Molecular detection and genetic characterization of Mycoplasma gallisepticum and Mycoplasma synoviae in selected chicken breeds in South Africa.

BACKGROUND: The impact of chickens on maintaining the economy and livelihood of rural communities cannot be overemphasized. In recent years, mycoplasmosis has become one of the diseases that affect the success of South African chicken production. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most prevalent strains of Mycoplasma in South Africa. MG and MS are significant respiratory pathogens affecting the productivity of chickens. The present study aimed to molecularly detect using qPCR and characterize the presence of MG and MS using phylogenetic analysis. The phylogenetic analysis was utilized to clarify general evolutionary relationships between related taxa of different MG and MS observed in tracheal swabs from South African chicken breeds. METHODS: Forty-five tracheal swabs of the Lohmann Brown (n = 9), Rhode Island Red (n = 9), Ovambo (n = 9), Venda (n = 9), and Potchefstroom Koekoek (n = 9) breeds were collected from symptomatic chickens present in the commercial farm. To detect MG and MS, DNA was extracted from tracheal swabs and faecal samples, and qPCR was performed with a 16 s rRNA (310 bp) and vlhA (400 bp) gene fragment. Following the sequencing of all the amplicons, MG, and MS dendrograms showing the evolutionary relationships among the five South African chicken breeds and the GeneBank reference population were constructed. RESULTS: The qPCR revealed the presence of MG and MS in 22% (2/9) of the tracheal swab samples tested for MS only in Rhode Island Red breeds; 66.6% (6/9) and 33% (3/9) of the tested samples in Ovambo breeds; and 11.1% (1/9) and 44.4% (4/9) of the tested samples in Venda breeds. No MG or MS were detected in the Lohmann Brown or Potchefstroom Koekoek breed. Furthermore, qPCR revealed the presence of MG in pooled faecal samples from Lohmann Brown and Ovambo breeds. Eight different bacterial isolates were recognized from both samples. Four isolates were of the 16 s ribosomal ribonucleic acid (rRNA) gene (named PT/MG51/ck/00, PT/MG48/ck/00, PT/MG41/ck/00 and PT/MG71/ck/00) gene of Mycoplasma gallisepticum, and the other was Mycoplasma Synoviae variable lipoprotein hemagglutinin A (vlhA) gene (named PT/MSA22/ck/01, PT/MS41/ck/01, PT/MS74/ck/01 and PT/MS46/ck/01) which were available in GenBank. These isolates were successfully sequenced with 95-100% similarity to the isolates from the gene bank. CONCLUSION: The study revealed the presence of both MG and MS in the chicken breeds sampled. Furthermore, the different breeds of chicken were found to be susceptible to infection under the intensive or commercial management system. Therefore, continuous surveillance is encouraged to prevent the spread and outbreak of MG and MS in the poultry industry in South Africa.

Characterisation of the tracheal transcriptional response of chickens to chronic infection with Mycoplasma synoviae.

Mycoplasma synoviae causes infectious synovitis and respiratory tract infections in chickens and is responsible for significant economic losses in the poultry industry. Effective attachment and colonisation of the trachea is critical for the persistence of the organism and progression of the disease it causes. The respiratory tract infection is usually sub-clinical, but concurrent infection with infectious bronchitis virus (IBV) is known to enhance the pathogenicity of M. synoviae. This study aimed to explore differentially expressed genes in the tracheal mucosa, and their functional categories, during chronic infection with M. synoviae, using a M. synoviae-IBV infection model. The transcriptional profiles of the trachea were assessed 2 weeks after infection using RNA sequencing. In chickens infected with M. synoviae or IBV, only 1 or 8 genes were differentially expressed compared to uninfected chickens, respectively. In contrast, the M. synoviae-IBV infected chickens had 621 upregulated and 206 downregulated genes compared to uninfected chickens. Upregulated genes and their functional categories were suggestive of uncontrolled lymphoid cell proliferation and an ongoing pro-inflammatory response. Genes associated with anti-inflammatory effects, pathogen removal, apoptosis, regulation of the immune response, airway homoeostasis, cell adhesion and tissue regeneration were downregulated. Overall, transcriptional changes in the trachea, 2 weeks after infection with M. synoviae and IBV, indicate immune dysregulation, robust inflammation and a lack of cytotoxic damage during chronic infection. This model provides insights into the pathogenesis of chronic infection with M. synoviae.

