Quadrivalent hemagglutinin and adhesion expressed on Saccharomyces cerevisiae induce protective immunity against Mycoplasma gallisepticum infection and improve gut microbiota.

Mycoplasma gallisepticum (MG) infection causes infectious respiratory diseases in poultry, causing economic losses to the poultry industry. Therefore, this study aims to develop a safe, convenient, and effective multivalent recombinant Saccharomyces cerevisiae vaccine candidate and to explore its potential for oral immunization as a subunit vaccine. Mycoplasma gallisepticum Cytadhesin (MGC) and variable lipoprotein and hemagglutinin (vlhA) are associated with the pathogenesis of MG. In this study, a quadrivalent recombinant Saccharomyces cerevisiae (ST1814G-MG) displaying on MGC2, MGC3, VLH5, and VLH3, proteins was innovatively constructed, and its protective efficiency was evaluated in birds. The results showed that oral immunization with ST1814G-MG stimulates specific antibodies in chickens, reshapes the composition of the gut microbiota, reduces the Mycoplasma loading and pulmonary disease injury in the lungs. In addition, we found that oral ST1814G-MG had better protection against MG infection than an inactivated vaccine, and co-administration with the inactivated vaccine was even more effective. The results suggest that ST1814G-MG is a potentially safer and effective agent for controlling MG infection.

STAT5-mediated transcription of miR-33-5p in Mycoplasma gallisepticum-infected DF-1 cells.

RESEARCH HIGHLIGHTS: MG-HS regulates the expression of transcription factor STAT5.Transcription factor STAT5 can target miR-33-5p promoter element.MG-influenced STAT5 regulates miR-33-5p and its target gene expression.

Rapid adaptation to a novel pathogen through disease tolerance in a wild songbird.

Animal hosts can adapt to emerging infectious disease through both disease resistance, which decreases pathogen numbers, and disease tolerance, which limits damage during infection without limiting pathogen replication. Both resistance and tolerance mechanisms can drive pathogen transmission dynamics. However, it is not well understood how quickly host tolerance evolves in response to novel pathogens or what physiological mechanisms underlie this defense. Using natural populations of house finches (Haemorhous mexicanus) across the temporal invasion gradient of a recently emerged bacterial pathogen (Mycoplasma gallisepticum), we find rapid evolution of tolerance (<25 years). In particular, populations with a longer history of MG endemism have less pathology but similar pathogen loads compared with populations with a shorter history of MG endemism. Further, gene expression data reveal that more-targeted immune responses early in infection are associated with tolerance. These results suggest an important role for tolerance in host adaptation to emerging infectious diseases, a phenomenon with broad implications for pathogen spread and evolution.

Isolation and characterization of an atypical Mycoplasma gallisepticum strain showing a new mgc2 variant.

Mycoplasma gallisepticum (MG) is an important pathogen of the poultry industry able to cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite the application of biosecurity measures and the availability of vaccines for chickens, monitoring systems routinely applied for MG detection are still essential for infection control. Pathogen isolation is time-consuming and not suitable for rapid detection, albeit it is a compulsory step for genetic typing and antimicrobial susceptibility evaluation of single strains. The mgc2 gene is a species-specific molecular target adopted by most of the PCR protocols available for MG diagnosis, which are also included in the WOAH Terrestrial Manual. We describe the case of an atypical MG strain, isolated in 2019 from Italian turkeys, characterized by an mgc2 sequence not detectable by common endpoint PCR primers. Considering the potential risk of false negative results during diagnostic screenings with the endpoint protocol, the authors propose an alternative mgc2 PCR endpoint protocol, named MG600, which should be considered as a further diagnostic tool.

Molecular detection of Mycoplasma gallisepticum in different poultry breeds of Abbottabad and Rawalpindi, Pakistan / Detecção molecular de Mycoplasma gallisepticum em diferentes raças de aves de Abbottabad e Rawalpindi, Paquistão

