Detection Of Mycoplasma Synoviae Research Articles

Development and Validation of Duplex PCR Assay for Simultaneous Detection and Differentiation of Mycoplasma gallisepticum and Mycoplasma synoviae in Poultry

Background: Mycoplasmosis caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically significant disease in both commercial and backyard poultry. The present work aimed at developing a duplex PCR assay for quick, accurate detection and differentiation of MG and MS and validating it for screening purpose. Methods: Duplex PCR assay was standardized with primers designed specific to the mgc2 gene for MG and hemagglutinin vlhA gene for MS with amplicon size of 150 bp and 787 bp, respectively. Validation of molecular detection of MG and MS with duplex PCR assay was performed with a total of 179 pooled oropharyngeal swab samples from backyard poultry flocks from different parts of India. Result: The duplex PCR assay showed, 133/179 (74.3%), 39/179 (21.78%) and 35/179 (19.55%) positivity for MG, MS and both MG and MS, respectively. Duplex PCR assay showed sensitivity of detection limit of 10-6 of 1 mg/ml of template DNA. Comparison of results of duplex PCR assay with single PCR of field samples showed perfect agreement between two assays (k= 0.90). The duplex PCR validated in the study would be useful in rapid screening/ surveillance, diagnosis and differentiation of MG and MS in field samples in cost-effective manner and to devise effective control strategies.

Comparative Study of Various Diagnostic Methods for Detection of Mycoplasma gallisepticum and Mycoplasma synoviae in Egyptian Chicken Flocks

The significance of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) in the poultry industry underscores the critical importance of diagnosing avian mycoplasmosis within the field of veterinary medicine. The present study aimed to compare various diagnostic methods for detecting MG and MS in Egyptian Chicken Flocks. A total of 360 samples were collected from breeder, layer, and broiler chickens from four governorates in Egypt. Conventional isolation methods and polymerase chain reaction (PCR) were used for the direct detection of MG and MS, while serum plate agglutination test (SPA) and Enzyme-linked immunosorbent assay (ELISA) were used for detecting antibodies against MG and MS. The highest detection rate of MG was found in commercial layers, followed by breeders, and broilers. Regarding MS, the highest detection rate was found in breeders, followed by commercial layers, and broilers. By comparing the used diagnostic methods, MG and MS were determined by the SPA test (40% and 31.1% respectively), ELISA test (31.7% and 23.6%), PCR (16.7% and 11.7%), and by the conventional culture method (10.8% and 3.9%). It could be concluded that the serological methods and PCR gave better sensitivity than culture methods and can be used in the diagnosis of avian mycoplasmosis.

Rapid and sensitive detection of Mycoplasma synoviae using RPA combined with Pyrococcus furiosus Argonaute

Mycoplasma synoviae (MS) is an important pathogen in laying hens and causes serious economic losses in poultry production. Rapid, accurate and specific detection is important for the prevention and control of MS. Argonaute from Pyrococcus furiosus (PfAgo) is emerging as a nucleic acid detector that works via "dual-step" sequence-specific cleavage. In this study, an MS detection method combining recombinase polymerase amplification (RPA) and PfAgo was established. Through elaborate design and screening of RPA primers and PfAgo gDNA and condition optimization, amplification and detection procedures can be completed within 40 min, whereas the results were superficially interpreted under UV and blue light. The sensitivity for MS detection was 2 copies/µL, and the specificity results showed no cross reaction with other pathogens. For the detection of 31 clinical samples, the results of this method and qPCR were completely consistent. This method provides a reliable and convenient method for the on-site detection of MS that is easy to operate without complex instruments and equipment.

Monitoring reveals a high prevalence of Mycoplasma synoviae in layer flocks in Croatia

Mycoplasma synoviae (MS), despite its lower pathogenicity, has the ability to cause significant losses in poultry production but is usually underdiagnosed. In egg layer production, losses could be significant because of the drop in egg production and poor eggshell quality. Problems with colibacillosis and other infections secondary to MS have been continuously reported on Croatian egg layer farms. As a result, regular monitoring of flocks also included screening of the seroprevalence and molecular detection of MS using ELISA and qPCR tests, respectively. During monitoring, altogether 1135 samples were tested, including 1067 serum samples and 68 tracheal swabs, in a total of 126 flocks and 83 longitudinally merged flocks on 15 farms during the period from 2017 to 2021. The results showed a high general prevalence of MS with 86.6 % positive layer farms, while grouped flock seroprevalence and prevalence were 98.6% and 85.7%, respectively. With age, seroprevalence and ELISA titers rise significantly compared to the rearing period, with a significant mutual correlation over the entire production period. Additionally, there is a significant correlation between ELISA titers and age in weeks. Several flocks covered longitudinally from the first week of age, over the rearing period to the end of production, showed low prevalence during the rearing period, with a later significant rise in titer and prevalence, which indicates the dominance of horizontal transmission during production. Overall results indicate the need for a prompt reaction regarding preventive measures, such as better flock management, biosecurity and vaccination, which would reduce the losses and improve production.

Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/μL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

Rapid and specific detection of Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry using single and duplex PCR assays

Avian mycoplasmosis, mainly caused by Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS), is an economically important disease of poultry industry. The present study was aimed to develop duplex PCR as a rapid, specific and economical method for accurate detection of MG and MS in poultry and its comparison with single (monoplex) MG/MS PCR. During present investigation, a total of 146 poultry flocks having clinical history of respiratory disease were screened. Pooled tissue samples (trachea, lungs and air sacs) from 4-5 birds of each flock were collected during necropsy at disease investigation laboratories, Hisar, Haryana, India. The single and duplex PCR assays were standardized using primers of intergenic spacer region (IGSR; 16S-23S rRNA) for MG and hemagglutinin vlhA gene for MS, with expected amplicon size of 812 bp and 1200 bp products, respectively. In single PCR, 6.85%, 2.74% and 2.74% tissue samples were found positive for MG, MS and both MG and MS, respectively. However, duplex PCR showed, 7.53%, 2.74% and 1.37% positivity for MG, MS and both MG and MS, respectively. Taking the results of monoplex PCR as a gold standard, sensitivity and specificity of the developed duplex PCR was found to be 94.44% and 100%, respectively. Moreover, Cohen's kappa statistic (k = 0.97) measured a ‘perfect’ agreement between monoplex and duplex PCR assays. The positive and negative predictive values of duplex PCR was found to be 1.0 and 0.9922, respectively at 95% confidence interval (CI), as compared to monoplex PCR. The simultaneous use of two genes in a duplex PCR was more rapid and economical than two separate single PCR reactions.

Reflected Light Spectrometry and AI-Based Data Analysis for Detection of Rapid Chicken Eggshell Change Caused by Mycoplasma Synoviae

Mycoplasma synoviae (MS) is a pathogen that causes economic losses in the poultry industry. It can be transmitted, amongst others, via the respiratory tract and spread relatively quickly. As such, MS infections are mainly controlled by maintaining MS-free breeder flocks. Routine diagnosis for the detection of MS may be based on serological, culture, and molecular tests. Here, we propose an optical solution where AI-based analysis of spectral data obtained from the light reflected from the eggshells is used to determine whether they originate from healthy or Mycoplasma synoviae-infected hens. The wavelengths proposed for spectral MS detection are limited to those of VIS and NIR DPSS lasers, which are freely accessible on market. The results are satisfactory: for white eggshells, the F-score is over 95% for five different combinations of wavelengths (using eight or nine wavelengths); for brown eggshells, the F-score is above 85%, also for five different combinations of 6–9 wavelengths.

Detection of Mycoplasma Synoviae and Other Pathogens in Laying Hens with Respiratory Signs in the Rearing and Production Phases

The present study aimed to investigate the occurrence of Mycoplasma synoviae (MS), M. gallisepticum (MG), Ornitobacterium rhinotracheale (OR), Avibacterium paragallinarum (AP), Pasteurella multocida (PM) and Infectious Bronchitis Virus (IBV) in laying hens with respiratory clinical signs in two phases of production. 140 tracheal swabs and 140 blood samples were collected from laying hens in the rearing and production phases, the chickens belonged to six farms (A-F) located in the state of São Paulo, Brazil. The samples were analyzed by PCR for MG, MS, OR, AP, PM and IBV and by ELISA for MG and MS. The highest frequencies observed by PCR were for MS at farms B and C with 95 and 100% positivity, followed by MG at farms D and E with 35% and 65%, IBV with 35% at farm F and ORT with 15% at farm A. All flocks were positive for MG and MS in serology. Although MG and IBV have been detected, this can be explained by the vaccination protocols, since live attenuated vaccines are widely used for immunization against these pathogens. It was also possible to detect OR and AP thorugh PCR in some flocks. The occurrence of several etiological agents that cause respiratory diseases in laying hens was confirmed by PCR and serology, with MS being the most prevalent and being present in all farms studied.

A simplified duplex real-time PCR incorporating TaqMan minor groove binder (MGB) probes and an exogenous internal positive control for the simultaneous detection of Mycoplasma gallisepticum and Mycoplasma synoviae cultures

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most pathogenic and economically important mycoplasma pathogens that infect chickens. The development of rapid and innovative molecular diagnostic techniques is of pivotal importance for their effective control. The aim of the present study was to develop a novel duplex TaqMan real-time PCR assay for the simultaneous detection of MG and MS. This duplex real-time PCR assay incorporates TaqMan (FAM/NED) labelled minor groove binder (MGB) probes that target the cytadhesin encoding surface protein (mgc2) gene and the haemagglutinin surface protein (vlhA) gene of MG and MS, respectively. The assay also contained a TaqMan exogenous internal positive control (Exo IPC), to avoid false negative results that might happen due to failure in DNA extraction/PCR inhibition. The TaqMan MGB probe-based duplex RT-PCR incorporating Exo IPC was then applied to DNA from culture isolates for the simultaneous detection of MG (mgc2 gene) and MS (vlhA gene). For duplex RT-PCR the sensitivity recorded was 10-3 CFU/ml and 10-2 CFU/ml for MG and MS template DNA, respectively. The specificity of the real-time PCR assay was 100% for MG- and MS-specific probes in detecting both single as well as double infections. In conclusion, the use of TaqMan MGB probes for the detection of mgc2 and vlhA genes confers extra specificity and the incorporation of Exo IPC simplifies the assay, allowing the detection of double infections with low-copy target DNAs.

