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.

Quantification of the effect of vaccination on the control of horizontal transmission of Mycoplasma synoviae under field conditions.

Beside biosecurity, vaccination is important for Mycoplasma synoviae (MS) control as it has been shown to contribute to the reduction of economic impact and, experimentally, also lessens horizontal transmission. In this study, the effect of MS live vaccination on horizontal transmission was quantified under field conditions by analysing 4-year MS monitoring data from non-MS-vaccinated broiler and layer breeders and MS-vaccinated broiler breeders with good biosecurity in single-age housing systems. Flocks were monitored at 20 and 30 weeks of age and every 12 weeks thereafter. At every sampling, 60 blood samples or 24 tracheal swabs were tested using rapid plate agglutination test and ELISA serially or MS DIVA PCR, respectively. The MS incidence rate was calculated and the association with vaccination was analysed by logistic regression. The average MS incidence rate per 1000 weeks was 11.6 cases for non-MS-vaccinated broiler breeders and decreased from 29.6 to 5.6 cases with successive vaccinated production cycles. In non-MS-vaccinated layer breeders it was 3.6. A significant negative association with MS incidence was found after vaccinating four to six successive production cycles compared to non-MS-vaccinated or only one production cycle vaccinated breeders (odds ratio (OR) = 0.23, P = 0.05 & OR = 0.12, P = 0.01, respectively). A significant negative association with MS in non-MS-vaccinated layer breeders (OR = 0.29, P = 0.00) was observed compared to non-MS-vaccinated broiler breeders, possibly due to more controlled contact structures within the layer breeder industry. The results suggest that vaccination and control of contacts contribute to the reduction of between-farm MS transmission.

Molecular assay for detecting MS-H vaccine strain and immune response mechanisms in chickens receiving one or two doses of live MS-H vaccine.

The MS-H vaccine, containing a live strain of Mycoplasma synoviae, is a feasible option for controlling M. synoviae infection in poultry flocks. A comprehensive understanding of vaccinated chickens, including strain differentiation and immune response mechanisms, is required to optimize vaccination strategy. This study aimed to verify the PCR-RFLP molecular assay as a convenient technique for detecting the MS-H vaccine strain and to characterize the immune response mechanisms in experimental layer-type chickens receiving one of three different vaccination programmes; a single dose at either 9 or 12 weeks of age or two doses at both 9 and 12 weeks of age. The PCR-RFLP assay, using restriction enzyme TasI to digest vlhA gene-targeted PCR amplicons, was performed to evaluate vaccine administration by detecting the MS-H vaccine strain in vaccinated chickens and differentiating it from non-vaccine strains such as WVU1853 reference strain and Thai M. synoviae field strains. Results demonstrated that vaccination in layer-type chickens, whether as one or two doses, stimulated immune response mechanisms with no significant advantages of two administrations over a single administration. Serological responses in vaccinated chickens, examined by RPA test and ELISA, were initially detected at 2 weeks post-vaccination, continuously increased, and then remained at the baseline levels from 6 to 9 weeks post-vaccination. Cellular immune responses against both homologous and heterologous antigens, examined by the MTS tetrazolium assay, were similar in the early period post-vaccination, whereas cellular immune response against the homologous MS-H antigen was improved in the late period post-vaccination.

The pharmacokinetics/pharmacodynamics integration of tilmicosin against Mycoplasma synoviae in vitro and in vivo.