The pathogenicity and immune effects of different generations of Mycoplasma synoviae on chicken embryos.

1. Mycoplasma synoviae (MS) is the primary causative agent of synovitis in avian species. In order to investigate the pathogenicity and immunological responses associated with MS in specific pathogen-free chicken embryos, a series of generations (F1, F95, F120, F160 and F200) of MS were introduced into 7-day-old SPF chicken embryos and subsequent mortality rates were recorded and analysed2. Reverse transcription-quantitative polymerase chain reaction was performed to detect expression of heat shock proteins HSP27, HSP40, HSP60, HSP70 and HSP90 and inflammatory factors interleukin (IL)-1ß, caspase-1 and IL-18 in the tracheal tissue.3. The results showed that the mortality rate of SPF chicken embryos decreased with an increase in the number of passages, with the highest being 80% (8/10) for F1 generation and the lowest being 10% (1/10) for F200. The expression of HSP27, IL-1ß, HSP40, caspase-1, HSP70 and HSP90 showed a significant downregulation trend with an increase in the generation (except IL-18; P < 0.05). The HSP60 expression was significantly upregulated with increasing generations (P < 0.05).4. A relationship between pathogenicity and the number of passages was observed and the decrease in pathogenicity appeared to be associated with HSP and genes related to inflammatory factors. The present work offers a scientific foundation for screening potential MS strains that might be employed to develop attenuated vaccines.

Mechanistic insights of magnolol antimicrobial activity against Mycoplasma using untargeted metabolomic analyses.

The unreasonable use of antibiotics is one of the important causes of antimicrobial resistance (AMR) that poses a huge public health threat. Magnolol is a traditional Chinese medicine exhibiting antibacterial-, antifungal-, anti-inflammatory-, and antioxidant activities. However, it is unclear whether magnolol has an inhibitory effect on mycoplasma. This study found that magnolol showed excellent inhibitory activity against various mycoplasmas. Magnolol showed dose-dependent inhibition of Mycoplasma synoviae growth and biofilm formation in vitro. Magnolol caused severely sunken and wrinkled M. synoviae cell membranes at the minimum inhibitory concentration, and an enlarged cell diameter. The chicken embryo infection model showed that magnolol significantly reduced M. synoviae pathogenicity in vivo. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the citrate cycle, glycolysis/gluconeogenesis, and pyruvate metabolism were significantly disturbed at the minimum inhibitory concentration of magnolol. Interestingly, 41% of differential metabolites were in the categories of lipids and lipid-like molecules. Protegenin A was up-regulated 58752-fold after magnolol treatment. It belongs to fatty acyls, and destroys cell membrane integrity and cell activity. Ghosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, and phosphatidylserine related to membrane maintenance and stress response were widely down-regulated. Collectively, our results illustrate the feasibility of magnolol as a phytochemical compound to treat mycoplasma infection.

Colloidal gold based immunochromatographic detection of Mycoplasmopsis synoviae infection and its prevalence in avian species of Karachi, Pakistan.