Abstract The poultry sector in Pakistan is contributing mainly in bridging gap between demand and supply for protein. Mycoplasma gallisepticum is an emerging bacterium causing serious problems in poultry industry of Pakistan. A cross-sectional study was conducted to evaluate the M. gallisepticum load in poultry populated regions of Pakistan. Total 600 serum and 600 swab samples were collected, 200 from each broiler, layers and breeders poultry in Rawalpindi and Abbottabad districts. Serum samples were analyzed through ELISA for seroprevalence. Swabs were cultured on Frey's medium followed by PCR and partial mgc2 gene sequencing. Results of seroprevalence of M. gallisepticum showed that layers (75%, n=150) are more positive as compared to breeders (70%, n=140) and broilers (50%, n=100). Typical colonies of the M. gallisepticum were observed in breeder (26.5%), followed by layer (21%) and broilers (9%). A total of 37.1% (n=42) samples were identified positive through PCR out of total 113 cultured based positive samples. A total of six M. gallisepticum isolates of current study showed 98-99 percent similarity with previously reported isolates on the basis of mgc2 gene partial sequencing. The M. gallisepticum was found highly prevalent in different poultry breads. Results of this study would add into basic data and provide a direction for livestock sector to strengthen a control strategy for mycoplasmosis in poultry farms.

Resumo O setor avícola do Paquistão está contribuindo principalmente para preencher a lacuna entre a demanda e a oferta de proteína. Mycoplasma gallisepticum é uma bactéria emergente que causa sérios problemas na indústria avícola do Paquistão. Um estudo transversal foi conduzido para avaliar a carga de M. gallisepticum em regiões de avicultura do Paquistão. Um total de 600 amostras de soro e 600 amostras de esfregaço foi coletado, 200 de cada frango de corte, poedeiras e aves reprodutoras nos distritos de Rawalpindi e Abbottabad. Amostras de soro foram analisadas por ELISA para soroprevalência. As zaragatoas foram cultivadas em meio Frey, seguido de PCR e sequenciação parcial do gene mgc2. Os resultados da soroprevalência de M. gallisepticum mostraram que as poedeiras (75%, n = 150) são mais positivas em comparação com matrizes (70%, n = 140) e frangos de corte (50%, n = 100). Colônias típicas de M. gallisepticum foram observadas em reprodutoras (26,5%), seguidas de poedeiras (21%) e frangos de corte (9%). Um total de 37,1% (n = 42) das amostras foi identificado como positivas por PCR de um total de 113 amostras positivas baseadas em cultura. Um total de seis isolados de M. gallisepticum do estudo atual mostrou 98-99% de similaridade com isolados relatados anteriormente com base no sequenciamento parcial do gene mgc2. O M. gallisepticum foi encontrado com alta prevalência em diferentes pães de aves. Os resultados deste estudo acrescentariam dados básicos e forneceriam orientação para o setor pecuário fortalecer uma estratégia de controle da micoplasmose em granjas avícolas.

Mycoplasma gallisepticum escapes the host immune response via gga-miR-365-3p/SOCS5/STATs axis.

A disruption in the expression of gga-miR-365-3p was confirmed in the Mycoplasma gallisepticum (MG)-infected Chicken primary alveolar type II epithelial (CP-II) cells based on previous sequencing results, but the role it plays in the infection was unclear. In the present study, we demonstrate that MG evaded cellular host immunity via a gga-miR-365-3p/SOCS5-JAK/STATs negative feedback loop. Specifically, we found that at the initial stage of MG infection in cells, gga-miR-365-3p was rapidly increased and activated the JAK/STAT signaling pathway by inhibiting SOCS5, which induced the secretion of inflammatory factors and triggered immune response against MG infection. Over time, though, the infection progressed, MG gradually destroyed the immune defences of CP-II cells. In late stages of infection, MG escaped host immunity by reducing intracellular gga-miR-365-3p and inhibiting the JAK/STAT pathway to suppress the secretion of inflammatory factors and promote MG adhesion or invasion. These results revealed the game between MG and host cell interactions, providing a new perspective to gain insight into the pathogenic mechanisms of MG or other pathogens. Meanwhile, they also contributed to novel thoughts on the prevention and control of MG and other pathogenic infections, shedding light on the immune modulating response triggered by pathogen invasion and their molecular targeting.

Role of DNA modifications in Mycoplasma gallisepticum.

