trachea swabs originated from live animals, tissue samples and FTA cards originated from animals found dead in the farms. The study did not involve endangered or protected animals. DNA was extracted from the strains, trachea swabs (in DNA pools, each containing 5 trachea swabs), tissue samples and FTA cards with the Qiamp DNA Mini kit (Qiagen GmbH, Hilden, Germany). The ts -MS-H re-isolate was identified by the MS-H1 and MS-H2 MAMAs, based on the point mutations at nt367 (G) and nt629 (C) in the obg gene [12].

Sequence analysis
The HIT family protein coding gene of the MS1 strain was amplified by conventional PCR using the primers MS-HL1 (5'-ACT GTA AAT GAC GCC TTT TCT AC-3') and MS-HL2 (5'-ACC GCT TAT GCA AGT AAA TTA TT-3'), designed in the present study. The reaction mixture contained 5 μl 5X Green GoTaq Flexi Buffer (Promega Inc., Madison, WI), 2.5 μl MgCl 2 (25mM, Promega), 0.5 μl dNTP (10 mM, Qiagen Inc., Valencia, CA), 1 μl of each primer (10 pmol/μl), 0.25 μl GoTaq DNA polymerase (5 U/μl; Promega) and 2 μl of target all samples were collected from the trachea of the animals; ch: chicken, t: turkey c farm, region and country of origin of the samples d DNA in a total volume of 25 μl. Thermocycling parameters consisted of 95˚C for 2 min, then 40 cycles of 95˚C for 30sec, 55˚C for 30 sec and 72˚C for 1 min and a final elongation step at 72˚C for 5 min. The amplicons were visualised in 1% agarose gel (Seakem Agarose, Lonza Group Ltd., Basel, Switzerland) under UV light. The amplicons were extracted from the gel using the QIAquick Gel Extraction kit (Qiagen) and submitted for Sanger sequencing on an ABI 3700 DNA Analyzer (Applied Biosystems, Foster City, CA). Sequence of the amplicon of the MS1 strain (660 bp) contains the complete sequence of the HIT family protein coding gene (321 bp) and fragments of the flanking regions (GenBank accession number: KY712765). Alignment of the sequences of the HIT family protein coding genes of the M. synoviae type strain (ATCC 25204, GenBank Accession Number: CP011096, region (complement): 195625-195945) and the MS1 strain (KY712765) revealed a point mutation at nucleotide (nt) 11 in accordance with the previous publication [9]. The described TCA (Ser) / TAA (stop codon) polymorphism results a stop codon in the MS1 vaccine strain (Fig 1).

Development of mismatch amplification mutation assays
MAMA is based on allele-specific competing primers and is used widely for SNP detection [15]. The allele-specific primers are SNP specific at the 3'end and a single base mismatch at the ante-penultimate (-3) position enhances the SNP discrimination capacity of the assay. A 15-20bp GC-clamp is added at the 5'end to one of the allele-specific primers which increases the melting temperature (T m ) and the size of the resulting PCR product. The difference between the T m and the product size is detectable by fluorescent dye on a real-time PCR platform (melt-MAMA) or by 3% agarose gel electrophoresis (agarose-MAMA), respectively.
In the current study melt-and agarose-MAMAs were developed to identify the point mutation at nt11 in the HIT family protein coding gene of M. synoviae (GenBank Accession Number: CP011096, region (complement): 195625-195945). Genome location, primer sequences, annealing and melting temperatures for the assays and PCR product sizes can be found in Table 2. The assays were optimised by altering primer ratios [15]. The MAMAs were designed to be used concurrently with the previously described MS-H1 and MS-H2 melt-and agarose-MAMAs [12].  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), 1 μl MgCl 2 (25mM), 0.3 μl dNTP (10 mM, Qiagen), 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 carried out on an Applied Biosystems Step-One Plus real-time PCR system with StepOne Software TM v2.2.2 and on Qiagen Rotor-Gene Q real-time PCR system, software version 2.3.1. Thermocycling parameters were 95˚C for 10 min, followed by 39 cycles of 95˚C for 15 sec and 58˚C for 1 min and 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.