Mycoplasma synoviae (MS) infection is a serious threat to poultry industry in China. Tilmicosin is a semisynthetic macrolide antibiotic used only in animals and has shown potential efficacy against MS, but there were no reported articles concerning the pharmacokinetics/pharmacodynamics (PK/PD) interactions of tilmicosin against MS in vitro and vivo. This study aimed to assess the antibacterial activity of tilmicosin against MS in vitro and in vivo using PK/PD model to provide maximal efficacy. The minimum inhibitory concentration (MIC) and killing rates of different drug concentrations were measured using the microdilution method in vitro. Then, tilmicosin was administered orally to the MS-infected chickens at doses of 7.5 and 60 mg/kg, and the PK parameters of tilmicosin in joint dialysates were determined using high-pressure liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) combined with the microdialysis technique. The antibacterial effect (△E) was calculated when the infected chickens were administered a single oral dose of tilmicosin at 4, 7.5, 15, 30, and 60 mg/kg b.w. The PK and PD data were fitted using the Sigmoid Emax model to evaluate the PK/PD interactions of tilmicosin against MS. The bactericidal activity of tilmicosin against MS was concentration dependent. Furthermore, the PK/PD index of AUC0-72h/MIC exhibited the most optimal fitting results (R2 = .98). The MS load decreased by 1, 2, and 3 Log10 CFU/mL, then AUC/MIC was determined as 13.99, 20.53, and 28.23 h, respectively, and the bactericidal effect can be achieved when the dose of MS-infected chickens is at 31.64 mg/kg b.w. The findings of this study hold significant implications for optimizing the treatment regimen for MS infection.

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.

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.

NADH oxidase of Mycoplasma synoviae is a potential diagnostic antigen, plasminogen/fibronectin binding protein and a putative adhesin.

BACKGROUND: Mycoplasma synoviae (MS) is an important pathogen causing respiratory diseases and arthritis in chickens and turkeys, thus, resulting in serious economic losses to the poultry industry. Membrane-associated proteins are thought to play important roles in cytoadherence and pathogenesis. NADH oxidase (NOX) is an oxidoreductase involved in glycolysis, which is thought to be a multifunctional protein and potential virulence factor in some pathogens. However, little is known regarding the NOX of MS (MSNOX). We previously demonstrated that MSNOX was a metabolic enzyme distributed in not only the cytoplasm but also the MS membrane. This study was aimed at exploring NOX's potential as a diagnostic antigen and its role in MS cytoadherence. RESULTS: Western blots and ELISAs indicated that recombinant MSNOX (rMSNOX) protein reacted with sera positive for various MS isolates, but not MG isolates or other avian pathogens, thus, suggesting that rMSNOX is a potential diagnostic antigen. In addition, rabbit anti-rMSNOX serum showed substantial complement-dependent mycoplasmacidal activity toward various MS isolates and MG Rlow. MSNOX protein was found not only in the cytoplasm but also on the membrane of MS through suspension immunofluorescence and immunogold electron microscopy assays. Indirect immunofluorescence assays indicated that rMSNOX adhered to DF-1 cells, and this adherence was inhibited by rabbit anti-rMSNOX, but not anti-MG serum. Furthermore, indirect immunofluorescence and colony counting assays confirmed that the rabbit anti-rMSNOX serum inhibited the adherence of various MS isolates but not MG Rlow to DF-1 cells. Moreover, plasminogen (Plg)- and fibronectin (Fn)-binding assays demonstrated that rMSNOX bound Plg and Fn in a dose-dependent manner, thereby further confirming that MSNOX may be a putative adhesin. CONCLUSION: MSNOX was identified to be a surface immunogenic protein that has good immunoreactivity and specificity in Western blot and ELISA, and therefore, may be used as a potential diagnostic antigen in the future. In addition, rMSNOX adhered to DF-1 cells, an effect inhibited by rabbit anti-rMSNOX, but not anti-MG serum, and anti-rMSNOX serum inhibited the adherence of various MS isolates, but not MG Rlow, to DF-1 cells, thus indicating that the inhibition of adherence by anti-MSNOX serum was MS specific. Moreover, rMSNOX adhered to extracellular matrix proteins including Plg and Fn, thus suggesting that NOX may play important roles in MS cytoadherence and pathogenesis. Besides, rabbit anti-rMSNOX serum presented complement-dependent mycoplasmacidal activity toward both MS and MG, indicating the MSNOX may be further studied as a potential protective vaccine candidate.

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.

Insights on Mycoplasma gallisepticum and Mycoplasma synoviae infection in poultry: a systematic review.