Avian mycoplasmosis is an infection that commonly prevails in birds, particularly in poultry chickens. Among mycoplasmosis causing organisms, Mycoplasmopsis synoviae is a predominant and lethal pathogen to the aves. Considering the increased incidence of infections by M. synoviae, the prevalence of M. synoviae was deduced in poultry chickens and fancy birds of Karachi region. The lungs and tracheal samples from chicken and dead fancy birds and swab samples from live fancy birds were collected and investigated by amplifying 16 s rRNA gene of M. synoviae. Biochemical characteristics of M. synoviae was also evaluated. Furthermore, surface-associated membrane proteins, that represent key antigens for diagnosis of M. synoviae infection was extracted by Triton X- 114 method. Results showed that M. synoviae was detected more frequently in lungs than in trachea, that could be due to its invasion capacity and tissue affinity. SDS PAGE analysis of extracted membrane proteins showed two prominent hydrophobic proteins of different molecular mass including proteins of 150 and 50 kDa. Protein of 150 kDa was purified by size exclusion chromatography and it exhibited agglutinogen activity. Purified protein was used in the development of one-step immunochromatographic (ICT) assay for the detection of antibodies against M. synoviae using gold nanoparticles coated with polyclonal antibodies. Low levels of antibodies were detected by the developed ICT kit, which has 88% sensitivity with 92% specificity.

Pathogenic bacteria associated with outbreaks of respiratory disease in Iranian broiler farms.

BACKGROUND: Multi-causal respiratory infections are more commonly observed than uncomplicated cases with single agents in the commercial poultry industry. Recently, increased mortality rates associated with respiratory clinical signs have been reported in Iranian broiler farms. OBJECTIVES: The present study aimed to determine the spectra of avian mycoplasmas (Mycoplasma gallisepticum, MG and Mycoplasma synoviae, MS) and Ornithobacterium rhinotracheale (ORT) in the broiler farms with the multi-causal respiratory disease (MCRD) from 2017 to 2020. METHODS: Trachea and lung tissue samples were collected from 70 broiler flocks presenting increased mortality and acute respiratory disease. MG, MS, and ORT were detected by performing polymerase chain reaction with primers complementary to the 16S rRNA, vlhA, and 16S rRNA genes, respectively. RESULTS: Genetic materials of MG, MS, and ORT were detected in five, three, and five of the 70 flocks. Based on the phylogenetic analysis of the complete mgc2 coding sequences, all MG strains formed a distinct cluster along with other Iranian MG isolates. According to the phylogenetic analysis of the partial vlhA gene of MS strains, two isolates were located along with Australian and European strains. In addition, one of them displayed an out-group association with MS isolates from Jordan. Phylogenetic analysis of Iranian ORT strains using a partial sequence of the 16S rRNA gene showed a distinct group among the other ORT strains. CONCLUSIONS: The results indicate that MG, MS, and ORT are not predominantly responsible for the MCRD. However, continuous monitoring of poultry flocks could be significant for obtaining valuable information related to different MG, MS, and ORT strains and designing effective control strategies.

Reversion of mutations in a live mycoplasma vaccine alters its metabolism.

The live attenuated temperature sensitive vaccine strain MS-H (Vaxsafe® MS, Bioproperties Pty. Ltd., Australia) is widely used to control disease associated with M. synoviae infection in commercial poultry. MS-H was derived from a field strain (86079/7NS) through N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced mutagenesis. Whole genomic sequence analysis of the MS-H and comparison with that of the 86079/7NS have found that MS-H contains 32 single nucleotide polymorphisms (SNPs). Three of these SNPs, found in the obgE, oppF and gapdh genes, have been shown to be prone to reversion under field condition, albeit at a low frequency. Three MS-H reisolates containing the 86079/7NS genotype in obgE (AS2), obgE and oppF (AB1), and obgE, oppF and gapdh (TS4), appeared to be more immunogenic and transmissible compared to MS-H in chickens. To investigate the influence of these reversions in the in vitro fitness of M. synoviae, the growth kinetics and steady state metabolite profiles of the MS-H reisolates, AS2, AB1 and TS4, were compared to those of the vaccine strain. Steady state metabolite profiling of the reisolates showed that changes in ObgE did not significantly influence the metabolism, while changes in OppF was associated with significant alterations in uptake of peptides and/or amino acids into the M. synoviae cell. It was also found that GAPDH plays a role in metabolism of the glycerophospholipids as well as an arginine deiminase (ADI) pathway. This study underscores the role of ObgE, OppF and GAPDH in M. synoviae metabolism, and suggests that the impaired fitness arising from variations in ObgE, OppF and GAPDH contributes to attenuation of MS-H.