Validation of mismatch amplification mutation assays
In order to test the intra-run repeatability at least four replicates of the two genotypes (wildtype and MS1 vaccine-type) were examined within the same run. For inter-run reproducibility test, the duplicate of at least ten samples from each genotype was examined in separate tests.
In order to test the sensitivity of the assays, 10 fold dilutions in replicates of four of the MS1 vaccine and M. synoviae type strain (WVU 1853, ATCC 25204, NCTC 10124) were used (Fig  2). Template copy number of the strains was calculated from the amount of DNA using the online tool developed by Staroscik [16]. The specificity of the assays was tested by including the following avian

Results
The developed melt-and agarose-MAMAs clearly identified the A-C polymorphism at the nt11 of the HIT family protein coding gene, distinguishing the wild, virulent M. synoviae strains and the MS-H vaccine strain from the MS1 vaccine strain, respectively. Due to the GC-clamp at the 5' end of the primer specific for the MS1 vaccine strain genotype, the melting temperature in the melt-MAMA for the MS1 vaccine genotype (T m : 79.1˚C, CI95: 78.9-79.3˚C on Step-One system and 79.3˚C, CI95: 79.2-79.5˚C on Rotor-Gene system) is higher by approximately 5˚C than for the wild-type or MS-H vaccine genotype (T m : 73.5˚C, CI95: 73.3-73.8˚C on Step-One system and 74˚C, CI95: 73.9-74.2˚C on Rotor-Gene system). For the same reason, the PCR product size in the agarose-MAMA for the MS1 vaccine genotype (83 bp) is longer by 16 bp than for the wild-type or MS-H vaccine genotype (67 bp) ( Table 2, Fig 2).
Consistent allele calls were observed by melt-MAMA until as low as 10 2 template number displaying a Ct range of 31-35. Negative controls or templates of other avian Mycoplasma species either were not amplified or generated non-specific products (with Ct above 37) with melt-profiles differing from the profiles of the expected two allelic states. Similarly, agarose-MAMAs worked reliable on samples containing 10 2 template number and neither of the tested other avian Mycoplasma species caused cross-reaction (no amplicons observed). The M. synoviae type strain (WVU 1835, ATCC 25204, NCTC 10124), the ts + MS-H vaccine strain (Vaxsafe1 MS-H), the ts -MS-H re-isolate, the 26 clinical M. synoviae isolates, the two tissue samples, the two FTA cards and the 11 DNA pools from the trachea swabs taken from clinically infected chickens and turkeys showed the wild-type strain profile while the MS1 vaccine strain (Nobilis1 MS Live) and the 32 DNA pools from the trachea swabs taken from vaccinated chickens showed the MS1 vaccine strain profile in the melt-and agarose-MAMAs ( Table 1). The results were confirmed by sequencing of the HIT family protein coding gene of these samples (data not shown).

Discussion
M. synoviae has great impact on the poultry industry and vaccination is a feasible and cost effective option to reduce economic losses. However, the differentiation of infected from vaccinated animals (DIVA) is crucial in the vaccination programs, animal trading and eradication programs.
The live, attenuated vaccine strains currently available against M. synoviae are the ts + MS-H vaccine (Vaxsafe1 MS-H) and the MS1 vaccine (Nobilis1 MS Live) strains. The MS-H vaccine strain was developed by chemical mutagenesis and the discriminatory phenotypic characteristic of this strain is its temperature sensitivity [8]. The MS1 vaccine strain has been commercially released recently, it was obtained by spontaneous mutation during in vitro passages and it does not require NAD for its growth [9]. The re-isolation of the vaccine strains and the examination of the phenotypic characteristics are time-consuming [17]. Genotyping based on the vlhA gene is used worldwide for the molecular differentiation of M. synoviae strains, but this method was proved to be unreliable in the discrimination of the MS-H vaccine strain [11,[18][19]. High resolution melt-curve analysis based assay was developed to identify specific SNPs in the obg gene in order to distinguish the ts + MS-H vaccine strain from its tsre-isolates and field strains [11]. In our previous study, the MS-H1 and MS-H2 MAMAs were designed to differentiate the SNPs in the obg gene to enable rapid, simple and cost efficient detection of the MS-H vaccine strain [12]. The purpose of the current study was to develop a DIVA-test for the recently released MS1 vaccine strain, which can be applied simultaneously with the assays used for the differentiation of the MS-H vaccine strain. Thus melt-and agarose-MAMAs were designed to identify the SNP at the nt11 in the HIT family protein coding gene with the same thermocycling profile as the MS-H1 and MS-H2 assays. The developed assay provides a reliable tool in routine diagnostics for the differentiation of the MS1 strain, as it is a sensitive and specific test and it can be performed directly on the clinical samples and in laboratories even with basic real-time PCR or conventional PCR equipment. The MS1 and MS-H vaccine strain specific MAMAs could efficiently support control programs against M. synoviae infections in one rapid, convenient and cost efficient step.