vlhA Gene 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.

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

BackgroundThe 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.MethodsForty-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.ResultsThe 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.ConclusionThe 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.

Mycoplasma gallisepticum and Mycoplasma synoviae in Turkeys in Poland.

The pathogenic mycoplasmas are among the bacteria causing significant losses in the poultry industry worldwide. Mycoplasma gallisepticum (MG) and M. synoviae (MS) are economically important pathogens causing chronic respiratory disease, decreased growth, egg production and hatchability rates, and significant downgrading of carcasses. Effective diagnosis of infection with these species in poultry is highly requisite considering their two routes of spreading-horizontal and vertical. Their prevalence and molecular epidemiology were investigated in 184 turkey flocks in Poland. Tracheal samples were selected from 144 broiler flocks and 40 turkey breeder flocks collected in 2015-2023. The prevalence of MG was determined by real-time PCR targeting the 16S rRNA gene and PCR targeting the mgc2 gene, and MS was determined by a 16-23S rRNA real-time PCR and a vlhA gene PCR. Further identification and molecular characterization were carried out using PCR and sequencing. M. gallisepticum and M. synoviae were found in 8.33% and 9.72% of turkey broiler flocks respectively. The phylogenetic analysis of MG isolates in most cases showed high similarity to the ts-11-like strains. MS isolates showed high similarity to strains isolated from flocks of laying hens causing EAA. Additional tests detected Ornithobacterium rhinotracheale, Gallibacterium anatis, Enterococcus faecalis and Enterococcus faecium, Staphylococcus aureus and Riemerella anatipestifer. These secondary pathogens could have significantly heightened the pathogenicity of the mycoplasma infections studied.

Development and validation of an insulated isothermal polymerase chain reaction assay for the rapid detection of Mycoplasma synoviae.

Mycoplasma synoviae, which causes the disease known as chicken synovitis, causes serious immunosuppression. We developed a rapid insulated isothermal polymerase chain reaction (iiPCR) assay for on-site detection of M. synoviae using a primer and probe set targeting the variable lipoprotein and haemagglutinin (vlhA) gene. In addition, the specificity, sensitivity, repeatability, and clinical detection of this method were evaluated. Our iiPCR assay detected M. synoviae clinical isolates and samples successfully and produced negative results on Mycoplasma galliscepticum, avian viral arthritis, Escherichia coli, Salmonella, Staphylococcus aureus and Corynebacterium, indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected the prepared positive standard plasmid diluted 10 times (1.00 × 10-1 - 1.00 × 10-10) as a template. The undiluted positive plasmid was positive and double distilled water was negative indicating that the PCR reactions were sensitive, respectively. Finally, the vlhA positive standard plasmid with dilution multiple of 1.00 × 10-4 - 1.00 × 10-6 was repeatedly detected three times to evaluate the repeatability of the iiPCR method established in this experiment showing that the iiPCR of M. synoviae is repeatable. The established iiPCR was also used to detect 50 chicken joint enlargement samples. The thermostatic detection PCR established in this experiment was comparable to a reference real-time PCR (qPCR).

Variability of pMGA/vlhA sequences among Mycoplasma gallisepticum field strains isolated from laying hens and their deformed eggs.

Mycoplasmosis, attributed to Mycoplasma gallisepticum, poses a significant challenge to poultry farming, leading to substantial economic losses and persistent infections within flocks. This bacterium harbours various surface proteins that are crucial for adhesion, transporter activity and evasion of the host immune response, facilitating its pathogenicity. One such key surface lipoprotein, referred to as pMGA or vlhA haemagglutinin, plays a pivotal role in adhesion processes. In this study, the clonal regions pMGA1.2 and pMGA1.3, as reported by Markham (M83178.1), were investigated to elucidate differences or similarities in the whole DNA sequences of M. gallisepticum field strains. The aim was to analyse sequence diversity within this region. Six internal primers were designed to amplify the target sequence, and isolates were obtained from both eggs and chickens sourced from laying hen flocks. Identification revealed 17 strains of M. gallisepticum and four strains of Mycoplasma synoviae, which were confirmed through the mgc2 and 16S rRNA genes, respectively. Positive and negative controls were established using the MGS6 and MSWUV1853 strains. Amplification results indicated a higher frequency of amplification proximal to the C-terminal region, with segments 4 (33.3 %) and 6 (27.8 %) being the most prevalent. Notably, none of the field strains exhibited the same amplification pattern as MGS6, and none of the strains characterized as M. synoviae amplified any primer set. Upon translation, the amino acid sequences from segments 4 and 6 were found to be compatible with conserved sequences within the Myco_haema protein domains of the genus Mycoplasma, specifically corresponding to Q7NAP3_MYCGA VlhA.3.04. The observed homology suggests a potential genetic transfer, while the variability identified in the pMGA or vlhA gene region of the field strains may have significant implications for protection against M. gallisepticum infection in chickens.