Avian mycoplasmosis mainly caused by Mycoplasma gallisepticum and M. synoviae is an economically important disease of poultry industry. It causes huge economic losses in terms of decrease in weight gain, feed conversion efficiency, egg production, hatchability; increase in embryo mortality, carcass condemnation, prophylaxis and treatment cost in broiler, layer and breeder flocks. The disease is caused by four major pathogenic mycoplasmas viz., M. gallisepticum (MG), M. synoviae (MS), M. meleagradis (MM) and M. iowae (MI). The MG and MS are World Organization for Animal Health listed respiratory pathogens. MG causes chronic respiratory disease in chicken and infectious sinusitis in turkey; however, MS causes synovitis and airsacculitis in birds. The infection is transmitted both horizontally and vertically. Prevention and control measures of avian mycoplasmosis mainly comprises of biosecurity, treatment and vaccination. For vaccination of birds, inactivated bacterins, live attenuated and/or recombinant live poxvirus vaccines are commercially available against MG and MS infection. The present systematic review summarizes the different epidemiological studies carried out on MG and MS infection in poultry in different geographical locations of India and abroad over the last decade (2010-2020), economic impact, diagnosis and prevention and control.

Multispectral Portable Fibre-Optic Reflectometer for the Classification of the Origin of Chicken Eggshells in the Case of Mycoplasma synoviae Infections.

The proper classification of the origins of food products is a crucial issue all over the world nowadays. In this paper, the authors present a device-a multispectral portable fibre-optic reflectometer and signal processing patch-together with a machine-learning algorithm for the classification of the origins of chicken eggshells in the case of Mycoplasma synoviae infection. The sensor device was developed based on previous studies with a continuous spectrum in transmittance and selected spectral lines in reflectance. In the described case, the sensor is based on the integration of reflected spectral data from short spectral bands from the VIS and NIR region, which are produced by single-colour LEDs and introduced to the sample via a fibre bundle. The measurement is carried out in a sequence, and the reflected signal is pre-processed to be put in the machine learning algorithm. The support vector machine algorithm is used together with three different types of data normalization. The obtained results of the F-score factor for classification of the origins of samples show that the percentages of eggs coming from Mycoplasma synoviae infected hens are up to 87% for white and 96% for brown eggshells.

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.

Mycoplasma synoviae induces serum amyloid A upregulation and promotes chicken synovial fibroblast cell proliferation.

Mycoplasma synoviae (MS) infection causes infectious synovitis and arthritis with hyperplasia of synovial cells in the chicken joint. However, its mechanism is unknown. We used primary chicken synovial fibroblast (CSF) as the research object to study the role of MS in the proliferation of MS-infected CSF and determine the mechanisms involved. Using integrated transcriptomic and proteomic analyses of the interaction between CSF and MS, we screened a proliferation-regulated factor, serum amyloid A (SAA), that may regulate proliferation of MS-infected CSF. SAA appears to be associated with MS-induced CSF proliferation. To study the role of SAA in MS-induced CSF proliferation, a eukaryotic expression vector overexpressing SAA and a small interfering RNA (siRNA) targeting Saa were constructed to manipulate the expression of SAA. Cell proliferation and apoptosis were detected via cell counting kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), or terminal deoxyribonucleotidyl transferase-mediated dUTP nick-dnd labeling (TUNEL) assays, respectively. Western blot analysis was used to examine the protein expression level of SAA, cyclin E1, and cyclin-dependent kinase 2 (CDK2). In vitro, MS significantly promoted the proliferation of CSF and increased the production of SAA. Overexpression of SAA accelerated the proliferative ability of CSF, whereas knockdown of SAA depressed the proliferative ability of CSF. A TUNEL assay indicated that MS did not induce apoptosis. Silencing of SAA suppressed the expression of cyclin E1 and CDK2. These results suggest that MS may upregulate the expression of SAA, accelerate the cell cycle, and promote proliferation of CSF.

Minimal inhibitory concentration of seven antimicrobials to Mycoplasma gallisepticum and Mycoplasma synoviae isolates from six European countries.

