Development and evaluation of semi-nested PCR for detection of the variable lipoprotein haemagglutinin (vlhA) gene of Mycoplasma Synoviae in chicken
Article information
Abstract
This study aimed to develop a semi-nested polymerase chain reaction assay for the direct detection of Mycoplasma synoviae (M. synoviae) from clinical samples using three newly designed oligonucleotide primers specific to the variable lipoprotein haemagglutinin (vlhA) gene and differentiate M. synoviae field strains based on a nucleotide deletion or the insertion of the proline-rich repeat (PRR) region of the vlhA gene. The developed semi-nested polymerase chain reaction (PCR) assay revealed positive results in 12 out of 100 clinical samples collected from chickens showing lameness and joint swelling. Six positive samples were selected randomly for sequencing, and sequence analysis revealed 96.3-100% nucleotide identities compared to the reference sequences. Phylogenetic analysis showed that sequences of the strains in this study were closely related to WVU1853 (Spain), CK.MS.UDL.PK.2014.2 (Pakistan), and F10-2AS (USA) strains, but they were distinct from the M. synoviae-H vaccine strain sequence. M. synoviae obtained from these samples were identified as types A and C with a length of 38 and 32 amino acids, respectively. These results indicated that the specific and sensitive semi-nested PCR could be a useful diagnostic tool for the direct identification of clinical samples, and the sequence analysis of the partial vlhA gene can be useful for typing M. Synoviae.
Introduction
Mycoplasma synoviae (M. Synoviae) is one of the most common Mycoplasma species causing infections in avian species worldwide [1]. M. synoviae causes a range of illnesses in infected birds, including chronic subclinical upper respiratory diseases, airsacculitis, eggshell apex abnormalities (EAA), and infectious synovitis and tenosynovitis [1,2]. These clinical signs adversely affect product performance and egg production and result in increased condemnation. Therefore, M. synoviae infections have caused numerous economic losses in the poultry industry throughout the world [3,4].
M. Synoviae infections can be transmitted vertically via eggs or horizontally via direct contact. Early detection of new infections and routine biosecurity monitoring is recommended for the elimination of M. synoviae transmission [4]. On the other hand, it is difficult to diagnose M. synoviae infections differentially from avian reovirus, Staphylococcus aureus, Mycoplasma gallisepticum (M. Gallisepticum), Escherichia coli, and Pasteurella multocida infections, based on the clinical signs [5]. Currently, the culture method is the gold standard for the identification of M. synoviae infection, but this method is laborious and time-consuming and can take up to 3-4 weeks to complete. The serological diagnosis of M. synoviae infection has several problems, including nonspecific reaction, cross-reactions with inactivated M. gallisepticum vaccination, and low sensitivity during the acute phase of infection [6,7]. A polymerase chain reaction (PCR) based on the 16S rRNA gene was developed for the rapid detection of M. synoviae infected flocks [8,9]. Moreover, other PCR-based assays, such as digitalized random amplified polymorphic DNA analysis and amplified fragment length polymorphism analysis, have also been used to genotype M. Synoviae [10]. With advantages over culture and serological methods, high sensitivity PCR assays, such as real-time PCR and nested PCR assays, have been used increasingly for the rapid, specific and sensitive detection, and identification of M. synoviae infections from clinical samples [3,4,11,12].
Although PCR assay targeting the 16S rRNA gene is now used routinely and extensively for the detection of avian Mycoplasma spp. infected flocks, the conserved nature of the 16S rRNA gene makes it unsuitable for M. synoviae strain differentiation by sequencing. Molecular detection and characterization of the variable lipoprotein hemagglutinin A (vlhA) gene has been used successfully for M. synoviae strain differentiation without the need for prior culture or isolation [13-15]. The vlhA gene of M. synoviae is cleaved post-translationally into an N-terminal lipoprotein (MSPB), which exhibits a high degree of antigenic variation [16]. M. synoviae strain typing based on nucleotide deletion or insertion within the proline-rich repeat (PRR) region of the vlhA gene is related to the invasiveness of M. synoviae associated with infectious synovitis [13]. This study aimed to develop a semi-nested PCR assay based on the vlhA gene for increased sensitivity of M. synoviae detection from clinical samples as well as to characterize the PRR region of the vlhA gene of M. synoviae for strain differentiation.