Molecular characterization and antimicrobial susceptibility profiles of Thai Mycoplasma synoviae isolates.

Mycoplasma synoviae (MS) infection is mainly controlled by pathogen-free flocks' maintenance, medication in infected flocks, and vaccination in high-risk flocks. The effective control strategy requires convenient approach for detecting and differentiating MS strains and reliable drug susceptible evidence for deciding on reasonable antimicrobial usage. This study aimed to characterize the partial vlhA gene of nine Thai MS isolates circulated in chickens in 2020, to verify the PCR-RFLP assay for strain differentiation, and to determine the eight antimicrobial susceptibility profiles using microbroth dilution method. Based on sequence analysis of the partial vlhA gene, Thai MS isolates in 2020 were classified as types E and L with 19 and 35 amino acid lengths, respectively. The developed PCR-RFLP assay could detect and differentiate vaccine and Thai field strains. Most Thai MS isolates in this study were susceptible to tylosin, tylvalosin, tiamulin, doxycycline, oxytetracycline, tilmicosin, and lincomycin-spectinomycin at MIC50 values of 0.0391, 0.0098, 0.0781, 0.1563, 0.1563, 0.625 and 0.625 µg/mL, respectively; and resistance to enrofloxacin at MIC50 value of 10 µg/mL. In conclusion, this study revealed diagnostic assays for differentiating MS strains and the antimicrobial susceptibility profiles of Thai MS, which are necessary to design suitable MS control procedures for poultry flocks.

Genomic Diversity of a Globally Used, Live Attenuated Mycoplasma Vaccine.

The Mycoplasma synoviae live attenuated vaccine strain MS-H (Vaxsafe MS; Bioproperties Pty., Ltd., Australia) is commonly used around the world to prevent chronic infections caused by M. synoviae in birds and to minimize economic losses in the poultry industry. MS-H is a temperature-sensitive strain that is generated via the chemical mutagenesis of a virulent M. synoviae isolate, 86079/7NS. 32 single nucleotide polymorphisms have been found in the genome of MS-H compared to that of 86079/7NS, including 25 in predicted coding sequences (CDSs). There is limited information on the stability of these mutations in MS-H in vitro during the propagation of the vaccine manufacturing process or in vivo after the vaccination of chickens. Here, we performed a comparative analysis of MS-H genomes after in vitro and in vivo passages under different circumstances. Studying the dynamics of the MS-H population can provide insights into the factors that potentially affect the health of vaccinated birds. The genomes of 11 in vitro laboratory passages and 138 MS-H bird reisolates contained a total of 254 sequence variations. Of these, 39 variations associated with CDSs were detected in more than one genome (range = 2 to 62, median = 2.5), suggesting that these sequences are particularly prone to mutations. From the 25 CDSs containing previously characterized variations between MS-H and 86079/7NS, 7 were identified in the MS-H reisolates and progenies examined here. In conclusion, the MS-H genome contains individual regions that are prone to mutations that enable the restoration of the genotype or the phenotype of wild-type 86079/7NS in those regions. However, accumulated mutations in these regions are rare. IMPORTANCE Preventative measures, such as vaccination, are commonly used for the control of mycoplasmal infections in poultry. A live attenuated vaccine strain (Vaxsafe MS; MS-H; Bioproperties Pty. Ltd., Australia) is used for the prevention of disease caused by M. synoviae in many countries. However, information on the stability of previously characterized mutations in the MS-H genome is limited. In this study, we performed a comparative analysis of the whole-genome sequences of MS-H seeds used for vaccine manufacturing, commercial batches of the vaccine, cultures minimally passaged under small-scale laboratory and large-scale manufacturing conditions, MS-H reisolated from specific-pathogen-free (SPF) chickens that were vaccinated under controlled conditions, and MS-H reisolated from vaccinated commercial poultry flocks around the world. This study provides a comprehensive assessment of genome stability in MS-H after in vitro and in vivo passages under different circumstances and suggests that most of the mutations in the attenuated MS-H vaccine strain are stable.