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

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. 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. 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. 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. 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.

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.

Percent positivity and phylogenetic analysis of Mycoplasma gallisepticum and Mycoplasma synoviae in commercial poultry from the different States of India.

Background and Aim:The Indian and global poultry industries suffer significant economic losses due to Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) infections, which adversely affect egg production, hatchability, weight gain, and feed efficiency in farms, thus decreasing the overall production efficiency. This study aimed to determine the percent positivity and phylogenetic analysis of MG, MS, and co-infection of both mycoplasmas in commercial poultry farms across different states of India from 2017 to 2021.Materials and Methods:A total of 3620 tracheal or choacal swabs were collected from breeder and layer farms showing clinical signs of avian mycoplasma infections from commercial poultry farms across India, and the percent positivity for MG, MS, and co-infection of both mycoplasmas were determined by Polymerase chain reaction using the 16S rRNA and vlhA genes amplification, respectively. Phylogenetic analysis was carried out by sequencing the mgc2 and vlhA genes of 2 samples of MG and 24 samples of M. synoviae to gain insight into the genetic variability of Indian strains. The data were then compared with other Indian strains, vaccines strains, and strains from other countries.Results:Our data shows the percent positivity of MG, MS, and co-infection of both MG and MS was 6.43%, 23.61%, and 15.49%, respectively. The phylogenetic relationship between MG and MS was determined using the vlhA and mgc2 genes, revealing two samples of MG and 24 samples of MS clustered with other Indian strains. M. synoviae MSM22 and previously studied M. synoviae MGS 482 clustered with vaccine strain M. synoviae MS-H.Conclusion:Mycoplasma synoviae infections in breeder, layer, and in both is predominant compared to MG across the states investigated in India. Sequenced samples of MG and MS showed evolutionary relationships with the previously studied Indian strains of MG and MS. These findings support our view that monitoring chickens for avian mycoplasma infections are of paramount significance. It further lends credence to the contention that such information will pave the way for the development of a home-grown vaccination control program and thus safeguard the poultry sector against mycoplasma infections.

Development of two novel SYBR green-based quantitative PCR assays for detection andquantification of Mycoplasma gallisepticum and Mycoplasma synoviae

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are considered the most pathogenic and economically devastating avian mycoplasmas. A definite MG and MS diagnosis depends on three main approaches, including isolation and identification, demonstration of specific antibodies, and detection of genetic materials. A fast and precise diagnosis technique is an indispensable prerequisite to prevent and control avian pathogens more effectively. This study aimed to develop and validate convenient SYBR green-based quantitative PCR (qPCR) assays using genome specific primers to rapidly detect and quantify MG and MS. Two sets of primers were designed based on the highly conserved regions of mgc2 and vlhA genes in MG and MS, respectively. The efficiencies of both reactions were calculated around 96% with a detection limit of as few as 10 copy numbers of DNA/μL in a sample. The standard curves showed linearity over a dynamic detection range of 6 log units from 10$^{1}$ to 10$^{6}$ DNA copy numbers/μL without any significant variations in inter- and intra-assays. The melting temperatures of the amplicons for MG and MS were determined to be 79.90 °C and 80.79 °C, respectively. The results showed that only MG and MS were detected as a single melt peak without cross reaction with other non-targeted pathogenic agents. The developed assays were further applied to the 60 swab specimens from commercial poultry with unknown MG and MS statuses and compared with the conventional PCR assays. The current assays detected 1 and 11 samples as MG and MS, respectively. However, five MG and three MS positive samples were tested positive in gel-based assays. The protocols developed here have the potential for use as a diagnostic research tool to detect and quantify MG and MS in clinical samples. Furthermore, the proposed qPCR techniques can provide an alternative approach for diagnosis and surveillance in epidemiological studies.