Materials and Methods
Positive and negative control DNA
M. synoviae DNA was kindly provided by the National Institute of Animal Health, Thailand, and the M. synoviae-H vaccine strain DNA positive control was derived from the modified-live vaccine Vanguard® plus 5/CV-L (Zoetis, A). The other pathogens, including the Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), and M. Gallisepticum, were local isolates and were proven by sequencing.
DNA preparation
The chromosomal DNA of M. synoviae-H and M. Gallisepticum vaccine strains, and DNA from clinical specimens were extracted using a previously published method [17] with some modifications. Briefly, DNA was extracted by placing swabs into 1 mL of PBS and twisting the swab vigorously. The suspension was then centrifuged at 13,000 rpm for 10 min. The pelleted cells were finally suspended in 50 µL of nuclease-free water to reduce the nuclease activity. The sample tubes were placed in a heating block at 100°C for 10 min to release DNA from the cells and were then refrigerated at -20°C for 10 min. After the cell lysate was centrifuged at 13,000 rpm for 10 min, the supernatant was collected and subjected to PCR. The viral nucleic acids of NDV, ILTV, and IBV were extracted using the Viral Nucleic Acid Extraction Kit (Invitrogen, USA) according to the manufacturer’s manual. The extracted nucleic acids were stored at - 20°C until used. The concentration of M. synoviae DNA was measured using a spectrophotometer (BioDrop, UK) at the A260/A280 ratio, and the DNA was serially diluted ten-fold from 0.7 ng/µL for the semi-nested PCR assay sensitivity detection.
Primers selection and semi-nested PCR reactions
Three primers targeting the highly conserved region of the M. synoviae vlhA gene were newly designed based on an alignment of the vlhA genes available in the GenBank database. Based on the nucleotide sequence of the M. synoviae strain T68W/IT1A GenBank accession numbers GU451303.1, the forward primer position was at 288nt to 308nt, while an outer reverse primer and an inner reverse primer positions were at 988nt to 1009nt and 550nt to 571nt, respectively. The nucleotide sequences of the primers were as follows: MS-SCF1 (5'-GGT GAT CAA ACT CCA GCA CCT-3') used as the forward outer primer of the first-round PCR and a forward inner primer of the second-round PCR. MS-SCR2 (5'-TCC ACT TGC ATT TTT AGA CCA T-3') and MS-SCR1 (5'-GTT ACC GAT CCG CTT AAT GCT T-3') were used as a reverse outer primer of the first-round PCR and a reverse inner primer of the second-round PCR, respectively. The sizes of the PCR products of the first- and second-round PCRs were at 724-base pairs (bp) and 283-bp, respectively.
Optimization of the semi-nested PCR
All semi-nested PCR reactions were performed in a total volume of 25 µL. The first-round PCR mixtures were composed of 12.5 µL of the 2x DreamTaq Green PCR Master Mix (Thermo Fisher Scientific, USA), 0.5 µL (25 µM) of MS-SCF1 and MS-SCR2 primer, 3 µL of DNA template, and 8.5 µL of RNase free water. The amplification steps were performed in a DNA thermal cycler (Major Science, USA) with initial heating at 94°C for 5 min and 35 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and polymerization at 72°C for 45 s, followed by a final elongation step of 10 min at 72°C. The second PCR reactions (nested-PCR) were composed of 12.5 µL of the 2x DreamTaq Green PCR Master Mix, 0.5 µL (25 µM) of MS-SCF1 and MS-SCR1, 3 µL from the first amplicons and 10.5 µL of RNase free water. The PCR amplification steps were conducted using the same conditions as described for the first-round PCR. Electrophoresis was performed to analyze the PCR products using 1.2% agarose gels containing 1X of GelRedTM Nucleic Acid Gel Stain (Biotium, USA). The gels were run at 100 volts for 30 min before visualization under ultraviolet light in the Gel Doc™ XR+ Gel Documentation System (Bio-Rad, USA).
Specificity test
The specificity of the primers developed in this study was tested against other pathogens that can cause respiratory diseases and infectious synovitis in chickens similar to M. Synoviae, including the Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), infectious bronchitis virus (IBV), and M. gallisepticum
Sensitivity test
A 0.7 ng/µL sample of purified M. synoviae DNA was serially diluted ten-fold for sensitivity testing, and the detection limits between the present semi-nested PCR and conventional PCR were compared [14]. The minimum concentrations showing positive results in both tests were noted.