The epigenetics of bacteria, and bacteria with a reduced genome in particular, is of great interest, but is still poorly understood. Mycoplasma gallisepticum, a representative of the class Mollicutes, is an excellent model of a minimal cell because of its reduced genome size, lack of a cell wall, and primitive cell organization. In this study we investigated DNA modifications of the model object Mycoplasma gallisepticum and their roles. We identified DNA modifications and methylation motifs in M. gallisepticum S6 at the genome level using single molecule real time (SMRT) sequencing. Only the ANCNNNNCCT methylation motif was found in the M. gallisepticum S6 genome. The studied bacteria have one functional system for DNA modifications, the Type I restriction-modification (RM) system, MgaS6I. We characterized its activity, affinity, protection and epigenetic functions. We demonstrated the protective effects of this RM system. A common epigenetic signal for bacteria is the m6A modification we found, which can cause changes in DNA-protein interactions and affect the cell phenotype. Native methylation sites are underrepresented in promoter regions and located only near the -35 box of the promoter, which does not have a significant effect on gene expression in mycoplasmas. To study the epigenetics effect of m6A for genome-reduced bacteria, we constructed a series of M. gallisepticum strains expressing EGFP under promoters with the methylation motifs in their different elements. We demonstrated that m6A modifications of the promoter located only in the -10-box affected gene expression and downregulated the expression of the corresponding gene.

Evaluation of culturing and molecular assay for detection of Mycoplasma gallisepticum in chicken suffering from chronic respiratory disease.

Mycoplasma gallisepticum (M. gallisepticum) is a bacterium that causes chronic respiratory disease (CRD) and infectious sinusitis (IS) in chicken and turkeys .Therefore, rapid and immediate diagnosis or regular detection of Mycoplasma may be of great help to early detection. 120 chicken layers, Within Karbala city was carried out during their laying period on breeding flocks. The study proposed a promising method for isolation of M. gallisepticum, 120 tracheal swabs and blood samples from chicken in different dairy farms were used to analyze M. gallisepticum utility of PCR and culture. Compared with ELISA anti-IgG M. gallisepticum, the clinical specificity of PCR detection is 89.66%, the sensitivity is 86.36%, and the kappa coefficient is 0.817. Compared with the culture method, the specificity is 100%, the specificity is 45%, and the kappa coefficient is 0.543. Demonstrate the method's effectiveness and applicability as a standard method for mycoplasmas field diagnosis.

Mycoplasma gallisepticum induced exosomal gga-miR-193a to disturb cell proliferation, apoptosis, and cytokine production by targeting the KRAS/ERK signaling pathway.

Mycoplasma gallisepticum (MG) is the main pathogen of chronic respiratory disease (CRD), an infectious disease in chickens with high morbidity. Exosomal miRNAs are emerging as important regulators in host immune response to microbial invasion. Previously, we found that gga-miR-193a was significantly up-regulated in exosomes from MG-infected primary chicken type II pneumocytes (CP-IIs). Therefore, the purpose of this study was to investigate the role of exosomal gga-miR-193a in MG infection. Exosomes were isolated and identified via ultracentrifugation, transmission electron microscopy, and nanoparticle-tracking analysis. Real-time quantitative PCR and Western blot were used to detect the gene expression. Enzyme-linked immunosorbent assay was used to examine the levels of the inflammatory cytokines. CCK-8 and flow cytometry assays were applied to analyze the cell functions. The results showed that MG infection induced high expression of gga-miR-193a in exosomes from CP-IIs. Moreover, exosomes secreted by MG-infected CP-IIs could selectively transport gga-miR-193a into DF-1 cells. Exosomal gga-miR-193a internalized by DF-1 cells inhibited cell proliferation, promoted apoptosis, and increased interleukin-1ß and tumor necrosis factor-α secretions by targeting the RAS/ERK signaling pathway. These results suggest that MG induced the secretion of gga-miR-193a by exosomes to damage the life activities of normal cells, which partially interpreted the mechanism of MG establishing systemic chronic infection in the body.

Detection of Mycoplasma gallisepticum in broiler chickens by PCR.

Background: Mycoplasma is a significant microorganism of poultry, which can cause respiratory infections and synovial inflammation, bringing about huge financial misfortunes to poultry workmanship worldwide. Aim: The goal of existing research was to determine the infection rate of Mycoplasma gallisepticum (MG) from chronic respiratory disease cases among broilers fields in Mosul/ Iraq using the polymerase chain reaction (PCR) technique. Methods: All 92 lungs samples were collected from broilers with classical respiratory signs in different regions of the Nineveh governorate for 3 months from February to April 2021. Results: PCR tests were performed using two couple primers, one for the qualitative amplification of 16S rRNA genes (285 base pairs) in Mycoplasma spp. and the second couple for the detection of M. gallisepticum (580 base pairs). Among the samples obtained from broilers, 87 (94.7%) were positive for Mycoplasma and 79 (85.9%) were positive for M. gallisepticum. Conclusion: Our results showed that MG infection in broiler chickens leads to serious clinical symptoms and severe lesions. The rate of Mycoplasma isolation in this study is high despite the short lifespan of broiler chickens.