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.

Antibiotic resistance of Mycoplasma Synoviae strains isolated in China from 2016 to 2019.

BACKGROUND: In the past decade, Mycoplasma synoviae (M. synoviae) infection has become widely prevalent in China, has caused serious economic losses and has become one of the most important diseases in the chicken industry. Medication is a general approach for the control of M. synoviae infection, but antibiotics are sometimes ineffective in clinical practice. To investigate the sensitivity of M. synoviae to antimicrobials commonly used in the treatment of M. synoviae infection, the antibiotic susceptibility of 32 M. synoviae strains isolated from China from 2016 to 2019 were determined using the minimum inhibitory concentration (MIC) method. RESULTS: All isolates had low MIC values for the combination of lincomycin and spectinomycin, pleuromutilin, and macrolides. However, the M. synoviae isolates displayed variance in MICs for doxycycline hydrochloride with a range of 0.25 to 8 µg/mL, and oxytetracycline hydrochloride with a range of 0.5 to 8 µg/mL. Three and one M. synoviae isolates showed intermediate MIC values to doxycycline hydrochloride and oxytetracycline hydrochloride, respectively. High MIC values for enrofloxacin were detected in all isolates with MICs ranging from 4 to 32 µg/mL. Furthermore, comparison of the parC QRDR identified a mutation at nucleotide position 254 (C254T) resulting in a Thr 85 Ile amino acid change in all M. synoviae isolates and the reference strain ATCC 25204 being resistant to enrofloxacin. Moreover, mutations at Glu 804 Gly and Thr 686 Ala of gyrA QRDR were identified in all M. synoviae isolates and ATCC 25204. The mutation in the QRDR of the parE gene resulted in amino acid changes at positions 197 (Pro to Ser) in 27/32 M. synoviae isolates. CONCLUSION: Three nonsynonymous mutations in gyrA and parE were first identified to be related to enrofloxacin resistance. Our results showed that M. synoviae resistance to enrofloxacin is widespread.

Transcriptional profiling of the chicken tracheal and splenic response to virulent Mycoplasma synoviae.

Mycoplasma synoviae (MS), an important avian pathogen, can cause chronic respiratory disease, eggshell apex abnormalities, infectious synovitis, and arthritis in avian species, leading serious economic losses in the global poultry industry. To date, studies have shown significant different transcript profiles using various chicken cells after MS infection. However, in vitro cell models cannot fully represent the complex in vivo regulations after adventitious infection. The objective of this study was to explore the nature of the host-pathogen interaction during MS infection. The tracheal and spleen tissues of chickens were collected at d 0, 1, 3, and 5 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A lot of significantly differentially expressed genes (DEGs) were observed in this analysis, and 861 DEGs were observed in trachea tissues and 753 DEGs were observed in spleen samples. Many of DEGs in trachea tissues participate in a variety of cellular activities, especially cellular metabolism. Immune-related DEGs were mainly enriched at d 3, and 5 postinfection in trachea tissues. While, DEGs in spleen tissues were significantly and mainly enriched into immune-related pathways. The results of this study show the direct interactions between MS and the chicken trachea and spleen for the first time. Early dysregulation of tissue-wide gene expression as observed here set the stage for persistent infection of MS.

Pooled molecular occurrence of Mycoplasma gallisepticum and Mycoplasma synoviae in poultry: A systematic review and meta-analysis.