Mycoplasma gallisepticum and Mycoplasma synoviae are bacterial pathogens that cause disease in poultry, adversely affecting their health and welfare, and are a financial burden on producers. This manuscript describes the results of the MycoPath project that is the first international antimicrobial susceptibility programme for mycoplasma pathogens isolated from poultry. Improved comparative analysis of minimal inhibitory concentration (MIC) results from participating countries was facilitated by using one laboratory determining all MICs. Chicken and turkey isolates were obtained from France, Germany, Great Britain, Hungary, Italy and Spain during 2014-2016. One isolate per farm was retained. The MIC of seven antimicrobial agents was determined using a broth microdilution method, with Friis Medium (M. gallisepticum) or Modified Chanock's Medium (M. synoviae). Of the 222 isolates recovered, 82 were M. gallisepticum and 130 were M. synoviae. M. gallisepticum MIC50/90 values were 0.12/0.5, 2/8, 0.5/4, 0.12/>64, 0.008/0.062, 0.008/32, 0.062/4 mg/l for doxycycline, enrofloxacin, oxytetracycline, spiramycin, tiamulin, tilmicosin and tylosin, respectively. For M. synoviae, the values were 0.5/1, 8/16, 0.5/1, 0.5/8, 0.25/0.5, 0.062/2 and 0.062/16 mg/l respectively. A bimodal MIC distribution for the fluoroquinolone (enrofloxacin) and the macrolides (spiramycin, tilmicosin and tylosin) indicate that both species have sub-populations that are less susceptible in vitro to those antimicrobials. Some differences in susceptibilities were observed according to host species, Mycoplasma species, and country of origin. This study provides a baseline of novel data for future monitoring of antimicrobial resistance in poultry Mycoplasma species. Additionally, this information will facilitate the selection of the antimicrobial agents most likely to be effective, thus ensuring their minimal use with targeted and correct therapeutic treatments.Highlights First large-scale pan-European collection of representative Mg and Ms isolates.MIC values assessed in central laboratory for Mg and Ms from chickens and turkeys.Range of MIC values for 82 Mg and 130 Ms isolates to seven licenced antibiotics shown.Data can be used to help determine Mg and Ms veterinary-specific breakpoints.

Preliminary comparative analysis of the genomes of selected field reisolates of the Mycoplasma synoviae vaccine strain MS-H reveals both stable and unstable mutations after passage in vivo.

BACKGROUND: Genomic comparison of Mycoplasma synoviae vaccine strain MS-H and the MS-H parental strain 86,079/7NS established a preliminary profile of genes related to attenuation of MS-H. In this study we aimed to identify the stability of mutations found in MS-H after passage in experimental or field chickens, and to evaluate if any reverse mutation may be associated with changes in characteristics of MS-H in vitro or in vivo. RESULTS: Whole genome sequence analysis of 5 selected MS-H field reisolates revealed that out of 32 mutations reported previously in MS-H, 28 remained stable, while four found to be reversible to the wild-type. Each isolate possessed mutations in one to three of the genes obg, oppF1 and gap and/or a non-coding region. Examination of the 4 reversible mutations by protein modeling predicted that only two of them (in obg and oppF1 genes) could potentially restore the function of the respective protein to that of the wild-type. CONCLUSIONS: These results suggest that the majority of the MS-H mutations are stable after passage in vaccinated chickens. Characterisation of stable mutations found in MS-H could be utilised to develop rapid diagnostic techniques for differentiation of vaccine from field strains or ts- MS-H reisolates.

Relative contribution of vertical, within-farm and between-farm transmission of Mycoplasma synoviae in layer pullet flocks.