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.

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.

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.

Mycoplasma gallisepticum ve Mycoplasma synoviae için Tanısal Kantitatif Gerçek Zamanlı PCR Geliştirilmesi

Avian mycoplasmas are associated with respiratory disease, synovitis, poor quality of day-old chicks and poor performance. The main approach used for the diagnosis of avian mycoplasmas is isolation and identification of the microorganism. Since the bacteria are slow growing fastidious organisms, conventional methods are time consuming, laborious and require experienced personnel. For this reason, we aimed to develop a rapid detection method for Mycoplasma gallisepticum and Mycoplasma synoviae by quantitative real-time polymerase chain reaction (qPCR). For this purpose, the lipoprotein (lp) and variable lipoprotein hemagglutinin (vlhA) genes were used to detect M. gallisepticum and M. synoviae, respectively. The limit of detection (LOD) of the assay was determined to be 100- 101 DNA/µl from artificially contaminated swab samples. The specificity and sensitivity ratios were 100%. Overall, these results indicate that this qPCR method can be accurately used for detection of MG and MS.

The influence of host tissue on M. gallisepticum vlhA gene expression

Mycoplasma gallisepticum, a significant poultry pathogen, has evolved rapidly in its new passerine host since its first reported isolation from house finches in the US in 1994. In poultry, M. gallisepticum infects the upper respiratory tract, causing tracheal mucosal thickening and inflammation, in addition to inflammation of the reproductive tract. However, in house finches M. gallisepticum primarily causes inflammation of the conjunctiva. Given that different tissues are primarily affected by the same pathogen in different hosts, we have compared the early changes in gene expression of the phase-variable lipoproteins (vlhA) gene family of M. gallisepticum collected directly from target tissues in both hosts. Previous data have demonstrated that vlhA genes may be related to virulence, exhibiting changes in expression in a non-stochastic, temporal progression and we hypothesize that this may be influenced by differences in the target host tissue. If this is true, we would expect M. gallisepticum to display a different vlhA gene expression pattern in the chicken trachea compared to its expression pattern in house finch conjunctiva.Here we report significant differences in vlhA gene expression patterns between M. gallisepticum collected from chicken tracheas compared to those collected from house finch conjunctiva. While many of the predominant vlhA genes expressed in the input population showed an increase in expression in the chicken trachea at day one postinfection, those same vlhA genes decreased in expression in the house finch. These data suggest that discrete suites of vlhA genes may be involved in M. gallisepticum pathogenesis and tropism for unique tissues in two disparate avian hosts.

Development and evaluation of semi-nested PCR for detection of the variable lipoprotein haemagglutinin (vlhA) gene of Mycoplasma Synoviae in chicken

Development and evaluation of semi-nested PCR for detection of the variable lipoprotein haemagglutinin (vlhA) gene of Mycoplasma Synoviae in chicken

Molecular detection and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae strains in backyard poultry in Italy