Worldwide, Mycoplasma gallisepticum (MG) and M. synoviae (MS) are the main agents responsible for chronic respiratory disease in poultry. Therefore, we conducted a systematic review and meta-analysis to estimate their occurrence. We searched electronic databases to find peer-reviewed publications reporting the molecular detection of MG and MS in poultry and used meta-analysis to estimate their pooled global occurrence (combined flock and individual), aggregating results at the regional and national levels. We performed a subgroup meta-analysis for subpopulations (broilers, layers, breeders and diverse poultry including turkeys, ducks and ostriches) and used meta-regression with categorical modifiers. We retrieved 2294 publications from six electronic databases and included 85 publications from 33 countries that reported 62 studies with 22,162 samples for MG and 48 studies with 26,413 samples for MS. The pooled global occurrence was 38.4% (95% CI: 23.5-54.5) for MS and 27.0% (20.4-34.2) for MG. Among regions, Europe and Central Asia had the lowest occurrence for both pathogens, while MG and MS were highly prevalent in South Asia and sub-Saharan Africa, respectively. At the national level, MG occurrence was higher in Algeria, Saudi Arabia and Sudan, whereas China, Egypt and Ethiopia reported higher values of MS. Among the poultry subpopulations, MS and MG were more prevalent in the breeders and layers (62.6% and 31.2%, respectively) than in diverse poultry. The year of publication, the sample size and the level of ambient air pollution (measured indirectly by PM2.5) were associated with the occurrence of both mycoplasmas. Our study revealed high and heterogeneous occurrence values of MG and MS and justifies the need for early detection and improved control measures to reduce the spread of these pathogens.

Clinical expression, epidemiology, and monitoring of Mycoplasma gallisepticum and Mycoplasma synoviae: an update.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are of clinical and economic importance for the global poultry industry. Many countries and integrations are involved in monitoring programmes to control both mycoplasma species. This review provides an extensive historic overview of the last seven decades on the development of the knowledge regarding the factors that influence the clinical expression of the disease, the epidemiology, and monitoring of both MG and MS. This includes the detection of new virulent strains, studies unravelling the transmission routes, survival characteristics, and the role of other avian hosts. Also the role of molecular typing tests in unravelling epidemiology and factors that complicate the interpretation of test results is discussed. The latter includes the presence of heterologous mycoplasma infections, the use of heterologous oil-emulsion vaccines, and the use of antibiotic treatments. Also the occurrence of MG and MS strains with low virulence and the use of live and/or inactivated MS and MS vaccines are discussed.

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.

Epidemiological investigations and locally determined genotype diversity of Mycoplasma synoviae in Central China from 2017 to 2019.

Mycoplasma synoviae (M. synoviae) has been identified worldwide to cause respiratory diseases, infectious synovitis, airsacculitis, and eggshell apex abnormalities (EAA) in commercial chickens, which results in substantial economic losses to the poultry industry. Therefore, in this study, 258 flocks were investigated between 2017 and 2019 for M. synoviae by screening samples from Central China. Subsequently, 129 M. synoviae strains were isolated, with a positive rate of 50%. Moreover, a higher incidence of M. Synoviae infections was in layers (74.1%) than in broilers (20%) in this study. The 5'-end conserved segment of the variable lipoprotein hemagglutinin A (vlhA) gene of these isolates was then cloned and sequenced because it is a common genomic target identified so far for M. synoviae genotyping. Genotyping of all isolates was based on the phylogenetic analysis and length analysis of the proline-rich-repeat (PRR) regions, respectively. Phylogenetic analysis based on 5'-end conserved segment of the vlhA gene (76-421 nt) assigned the majority of the occurring strains as being from group 6, and others from groups 2 and 3. Results identified that these isolates were of 6 types: A (38aa), D (23aa), E (19aa), I (28aa), J (20aa), and L (35aa), based on the size of the PRR region analysis. Furthermore, most of the isolates (81.4% were identified as type L. Additionally, the epidemic types included only I and L in 2017; however, the types rose to 5 (A, D, E, I, L) in 2018 and rose to 6 (A, D, E, I, J, L) in 2019. These data showed the genotype diversity of M. synoviae in Central China. The high rate of positive flocks suggests the urgent need to take real-time supervisory controls of this Mycoplasma species in avian flocks.

Multi-locus sequence typing analysis of Mycoplasma synoviae isolates reveals unique sequence types in China.