In this study, the relative contribution of vertical transmission, within-farm transmission and between-farm transmission of Mycoplasma synoviae in layer pullet flocks was quantified using logistic regression analysis. Data from 311 Dutch pullet flocks, of which 172 (55%) were positive for M. synoviae, were included in the study. Also the M. synoviae status of the parent stock of these flocks was included. The M. synoviae status was determined with the M. synoviae rapid plate agglutination test. Data analysis showed that vertical transmission was the most important transmission route for M. synoviae in layers as is demonstrated by an odds ratio of 5.8 (P = 0.000). A positive association with M. synoviae infections was found for layer pullet flocks on a multi-house farm where at least one other flock was M. synoviae-positive compared to single-house farms (odds ratio 3.1, P = 0.022), while a negative association was found when no other M. synoviae-positive flocks were present (odds ratio = 0.2, P = 0.003). No association was found between M. synoviae status of pullet flocks and poultry farm density. Odds ratios were 0.54 (P = 0.288) and 0.34 (P = 0.073), respectively, for medium and highest poultry farm density compared to lowest poultry farm density. This is the first time that the relative contribution of horizontal and vertical transmission of M. synoviae has been quantified. These results can be extrapolated to M. synoviae control in general, and emphasize the importance of M. synoviae control in parent stock and practical channelling.

Influences of swab types and storage temperatures on isolation and molecular detection of Mycoplasma gallisepticum and Mycoplasma synoviae.

Routine diagnosis of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) is performed by collecting oropharyngeal swabs, followed by isolation and/or detection by molecular methods. The storage temperature, storage duration and the type of swab could be critical factors for successful isolation or molecular detection. The aim of this study was to compare the influence of different types of cotton-tipped swab stored at different temperatures, on the detection of MG and MS. To achieve this, combined use of traditional culture analysis (both agar and broth), with modern molecular detection methods was utilized. Performances of wooden and plastic shaft swabs, both without transport medium, were compared. Successful culture of M. gallisepticum was significantly more efficient from plastic swabs when compared to wooden, whereas no difference was seen for the re-isolation of M. synoviae. Storage at 4°C compared to room temperature also increased the efficiency of culture detection for both Mycoplasma species. When stored at room temperature, PCR detection limits of both MG and MS were significantly lower for wooden compared to plastic swabs. The qPCR data showed similar detection limits for both swab types when stored at both temperatures. The results suggest that swabs with a plastic shaft are preferred for MG and MS detection by both culture and PCR. While a lower storage temperature (4°C) is optimal for culture recovery, it seems that both temperatures investigated here are adequate for molecular detection and it is the swab type which carries a greater influence.

Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF1 (oligopeptide permease transporter) which results in expression of a truncated OppF1. The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF1 gene in the MS-H genome. Wild-type oppF1 was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF1-tetM or with a similar vector lacking oppF1 coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF1-tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF1 detected bands of expected size for full-length OppF1 in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF1-tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF1-tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF1-tetM (passage 27) showed that the chromosomal copy of the mutated oppF1 had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF1 impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species.RESEARCH HIGHLIGHTS The full-length OppF1 was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF1 impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF1 in MS-H was replaced with wild-type oppF1.

Investigation on eggshell apex abnormality (EAA) syndrome in France: isolation of Mycoplasma synoviae is frequently associated with Mycoplasma pullorum.