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) represent the most important avian Mycoplasma species in the poultry industry, causing considerable economic losses. In Italy, the presence of MG or MS has been investigated especially in commercial poultry farms. To our knowledge, no systematic investigations on MG or MS presence using highly specific diagnostic assays have been performed in backyard poultry. The aim of this study was to detect and molecularly characterize MG and MS strains in 11 backyard poultry flocks located in different regions of Italy. Tracheal swabs were collected and DNA was extracted. For MS, a PCR targeting a vlhA gene fragment was performed, and typing and subtyping was attempted. The presence of MG was investigated by a screening PCR, then MG typing by gene-targeted sequencing (GTS). All the amplicons were sequenced, then MG and MS dendrograms were constructed. All the flocks examined resulted Mycoplasma positive: 5 out of 11 (45.45%) were MG and MS positive, 3 (27.27%) were MG positive, and the remaining 3 (27.27%) were MS positive. The MS detections were assigned to types C, D, and F. All strains of type D belonged to subtype D1 and 2 unknown subtypes were identified. A MS sequence showed peculiar characteristics, which did not allow assignment to a known MS type or subtype. MG GTS analysis identified 6 MG strains belonging to 5 subclusters circulating in Italian backyards chicken flocks. The results of this study provide evidence of a risk for commercial poultry farms, especially in areas where backyard and commercial farms are close, suggesting the implementation of biosecurity measures.

Detection and antibiotic resistance of Mycoplasma gallisepticum and Mycoplasma synoviae among chicken flocks in Egypt.

Background and Aim:Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most significant pathogens of avian mycoplasmosis. This study aimed to isolate and identify MG and MS from chickens and detect the various virulence genes in the isolates. Moreover, the efficacies of different antibiotics were tested to identify suitable treatment regimens.Materials and Methods:We isolated MG and MS from 487 chicken samples of different ages located in different Governorates in Egypt using conventional isolation methods. The isolates were characterized by polymerase chain reaction (PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method.Results:The prevalence of MG among the isolates was 9.85%, with the highest percentage isolated from air sacs, while the prevalence of MS among the isolates was 1.6%. Moreover, the highest levels of the prevalence of both MG and MS were during the winter and autumn sampling, while the lowest levels were in the summer and spring. Following the 16S rRNA-based detection of Mycoplasma isolates, 14 MG and 5 MS isolates were identified by different PCR-based detection methods for various virulence genes. Nine MG isolates contain the mgc2 gene, six MG isolates contain the gapA gene, and three MS isolates contain the vlhA gene. We validated a duplex PCR method for the simultaneous identification of MG and MS, based on 100% of the MG and MS isolates generating common bands at 55 and 17 kDa, respectively. The MIC method identified tiamulin and spiramycin as the antibiotics of choice for the treatment of MG and MS infections, respectively.Conclusion:For more precise diagnosis of Mycoplasma infections in chicken flocks, conventional isolation methods must be confirmed by PCR. SDS-PAGE analysis helps in epidemiological studies and vaccine preparation. The MIC method can be used to help develop therapies to control avian mycoplasmosis infections.

In vitro evaluation of various antimicrobials against field mycoplasma gallisepticum and mycoplasma synoviae isolates in Egypt

Among many avian mycoplasmas, Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are recognized as the main etiological agents of respiratory diseases and infectious synovitis in chickens and turkeys causing tremendous economic losses worldwide. Therefore, proper treatment is promoted for the control of these diseases. This study was the first in Egypt to evaluate the in vitro efficacy of various antimicrobials against field MG and MS isolates recovered from chicken and turkey flocks using both conventional broth microdilution and quantitative real-time polymerase chain reaction assays. Totally, 47 mycoplasma isolates were recovered from 160 collected tracheal samples (29.4%). Of these, 44 MG (27.5%) and 3 MS (1.9%) were identified using conventional and molecular assays. The in vitro susceptibilities of 4 representative mycoplasma field isolates (3 MG and one MS) to 8 antibiotics and 4 essential oils were investigated. The tested isolates showed various susceptibilities to tested antimicrobials. Toldin CRD, followed by clove, cumin, and cinnamon oils were effective against both MG and MS clinical isolates with minimum inhibitory concentration (MIC) values ranging from 0.49 to 15.63 µg/mL. Similarly, tylvalosin was the most active antibiotic against MG and MS isolates with the lowest MIC values (0.015 to 0.03 µg/mL). DNA loads of both MG mgc2 and MS vlhA genes were markedly decreased upon treatment with majority of the tested antimicrobials confirming their effectiveness as was also evaluated by conventional MIC results. In conclusion, Toldin CRD and tylvalosin were found to be the most effective antimicrobials in this study. This finding highlights the importance of using these antimicrobials in controlling mycoplasma infections in chickens and turkeys.