Lnc90386 Sponges miR-33-5p to Mediate Mycoplasma gallisepticum-Induced Inflammation and Apoptosis in Chickens via the JNK Pathway.

Mycoplasma gallisepticum (MG) is one of the most important pathogens, that causes chronic respiratory disease (CRD) in chickens. Long non-coding RNAs (lncRNAs) are emerging as new regulators for many diseases and some lncRNAs can function as competing endogenous RNAs (ceRNAs) to regulate mRNAs by competitively binding to miRNAs. Here, we found that miR-33-5p was significantly up-regulated both in MG-infected chicken embryonic lungs and chicken embryo fibroblast cells (DF-1), and Lnc90386 negatively correlated with miR-33-5p. miR-33-5p, as a new regulator for MG infection, repressed apoptosis, inflammatory factors in DF-1 cells by targeting JNK1. Further analyses showed that Lnc90386 sponged miR-33-5p to weaken its inhibitory effect on JNK1, forming the ceRNA regulatory network. Furthermore, knockdown of Lnc90386 significantly inhibited apoptosis and inflammatory factors, and promoted DF-1 cells proliferation. However, co-treatment with miR-33-5p inhibitor and Lnc90386 siRNA showed that knockdown of Lnc90386 could partially eliminate the inhibiting effect of miR-33-5p inhibitor on inflammation, cell apoptosis and proliferation. In conclusion, Lnc90386 sponges miR-33-5p to defend against MG infection by inhibiting the JNK signaling pathway.

Targeted sequencing analysis of Mycoplasma gallisepticum isolates in chicken layer and breeder flocks in Thailand.

Mycoplasma gallisepticum (MG) is one of the most economically important pathogens worldwide. MG affects the respiratory system and impairs growth performance in poultry. In developing countries, the most widely used technique to identify MG is the conventional PCR assay. In this study, 24 MG isolates collected from Thailand farms with unvaccinated chickens during 2002-2020 were characterized by gene-targeted sequencing (GTS), followed by phylogenetic analysis using unweighted pair group method with arithmetic mean. These 24 Thai MG isolates differed from vaccine strains, including the F, ts-11 and 6/85 strains. One isolate showed 99.5-100% genetic similarity to the F strain with 4 partial gene analyses. This result may have been due to contamination from vaccinated flocks because the F strain is the most commonly used vaccine strain in Thailand. However, the GTS analysis using the partial MG genes in this study showed that the isolates could be grouped into different patterns based on individual gene sequences. The phylogenetic analysis of partial mgc2, gapA, pvpA and lp gene sequences classified the Thai MG isolates into 7, 11, 7 and 2 groups, respectively. In conclusion, at least 2 partial MG genes, especially partial gapA and mgc2 genes, are needed to differentiate MG isolates.

Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.

Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants. A guide RNA plasmid, pKM-CRISPR, was constructed, with spacers targeting the ksgA and mnuA genes transcribed under the control of the vlhA1.1 promoter in a backbone plasmid carrying the oriC of M. imitans, and this plasmid was introduced into electrocompetent M. gallisepticum strain S6 cells. PCR assays targeting the ksgA gene, followed by Sanger sequence analyses of the phenotypically resistant transformants, detected polymorphisms within the targeted region of ksgA, confirming the activity of the endogenous CRISPR/Cas system. The nuclease activity of the kasugamycin resistant colonies was then assessed using zymogram assays. The complete or partial loss of nuclease activity in the majority of kasugamycin resistant isolates transformed with the CRISPR plasmid confirmed that the endogenous CRISPR/Cas system had effectively interfered with the function of both ksgA and mnuA genes. Sanger sequencing and RT-qPCR analyses of the mnuA gene suggested that the M. gallisepticum CRISPR/Cas system can be programmed to cleave both DNA and RNA.

Glycyrrhizic Acid against Mycoplasma gallisepticum-Induced Inflammation and Apoptosis Through Suppressing the MAPK Pathway in Chickens.