Detection of M. synoviae DNA from lameness chickens
From June to August 2016, choanal and tracheal swabs were collected from 100 lame chickens raised in six commercial farms located in the eastern and western regions of Thailand. These chickens had joint problems, such as infectious synovitis and tenosynovitis. Their ages ranged from 36 to 42 weeks and 40 days for broiler breeders and broilers, respectively. The swabs were placed in phosphate-buffered saline (PBS) and kept at -80°C until used. The swab samples were submitted for DNA detection by a semi-nested PCR assay, as described above, and the presence of M. synoviae DNA was recorded.
Detection of anti- M. synoviae antibody from lameness chickens
All 100 serum samples collected from the lame chickens described above were examined for antibodies against M. synoviae using a commercial test kit, ProFLOK® (Synbiotics Corporation, USA) according to the manufacturer’s recommendation. The diluted sera were added to M. synoviae antigen-coated plates. The optical density was read using an ELISA reader at 405 nm and was calculated and interpreted according to the manufacturer’s recommendation. Antibody titer of 0-269, 270-743, and higher than or equal to 744 were considered negative, suspect, and positive results, respectively.
Sequence analysis of partial vlhA gene of M synoviae
Positive PCR samples were purified using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany), and the purified DNA was subjected to DNA sequencing (First BASE Laboratories Sdn Bhd, Malaysia). DNA sequences of the PCR products were analyzed for sequence identity and similarity compared to positive M. synoviae and M. SynoviaeH. The BLAST program (www.ncbi.nlm.nih.gov/BLAST) and the BioEdit Sequence Alignment Editor Version 7.0.9.0 software were used for nucleotide sequence similarity analysis.
Phylogenetic tree analysis
The partial vlhA gene sequences identified were then analyzed by comparing with the sequences of other M. synoviae strains obtained from the GenBank database. The nucleotide (nt) and amino acid sequence alignment and relationship analysis were performed using MEGA6 software (http://www.megasoftware.net). Phylogenetic tree and distance analyses of the nucleotide sequences were constructed using MEGA6 software version 6.06 by Neighbor-joining with the maximum likelihood method based on the Kimura 2-parameter model with the neighbor-joining (NJ) method and 1,000 replications of bootstrap values [18]. Table 1 and Table 2 list the M. synoviae strains in this study and the reference M. synoviae strains, respectively.
Historic details of the M. synoviae isolates used for sequence comparison and phylogenetic analysis
Reference M. synoviae strains used for sequence comparison and phylogenetic analysis
Results
The expected PCR product sizes were 724 bp and 283 bp for the first and second rounds of semi-nested PCR, respectively. The primers designed in this study were based on the vlhA gene of M. synoviae accession number GU451303.1. The specificity of semi-nested PCR was tested against other pathogen species that commonly cause respiratory diseases in chickens. The results revealed the nonspecific bands of other pathogens DNA (Fig. 1). The semi-nested PCR sensitivity test results were 10-5, 10-5, and 10-9 for the conventional PCR, outer primers, and inner primers, respectively (Fig. 2).
Specificity determination: A total of 4 common respiratory pathogens were tested. M, molecular weight ladder; 1-2, infectious bronchitis virus; 3-4, infectious laryngotracheitis; 5-6, Newcastle disease virus; 7-8, M. gallisepticum; 9-10, negative control; and 11-12, positive control.
Sensitivity detection of the conventional PCR (A), semi-nested PCR procedure using outer primers (B) and inner primers (C) detecting 10-fold dilution of the extracted genomic DNA of M. synoviae. M, molecular weight ladder; 1-9, 10-1-10-9 dilution of M. synoviae positive stock, respectively; NC, negative control; PCR, polymerase chain reaction.
Detection of M. Synoviae from clinical samples
One hundred choanal cleft swabs were detected for M. synoviae DNA using the semi-nested PCR. M. synoviae DNA was observed in 12 out of 100 swab samples (12%) tested, which gave 283 bp of M. synoviae specific bands by semi-nested PCR (Fig. 3).
Polymerase chain reaction products from choanal and tracheal swabs for detection of M. synoviae in clinical samples, the sizes of 724 bp with the primers MS-SCF1&MS-SCR2 (outer primer) and 283 bp with the primers MS-SCF1&MSSCR1 (inner primer). M, molecular weight ladder; 1-12, DNA of clinical samples; NC, negative control; PC, positive control.