BACKGROUND: Mycoplasma synoviae (MS) is known to cause Eggshell Apex Abnormality (EAA) syndrome characterized by an altered shell surface with increased translucency on the apex. However, no large-scale studies have been conducted to obtain prevalence data of EAA and MS isolates associated to this syndrome. This manuscript reports the results of two field studies performed in the French poultry industry (2015-2017): focusing mainly on investigation of presence and prevalence of EAA in different types of laying hen flocks (phase 1), and isolation of MS strains from EAA-infected flocks (phase 2). RESULTS: The first survey included 77 farms of commercial layers in three French egg-production regions, hosting 40 flocks in alternative systems (ALT) and 56 in furnished cages (FC). Seven flocks (4 FC and 3 ALT) presented EAA clinical signs, giving a prevalence of 7.3% in this studied sample. A second independent field study was conducted to identify MS by in vitro cultivation and PCR in samples from 28 flocks with clinical signs of EAA. Different types of biological specimens were collected in EAA-affected flocks and submitted to the laboratory. M. synoviae was detected in 25/28 flocks, from both production systems (5/5 ALT and 20/23 FC). Detection of MS was significantly higher in tracheal swabs (59%) than in cloacal (10.5%), albumen (3.6%) and egg yolk (1.1%) swabs. It is worth to mention that attempts to clone MS from positive samples were often hampered by the presence of another Mycoplasma species, which showed fast growing behaviour in the selective media used in this study (Frey Medium 4 and Frey Medium 4 supplemented with erythromycin). The use of MALDI-TOF mass spectrometry in combination with next-generation sequencing (NGS) results allowed the identification of this fast growing mycoplasma as Mycoplasma pullorum, which was detected in 14 of the 25 (56%) MS-positive flocks. CONCLUSIONS: These results confirmed the presence of the EAA syndrome in MS-positive flocks of layers in France, reared in different regions and in different production systems (ALT and FC). Studies need to be conducted to test whether M. pullorum may influence the expression of clinical signs of EAA in MS-infected layer farms.

Molecular detection and genetic characterization of Mycoplasma gallisepticum, Mycoplama synoviae, and infectious bronchitis virus in poultry in Myanmar.

BACKGROUND: In Southeast Asian countries, including Myanmar, poultry farming is a major industry. In order to manage and maintain stable productivity, it is important to establish policies for biosecurity. Infectious respiratory diseases are a major threat to poultry farming. Avian influenza and Newcastle disease have been reported in Myanmar, but no scientific information is available for other respiratory pathogens, such as mycoplasmas and infectious bronchitis virus (IBV). Identifying the genotypes and serotypes of IBVs is especially important to inform vaccination programs. In this study, we detected Mycoplasma gallisepticum (MG), M. synoviae (MS), and IBV in several poultry farms in Myanmar. RESULTS: Samples were collected from 20 farms in three major poultry farming areas in Myanmar, and MG, MS, and IBV were detected on two, four, and eight farms, respectively, by polymerase chain reaction. Phylogenetic analysis revealed that the observed MG and MS isolates were not identical to vaccine strains. Three different genotypes of IBV were detected, but none was an unknown variant. CONCLUSIONS: Mycoplasmas and IBV were detected on poultry farms in Myanmar. Periodic surveillance is required to establish the distribution of each pathogen, and to institute better vaccine protocols.

Eggshell apex abnormalities caused by two different Mycoplasma synoviae genotypes and evaluation of eggshell anomalies by full-field optical coherence tomography.

BACKGROUND: Mycoplasma synoviae (MS) is an important poultry pathogen worldwide. This bacterium may cause eggshell changes including an altered shell surface, thinning, and increased translucency in different areas, which leads to a greater incidence of eggshell cracks and breaks. In the present study the association between experimental infection of birds with two field strains of MS from different genotypes and the production of abnormal eggs is described. The analysis of those eggshells using a full-field optical coherence tomography (FF OCT) scanner is also reported. RESULTS: Eggshell samples were obtained from three experimental groups of chickens: one control and two infected tracheally with field strains of MS which produced abnormal eggs. In both experimental groups infected with MS a reduction of mean daily egg production by 11% was observed compared to the control group, which started at 21 to 42 dpi. Eggshell apex abnormalities increased to 24.5% of eggs and in some cases, soft-shelled eggs were produced. This study provides the first analysis of shells from anomalous eggs carried out using FF OCT, which allows three-dimensional structural imaging of an investigated sample at micrometre scale. FF OCT showed ultrastructural changes in eggshells and a smaller number of pores on the entire surface of the affected shells. CONCLUSIONS: The eggshell pathology and the concomitant egg production losses that result from infections highlight the economic significance of MS in commercial poultry. There are differences in the strains of MS which may induce eggshell apex abnormalities (EAA) and egg production losses. The use of FF OCT, which is a noninvasive measurement method based on analysis of the light backscattered from the measured object, will confer the ability to control the quality of eggshells in flocks infected with MS.