Rapid and visible detection of Mycoplasma synoviae using a novel polymerase spiral reaction assay

In this study, a rapid, specific, and sensitive detection assay for Mycoplasma synoviae (MS) was established using a polymerase spiral reaction (PSR) method. A pair of primers were designed according to the conserved region of the vlhA gene of MS, and PSR results were assessed using agarose gel electrophoresis and color rendering with a dye indicator. The optimum reaction temperature and time for PSR using the specific primers were 62°C and 40min in a water bath, respectively. The sensitivity of the PSR assay for MS detection was 100 times more than that of the polymerase chain reaction assay based on agarose gel electrophoresis results and color change detected by the naked eye. Further experiments demonstrated that the primers specifically detected MS and showed no cross-reaction with other prevalent avian pathogens. Clinical sample testing confirmed that the MS-PSR assay is simple, rapid, specific, and sensitive, and thereby very suitable for application and promotion in the field and laboratories of grassroots units.

Molecular identification of Mycoplasma synoviae from breeder chicken flock showing arthritis in Egypt.

Aim:Arthritis is one of the most economic problems facing poultry industry worldwide. The study was done to detect possible causes of arthritis in breeder chicken flock with emphasis on molecular identification of Mycoplasma synoviae (MS).Materials and Methods:This study was carried on chicken from broiler breeder flock of 57 weeks’ age in Dakahlia, Egypt, suffered from arthritis with frequently 5-7% decrease in egg production, reduced fertility, and hatchability. Forty blood samples were randomly collected from individual birds in sterile tubes and used for serum separation. Serum samples were tested using serum plate agglutination (SPA) test against colored antigens for Mycoplasma gallisepticum (MG), MS, and Salmonella gallinarum-pullorum (SGP). On the other hand, 24 joint samples were collected. Of those 24 samples, 12 joint samples were subjected to bacteriological examination, while the other 12 were utilized for molecular diagnosis by polymerase chain reaction (PCR) for MS and avian reovirus (ARV).Results:SPA test results revealed the presence of antibodies against MG, MS, and SGP in tested sera in rates of 14/40 (35%), 35/40 (87.5%), and 9/40 (22.5%), respectively. Furthermore, 19 bacterial isolates were recognized from joint samples and identified as five Staphylococcus spp., nine Escherichia coli, three SGP, one Citrobacter, and one Proteus. The identified Staphylococcal isolates were three coagulase-positive staphylococci (two Staphylococcus aureus and one Staphylococcus hyicus) and two coagulase-negative staphylococci (one Staphylococcus epidermidis and one Staphylococcus lentus), while E. coli isolate serotypes were 1 O11, 2 O55, 3 O78, 1 O124, 1 O125, and 1 untyped. PCR proved that 12/12 (100%) samples were positive for MS variable lipoprotein hemagglutinin A (vlhA) gene, while ARV was not diagnosed in any of the examined samples. Four amplified vlhA gene of MS isolates (named MS-2018D1, MS-2018D2, MS-2018D3, and MS-2018D4) was successfully sequenced.Analysis of phylogenetic tree revealed the presence of 100% identity between each two sequenced isolates(isolates MS-2018D1 and MS-2018D4 and also isolates 2018D2 and MS-2018D3). However, the nucleotide similarity between four isolates was 88.6%. On the other hand, our field isolates MS-2018D1, MS-2018D4, MS-2018D2, and MS-2018D3 showed nucleotide identity with vaccine strain MS-H 98.4%, 98.4%, 88.1%, and 88.1%, respectively. Furthermore, the nucleotide similarities with field strains from Argentina ranged between 87.8% and 98.6%.Conclusion:Four field isolates of MS were identified in examined broiler breeder flock. A phylogenetic study of these isolates revealed the variation between isolated MS strains and vaccine strain. Therefore, further studies are required for evaluating the vaccine efficacy against the present field isolates of MS. In addition, application of MS immunization of breeder flocks is necessary for proper control of the disease.