Mycoplasma synoviae is a common pathogen affecting poultry and has important economic significance. Infectious synovitis is the most common clinical effect. Since 2010, the incidence of M. synoviae infection in China has rapidly risen, causing significant economic losses to the chicken industry; however, the cause of the disease outbreak remains unclear. Phylogenetic and evolutionary analyses of field strains will help unravel the mystery. The multi-locus sequence typing (MLST) method is typically utilized to conduct genotyping and traceability analysis of microorganisms. MLST of M. synoviae has previously been established and shown strong discriminatory power. In this study, 54 Chinese M. synoviae strains isolated from 2016 to 2020 were genotyped by MLST based on seven housekeeping genes. This study aimed to investigate the dominant genotypes of M. synoviae in China and reveal the genetic and evolutionary relationships of these isolates. All 54 isolates were found to have new allelic sequences, which may indicate new sequence types. The results of BURST analysis indicated that all 54 strains belonged to group 11, which is an independent phylogenetic branch, and were separated from any other reference strains (189 isolates) in the PubMLST database. In conclusion, the results of this study suggest that the M. synoviae strains circulating in China are relatively independent in terms of transmission and evolutionary relationships.

Characterization of pyruvate dehydrogenase complex E1 alpha and beta subunits of Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an important pathogen which causes huge economic losses to the poultry industry worldwide, and research on MS can provide the foundation for diagnosis, prevention, and treatment of MS infection. In this study, primers designed based on the sequences of pyruvate dehydrogenase complex (PDC) E1 alpha and beta subunit genes (pdhA and pdhB, respectively) of MS 53 strain(AE017245.1) in GenBank were used to amplify the pdhA and pdhB genes of MS WVU1853 strain through PCR. Subsequently, the prokaryotic expression vectors pET-28a(+)-pdhA and pET-28a(+)-pdhB were constructed and expressed in Escherichia coli BL21(DE3) cells. The recombinant proteins rMSPDHA and rMSPDHB were purified, and anti-rMSPDHA and anti-rMSPDHB sera were prepared by immunizing rabbits, respectively. Subcellular localization of PDHA and PDHB in MS cells, binding activity of rMSPDHA and rMSPDHB to chicken plasminogen (Plg) and human fibronectin (Fn), complement-dependent mycoplasmacidal assays, and adherence and adherence inhibition assays were accomplished. The results showed that PDHA and PDHB were distributed both on the surface membrane and within soluble cytosolic fractions of MS cells. The rMSPDHA and rMSPDHB presented binding activity with chicken Plg and human Fn. The rabbit anti-rMSPDHA and anti-rMSPDHB sera had distinct mycoplasmacidal efficacy in the presence of guinea pig complement, and the adherence of MS to DF-1 cells pretreated with Plg was effectively inhibited by treatment with anti-rMSPDHA or anti-rMSPDHB sera. These findings indicated that surface-associated MSPDHA and MSPDHB were adhesion-related factors of MS and that the binding between MSPDHA/MSPDHB and Plg/Fn contributed to MS adhesion to DF-1 cells.

Prevalence of Mycoplasma synoviae and Its Impact on Productivity in Commercial Poultry Farms in Quebec, Canada.

Mycoplasma synoviae (MS) is associated with upper respiratory disease, joint, and reproductive system disease in poultry. Economic losses are due to stunting, increased mortality, lower egg production, and higher slaughterhouse condemnations. The seroprevalence of MS is increasing worldwide, and more pathogenic strains have emerged over the past few years. Where this increase is noted, the economic consequences are considerable, even when there are no obvious clinical signs. The best control strategy is to maintain mycoplasma-free flocks. Since 2014 in Quebec, Canada, MS has been isolated with greater frequency in poultry farms and at times, as a primary pathogenic agent. The aim of this study was to evaluate the prevalence and impact of MS in commercial poultry farms in Quebec because the poultry industry was considering an insurance program that would cover losses in case of an outbreak. MS was shown to be present in all types of commercial production, although egg layers were principally affected with over 50% of flocks sampled being MS-positive in all producing regions of the province. On the basis of vlhA gene sequencing, several strains were identified with the most prevalent ones being type E, followed by Qc-1, a strain specific to Quebec. On average, the impact of MS on production parameters were not significant for any of the different types of commercial poultry production.