Determination of M. synoviae-antibody
Six out of 100 serum samples had antibodies against M. synoviae and were positive for M. synoviae DNA by semi-nested PCR.
Sequence analysis of M. synoviae vlhA gene
The partial vlhA fragments of six randomly selected positive PCR samples were sequenced, and all six nucleotide sequences were submitted to GenBank (National Center for Biotechnology Information, Bethesda, MD). The GenBank accession numbers were KY930895, KY930896, KY930897, KY930898, KY930899, KY930900 for KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, KKUMSP49, and KKUMSTF strain, respectively. Sequence analysis showed that these six strains had 98.9 to 100% nucleotide sequence homology. The sequences of five strains, designated KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49, had 89.5 to 100% homology with the reference strains and had complete identity with the WVU1853, F10-2AS and CK. MS.UDL.PK.2014.2 strains. The KKUMSTF strain of this study showed 90.7 to 98.9% homology with the sequences of the reference strains (Table 3).
Sequence homology of nucleotides and amino acids of vlhA gene in this study and reference strains
A partial vlhA sequence alignment of 19 different strains revealed several nucleotide substitutions among these strains. The nucleotide sequence position 177 of five strains in this study (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49) was A (adenine) nt similar to the WVU1853, K1968, F10-2AS, and CK.MS.UDL.PK.2014.2 strains. In contrast, the other strains, including KKUMSTF, had G nt in this position. The nucleotide sequence position 225 of five strains (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49) was C (cytosine) nt, which was similar to the M. synoviae-H, F10-2AS, AHRU2014CU5801.2, and CK.MS.UDL.PK.2014.2 strains, whereas the KKUMSTF, K1968, FMT, AHRU2015CU2807.1, CHN-QZ114-1-2013, JBSJ_Br, EAA, and CBU080258 strains had T (thymine) nt. The nucleotide sequence position 238 of the KKUMSTF strain was G, which was similar to the reference strains, K1968, AHRU2015CU2807.1, CHN-QZ114-1-2013, JBSJ_Br, and CBU080258. The other reference strains and five strains of this study (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49) were T nt. Furthermore, the nucleotide sequence positions 308 and 336 of all six strains of this study were A and C similar to the WVU1853 and F10-2AS strains, while the other reference strains were C and A nt, respectively (Fig. 4).
Comparison of the deduced amino acid sequences of the 19 MS strains. The comparison was performed by the BioEdit software and the results are displayed graphic view. Symbols that are identical to the consensus are displayed in upper case. In comparison with the published sequence of the K1968 strain had an insertion of seven amino acids in the PRR (DNPQNPN) whereas the 5 strains (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3 and KKUMSP49) are the WVU1853 strain. The KKUMSTF strain revealed deletions of 13 amino acids in the PRR region comparing with K1968 strain.
The deduced amino acid sequence analysis revealed 98.9 to 100% homology for all strains in this study, which was similar to the nucleotide sequence analysis. These six strains showed 93.9 to 100% sequence homology compared to the reference strains. The amino acid sequences of the five strains in this study (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49) showed 100% identity with the reference WVU1853 and CK.MS.UDL.PK.2014.2 strains (Table 3).
Based on the vlhA sequence analyses, the six M. synoviae field strains were identified as PRR type group A (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49) and C (KKUMSTF), with types A and C containing 38 and 32 amino acids, respectively (Fig. 4).
Phylogenetic analysis of the deduced amino acid sequences of the six strains and 13 reference strains showed that the present six strains belonged to two groups (Fig. 5). The five strains (KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49) were allocated to the same group as the F10-2AS, CK.MS.UDL.PK.2014.2 and WVU1853 strains.
Phylogenetic tree constructed by neighbor-joining method based on the partial sequence of vlhA gene of M. synoviae. The sequences were obtained from the 6 samples in this study (●), and 13 sequences from GenBank. Values at nodes indicate bootstrap probabilities, as determined for 1,000 re-samplings.