Rapid detection of Mycoplasma synoviae by loop-mediated isothermal amplification.

Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green® fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10−1 CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.

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.

Validation of a previously developed quantitative polymerase chain reaction for the detection and quantification of Mycoplasma synoviae in chicken joint specimens

A quantitative polymerase chain reaction (qPCR) was validated for the detection of Mycoplasma synoviae (PCR equivalents of colony-forming units [CFU eq.]) in chicken joint specimens with time and compared with direct mycoplasma culture. Joint specimens were obtained from 70 layer pullets inoculated intravenously with M. synoviae at 6 weeks of age. Ten control birds were injected intra-articularly with Freund's complete adjuvant. Macroscopic joint lesions were observed in 54 infected birds, of which 11 showed positive M. synoviae culture. The specificity of direct mycoplasma culture was high (100%, 95% confidence interval [CI] = 74 to 100), but its sensitivity low (16%, 95% CI = 8 to 26). Most positive results were obtained during the first 2 weeks after onset of joint swelling using synovial fluid. The qPCR was positive in 26 of 28 synovial fluid samples and in 51 of 70 joint swabs. The sterile joint samples obtained from Freund's complete adjuvant-injected birds were negative in the mycoplasma culture. The specificity and sensitivity of the qPCR for synovial fluid samples were 100% (95% CI = 65 to 100) and 93% (95% CI = 77 to 99); for joint swabs they were 100% (95% CI = 74 to 100) and 73% (95% CI = 61 to 83), respectively. Positive qPCR results (100.3 to 4.6 CFU eq./ml) were found until the end of the experiment (12 weeks post inoculation). At the end of the study, eight out of 16 joint swabs from birds without macroscopic joints lesions were positive in the qPCR (102.0 to 2.8 CFU eq./ml). Under the conditions of this study, the sensitivity of the qPCR was higher than that of direct mycoplasma culture (P< 0.0001) during the acute, subacute and chronic stages of arthritis.

Development and evaluation of an improved diagnostic PCR for Mycoplasma synoviae using primers located in the haemagglutinin encoding gene vlhA and its value for strain typing

Using published primers, detection of Mycoplasma synoviae and strain identification using the vlhA gene sequence was attempted. However, of 21 M. synoviae strains examined, three could not be amplified, so a new reverse primer was designed with a target in the conserved region of the vlhA gene. This allowed all 21 M. synoviae strains, a further nine strains and also material from 11 swab samples from M. synoviae-positive birds, to produce a PCR product, suggesting that the method could also be suitable for clinical specimens. The protocol was then tested on the type strains of M. synoviae and the other 22 recognised avian Mycoplasma species, with amplification of M. synoviae only. Further testing demonstrated that this PCR was equally or more sensitive than other PCR tests used to detect M. synoviae. Subsequent DNA sequence analysis of the PCR product based on percent similarity and evolutionary relationship appeared to be a useful tool for strain differentiation.

Detection of Mycoplasma synoviae in clinically normal pheasants.

Mycoplasma synoviae was isolated from the tracheas of seven clinically normal pheasants found in the vicinity of a chicken farm infected with M synoviae, but not from 120 pheasants and partridges with respiratory disease. When specimens were examined by the polymerase chain reaction only two additional pheasants infected with M synoviae were identified, one healthy and one diseased.

Cloning of Mycoplasma synoviae genes encoding specific antigens and their use as species-specific DNA probes.

A genomic library of Mycoplasma synoviae (MS) was generated by using bacteriophage lambda gt11 as a cloning and expression vector. Identification of recombinant clones highly specific to MS was achieved by screening the library for expression of MS proteins with polyclonal antiserum that had been preadsorbed with 6 heterologous avian mycoplasma species antigens. Expression of the recombinant clones in Escherichia coli followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total cell lysates and immunoblot yielded a predominant reactive fusion protein of 165 kD. Two clones (MS2/28 and MS2/12) that yielded inserts of different size were selected. The 2 MS DNA inserts were subcloned in a plasmid vector, labeled with digoxigenin, and used as probes for the specific recognition of several MS strains. A high degree of conservation was demonstrated for the MS2/12 and MS2/28 genes in tested MS strains. In addition, neither DNA fragment recognized any other avian mycoplasma species (M. gallisepticum, M. meleagridis, M. gallinarum, M. iners, M. anatis, and M. iowae), thus indicating their high specificity to MS. The sensitivity of the slot blot hybridization method using digoxigenin-labeled MS2/12 and MS2/28 probes for direct detection of MS from broth cultures of field isolates was 10(5) colony-forming units/ml. These results demonstrate the effectiveness of adsorbed antisera for the isolation of species-specific mycoplasma DNA and the potential for its use as probes for the specific and direct detection of MS from broth cultures of field isolates.