Prevalencia de Mycoplasma synoviae y su impacto en la productividad en granjas avícolas comerciales en Quebec, Canadá. Mycoplasma synoviae (MS) se asocia con enfermedades de las vías respiratorias superiores, problemas articulares y del sistema reproductivo en la avicultura comercial. Las pérdidas económicas se deben al retraso en el crecimiento, aumento de la mortalidad, menor producción de huevo y mayores decomisos en plantas de procesamiento. La seroprevalencia de M. synoviae está aumentando en todo el mundo y en los últimos años han surgido cepas más patógenas. En los lugares donde se ha observado este aumento, las consecuencias económicas han sido considerables, incluso cuando no se presentan signos clínicos evidentes. La mejor estrategia de control es mantener las parvadas libres de micoplasmas. Desde 2014 en Quebec, Canadá, M. synoviae se ha aislado con mayor frecuencia en granjas avícolas y en ocasiones como agente patógeno primario. El objetivo de este estudio fue evaluar la prevalencia y el impacto de M. synoviae en granjas avícolas comerciales en Quebec porque la industria avícola estaba considerando un programa de seguros que cubriría las pérdidas en caso de un brote. Se demostró que M. synoviae estaba presente en todos los tipos de producción comercial, aunque las aves de postura de huevo se vieron afectadas principalmente, con más del 50% de las parvadas muestreadas positivas a M. synoviae en todas las regiones productoras de la provincia. Con base en la secuenciación del gene vlhA, se identificaron varias cepas, siendo las más prevalentes el tipo E, seguidas de la cepa QC-1, que es específica de Quebec. En promedio, el impacto de M. synoviae en los parámetros de producción no fue significativo para ninguno de los diferentes tipos de producción avícola comerciales.

Development of molecular assays for the rapid and cost-effective determination of fluoroquinolone, macrolide and lincosamide susceptibility of Mycoplasma synoviae isolates.

Mycoplasma synoviae infection occurs worldwide, leading to considerable economic losses in the chicken and turkey industry due to infectious synovitis, respiratory diseases and eggshell apex abnormalities. Control programs against M. synoviae infection are based on eradication, vaccination and medication with antimicrobial agents. Prudent use of antibiotics can be improved greatly by the determination of antibiotic susceptibility prior to the treatment. However, the conventional broth or agar microdilution is very labor-intensive and time-consuming method. Thus, there is an increasing need for rapid antimicrobial susceptibility tests in order to guide antibiotic therapy more effectively. The aim of this study was to develop mismatch amplification mutation assays (MAMAs) to detect resistance-associated mutations in M. synoviae. M. synoviae strains with previously determined minimal inhibitory concentrations (MICs) and whole genomes (n = 92) were used for target selection and assay specification. For the evaluation of the developed assays, 20 clinical samples and an additional 20 M. synoviae isolates derived from these specimens were also included in this study. MIC values of these 20 isolates were determined by broth microdilution method. Five MAMAs were designed to identify elevated MICs of fluoroquinolones, while three MAMAs were developed to detect decreased susceptibility to macrolides and lincomycin. The sensitivity of the MAMA tests varied between 102-104 template copy number/reaction depending on the assay. Clinical samples showed identical genotype calls with the M. synoviae isolates derived from the corresponding specimens in each case. Supporting the results of conventional in vitro sensitivity tests, our approach provides a feasible tool for diagnostics. Rapidity, robustness and cost-effectiveness are powerful advantages of the developed assays. Supporting prudent antibiotic usage instead of empirical treatment, the use of this method can reduce significantly the economic impact of M. synoviae in the poultry industry and decrease bacterial resistance-related public health concerns.