Mycoplasma gallisepticum (MG) is the primary pathogen of chronic respiratory diseases (CRDs) in chickens. In poultry production, antibiotics are mostly used to prevent and control MG infection, but the drug resistance and residue problems caused by them cannot be ignored. Glycyrrhizic acid (GA) is derived from licorice, a herb traditionally used to treat various respiratory diseases. Our study results showed that GA significantly inhibited the mRNA and protein expression of pMGA1.2 and GapA in vitro and in vivo. Furthermore, the network pharmacology study revealed that GA most probably resisted MG infection through the MAPK signaling pathway. Our results demonstrated that GA inhibited MG-induced expression of MMP2/MMP9 and inflammatory factors through the p38 and JUN signaling pathways, but not the ERK pathway in vitro. Besides, histopathological sections showed that GA treatment obviously attenuated tracheal and lung damage caused by MG invasion. In conclusion, GA can inhibit MG-triggered inflammation and apoptosis by suppressing the expression of MMP2/MMP9 through the JNK and p38 pathways and inhibit the expression of virulence genes to resist MG. Our results suggest that GA might serve as one of the antibiotic alternatives to prevent MG infection.

Genome Engineering in Mycoplasma gallisepticum Using Exogenous Recombination Systems.

Mycoplasma gallisepticum (Mgal) is a common pathogen of poultry worldwide that has recently spread to North American house finches after a single host shift in 1994. The molecular determinants of Mgal virulence and host specificity are still largely unknown, mostly due to the absence of efficient methods for functional genomics. After evaluating two exogenous recombination systems derived from phages found in the phylogenetically related Spiroplasma phoeniceum and the more distant Bacillus subtilis, the RecET-like system from B. subtilis was successfully used for gene inactivation and targeted replacement in Mgal. In a second step, the Cre-lox recombination system was used for the removal of the antibiotic resistance marker in recombinant mutants. This study therefore describes the first genetic tool for targeted genome engineering of Mgal and demonstrates the efficiency of heterologous recombination systems in minimal bacteria.

Mycoplasma gallisepticum induced inflammation-mediated Th1/Th2 immune imbalance via JAK/STAT signaling pathway in chicken trachea: Involvement of respiratory microbiota.

The respiratory microbiota plays a significant role in the host defense against Mycoplasma gallisepticum (MG) infection. The results showed that MG infection changed respiratory microbiota composition, which lead to the tracheal inflammation injury and oxidative stress. MG infection significantly induced immunosuppression in chickens at day 3 and 5 post-infection. In addition, MG infection increased the expressions of pro-inflammatory cytokines in tracheal tissues and activated TLR4 mediated JAK/STAT signaling pathway at day 3 post-infection compared to the control group. Meanwhile, the expressions of pro-inflammatory cytokines were decreased and the expressions of JAK/STAT signaling pathway were decreased at day 5 and day 7 post-infection. On the contrary, the expressions of anti-inflammatory cytokines were significantly decreased at day 3 post-infection and were increased at day 5 and day 7 post-infection in the MG infection group. The antibiotic cocktail group received the respiratory microbiota from the MG infection group, which induced inflammatory injury and oxidative stress, induced mucosal barrier damage by down regulating tight junction-related genes and altered the expressions of mucin, which could be the possible causes of dysregulated immune responses. Importantly, the expressions of pro-inflammatory cytokines were significantly decreased and TLR4 mediated JAK/STAT signaling pathway was downregulated at day 1 and 3 post-transplantation. While, respiratory microbiota transplanted from MG infection significantly increased the expressions of pro-inflammatory cytokines and activated JAK/STAT signaling at day 7 post-transplantation. These results highlighted the role of respiratory microbiota in MG-induced tracheal inflammation injury, and offered a new strategy for the preventive intervention of this disease.

Comparative Proteomic Analysis of the Mycoplasma gallisepticum Nucleoid Fraction before and after Infection.

Mycoplasma gallisepticum belongs to the class Mollicutes and induces severe chronic respiratory disease in chickens. It lacks the cell wall and contains a very small genome and, accordingly, a reduced set of regulatory proteins. It is assumed that one of the regulatory mechanisms in mycoplasmas may be the dynamics of the spatial organization of the chromosome. M. gallisepticum has only two known nucleoid-associated (NAP) histone-like proteins (Hup_1 and Hup_2). To search for new potential NAP that may play a role in the infection process, we isolated nucleoid fractions from M. gallisepticum cells before and after infection of HD3 chicken erythroblast cell line and performed a comparative proteomic analysis of these fractions. We identified several potential NAP that included the components of the terminal organelle and adhesion, VlhA antigen, NADH oxidase, and PykF pyruvate kinase.

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.