Discussion
Although conventional PCR has been used increasingly for M. synoviae detection over the past decade [14], few studies on nested PCR have been reported. Therefore, this semi-nested PCR assay may provide an alternative, sensitive, and useful diagnostic tool for M. synoviae detection. Semi-nested PCR was approximately 10,000 times more sensitive than conventional PCRs with the previously reported primers [14]. Therefore, semi-nested PCR enhances the sensitivity of M. synoviae determination. In addition, it was specific to M. synoviae detection, indicating that the primers were particular to M. synoviae nucleic acid. The DNA extracted from field samples collected from chickens showing clinical signs associated with M. synoviae infections was tested directly by the semi-nested PCR, and positive results were observed.
Moreover, the infection rate, according to the ELISA results, revealed lower sensitivity than the direct detection of swab samples using semi-nested PCR. On the other hand, M. synoviae infection diagnosis and monitoring should use ELISA for disease confirmation and screening the status of chicken flocks. In addition, partial vlhA gene amplification is useful for M. synoviae strain identification [19]. The primers specific to the vlhA gene had been designed [13,14,20], in which the resulting PCR products could be analyzed further by sequencing, and the genotype of M. Synoviae could be identified.
Several reports on molecular and evolutionary analysis of M. synoviae strains based on the vlhA gene have been published [2,13,15,21-23,28]. Sequence alignment and phylogenetic analysis based on the partial vlhA gene of this study demonstrated that the field strains were different from other M. synoviae strains and the live commercial vaccine strain that has been reported earlier [21,22,24,25]. Phylogenetic analysis indicated that the KKUMSB1, KKUMSB7, KKUMSB9, KKUMSP3, and KKUMSP49 were closely related to the WVU1853, CK.MS.UDL.PK.2014.2, and F10-2AS strains isolated from Spain, Pakistan, and the USA, respectively. Based on vlhA gene sequencing and analysis, Ogino et al. [22] identified the vaccine strain MS-H and field strains rapidly without any cultures or isolation methods. Similar to previous studies [14,15,22], the sequence analysis results of this study could differentiate the field strains from the M. synoviae-H strain based on the vlhA gene, without prior cultures. In addition, the six strains obtained from lame chickens in this study displayed a high identity of their nucleotide and amino acid sequences. Sequencing analysis results revealed sequence homologies indicating that these strains were closely related to the WVU1853, F10-2AS, and CK.MS.UDL.PK.2014.2 strains.
Currently, eleven types of M. synoviae, type A-K, have been classified based on the insertion or deletion of the PRR region [13,14,23,26]. In Thailand, previous studies on M. synoviae differentiation based on partial vlhA gene sequence analysis reported at least three types, including types C, E, and L. Type C and E were obtained from chickens with respiratory signs and type L were obtained from chickens showing clinical lameness and joint swelling without respiratory signs [7]. The current M. synoviae field strains could be classified as types A and C similar to the reference WVU1853 (type A) and MS-H (type C) strains [14]. These strains were detected and identified from choanal cleft swabs collected from chickens showing clinical joint swelling without respiratory signs, indicating that the developed semi-nested PCR was useful for identifying and screening M. synoviae infections in commercial chicken farms. Although the KKUMSTF strain was classified as type C (32 AAs) similar to the MS-H strain, the sequences of the deduced amino acids were a different deletion and insertion compared to the reference strain, K1968. This result indicated that KKUMSTF could be classified further into subtypes [13,18]. In addition, this type was associated with infectious synovitis, with the chickens showing clinical lameness and joint swelling without respiratory signs similar to the previous report of Thai M. synoviae isolates type L [27]. The six clinical samples found to be PCR-positive but did not present the antibody reactor detected by ELISA may indicate an early MS infection. This finding is similar to a previous study showing that the PCR procedure could first detect the MS DNA, but did not reveal the antibody reactor detected by either the SPA test or ELISA or show any clinical signs [29].
In conclusion, the specific and sensitive semi-nested PCR was developed based on the vlhA gene for M. Synoviae detection and identification. This PCR enables the rapid detection of M. Synoviae and molecular characterization of M. synoviae directly from clinical samples that may be useful for a further study of M. synoviae evolution and differentiation.
Notes
The authors declare no conflict of interest.
Acknowledgements
This study was supported by the Research Fund of the Faculty of Veterinary Medicine, Khon Kaen University, Thailand and Research Group for Animal Health Technology, Faculty of Veterinary Medicine, Khon Kaen University, Khon Kaen, Thailand. The author wishes to thank Dr. Patcharee Thongkamkoon and Zoetis (Thailand) Limited for supporting this research.