Real-time Loop-mediated Isothermal Amplification (LAMP) of mgc2 Gene of Mycoplasma Gallisepticum.
ABSTRACT: Introduction:Mycoplasma gallisepticum is considered the most pathogenic and economically significant avian Mycoplasma spp. for the worldwide poultry industry. The aim of this study was to develop a novel and sensitive real-time loop-mediated isothermal amplification (LAMP) assay based on the amplification of its mgc2 gene sequence for its rapid molecular detection in poultry. Material and Methods:Blood samples from 300 broiler and layer chickens were screened using a rapid serum agglutination (RSA) test. A real-time LAMP reaction was conducted with seropositive swab samples at 60ºC for 90 min in an ESEQuant tube scanner using 6-carboxyfluorescein as the reporting dye. Results:The sensitivity of the developed assay was 10 fg/µL of DNA. The assay was found 100% specific, showing no cross-reactivity with other avian Mycoplasma species. The proportion found of the positive samples by the real-time LAMP was 58%. In comparison, the RSA was found to detect 52% of positive cases. Conclusion:The mgc2 real-time LAMP emerged as a more sensitive and accurate method for molecular detection of M. gallisepticum than RSA. Robustness and precision give it applicability as a potential field diagnostic tool for M. gallisepticum control. The study will be beneficial in reducing economic losses that M. gallisepticum inflicts on the poultry industry. This is the first reported development of a real-time LAMP assay based on the amplification of the mgc2 gene sequence using an ESEQuant tube scanner for galline M. gallisepticum detection.
Project description:Mycoplasma gallisepticum, the most pathogenic mycoplasma in poultry, is able to glide over solid surfaces. Although this gliding motility was first observed in 1968, no specific protein has yet been shown to be involved in gliding. We examined M. gallisepticum strains and clonal variants for motility and found that the cytadherence proteins GapA and CrmA were required for gliding. Loss of GapA or CrmA resulted in the loss of motility and hemadsorption and led to drastic changes in the characteristic flask-shape of the cells. To identify further genes involved in motility, a transposon mutant library of M. gallisepticum was generated and screened for motility-deficient mutants, using a screening assay based on colony morphology. Motility-deficient mutants had transposon insertions in gapA and the neighbouring downstream gene crmA. In addition, insertions were seen in gene mgc2, immediately upstream of gapA, in two motility-deficient mutants. In contrast to the GapA/CrmA mutants, the mgc2 motility mutants still possessed the ability to hemadsorb. Complementation of these mutants with a mgc2-hexahistidine fusion gene restored the motile phenotype. This is the first report assigning specific M. gallisepticum proteins to involvement in gliding motility.
Project description:Currently, there are no FDA-approved nucleic acid amplification tests (NAATs) for the detection or confirmation of HIV-2 infection. Here, we describe the development of a real-time assay for the detection of HIV-2 DNA and RNA using reverse transcription-loop-mediated isothermal amplification (RT-LAMP) and the ESEQuant tube scanner, a portable isothermal amplification/detection device.
Project description:Mycoplasma gallisepticum (MG) is among the most significant problems in the poultry industry worldwide, representing a serious threat to international trade. Despite the fact that the mgc2 gene has been widely used for diagnostic and molecular characterization purposes, there is a lack of evidence supporting the reliability of this gene as a marker for molecular epidemiology approaches. Therefore, the current study aimed to assess the accuracy of the mgc2 gene for phylogenetic, phylodynamic, and phylogeographic evaluations. Furthermore, the global phylodynamic expansion of MG is described, and the origin and extension of the outbreak caused by MG in Ecuador were tracked and characterized. The results obtained strongly supported the use of the mgc2 gene as a reliable phylogenetic marker and accurate estimator for the temporal and phylogeographic structure reconstruction of MG. The phylodynamic analysis denoted the failures in the current policies to control MG and highlighted the imperative need to implement more sensitive methodologies of diagnosis and more efficient vaccines. Framed in Ecuador, the present study provides the first piece of evidence of the circulation of virulent field MG strains in Ecuadorian commercial poultry. The findings derived from the current study provide novel and significant insights into the origin, diversification, and evolutionary process of MG globally.
Project description:Background and Aim:Respiratory bacterial agents represent one of the most harmful factors that ordinarily threaten the poultry industry and usually lead to great economic losses. Meanwhile, there is a global demand to avoid the highly emerging antibiotic resistance and antibiotic residues in edible meat. Whereas, the use of alternatives became of great priority, especially for those substances extracted from natural plant origin. The study aimed to evaluate the antibacterial effect of cinnamon oil as a herbal extract on different respiratory bacterial agents. Materials and Methods:One hundred and fifty biological samples were collected through targeted surveillance for respiratory diseased poultry farms representing three governorates, from which bacterial isolation and identification, DNA sequencing of representative strains were performed. Furtherly, phenotypic and genotypic evaluation of the antibacterial effect of cinnamon oil was performed by minimum inhibitory concentration, agar disk diffusion, and virulence genes expression real-time polymerase chain reaction. Results:Cinnamon oil gave rise to acceptable degrees of virulence genes downregulation of 0.15, 0.19, 0.37, 0.41, 0.77, and 0.85 for Staphylococcus aureus sed gene, Escherichia coli stx1 gene, Avibacterium paragallinarum HPG-2 gene, Pasteurella multocida ptfA gene, Mycoplasma gallisepticum Mgc2 gene, and Ornithobacterium rhinotracheale adk gene, respectively. Phenotypically, using agar disk diffusion assay and broth microdilution susceptibility, cinnamon oil showed also tolerable results as it stopped the growth of S. aureus, E. coli, P. multocida, and A. paragallinarum with varying zones of inhibition. Conclusion:The encountered results declared the successful in vitro effect of cinnamon oil that recommends its application for living birds for future use as a safe antibacterial in the poultry industry.
Project description:A second cytadhesin-like protein, MGC2, was identified in the avian respiratory pathogen Mycoplasma gallisepticum. The 912-nucleotide mgc2 gene encodes a 32.6-kDa protein with 40.9 and 31.4% identity with the M. pneumoniae P30 and M. genitalium P32 cytadhesins, respectively. Functional studies with reverse transcription-PCR, immunoblotting, double-sided immunogold labeling, and attachment inhibition assays demonstrated homology to the human mycoplasmal P30 and P32 cytadhesins. These findings suggest that there is a family of cytadhesin genes conserved among pathogenic mycoplasmas infecting widely divergent hosts.
Project description:Mycoplasma synoviae (MS) remains a serious concern in production of poultry and affects world production of chickens and turkeys. Loop-mediated isothermal amplification (LAMP) of DNA has been recently used for the identification of different economically important avian pathogens. The aim of this study was to develop LAMP for simple and inexpensive detection of MS strains in poultry using specifically designed primers targeting hemagglutin A (vlh) gene. The assay was conducted in a water bath for 1 h at 63 °C. The results were visualized after addition of SYBR Green(®) fluorescent dye. LAMP was specific exclusively for MS without cross-reactivity with other Mycoplasma species. The sensitivity of LAMP was determined as 10(-1) CFU/ml and was 1,000 times higher than MS-specific polymerase chain reaction. LAMP assay was conducted on 18 MS field strains to ensure its reliability and usefulness. This is the first report on LAMP development and application for the rapid detection of MS isolated from chickens. This simple method may be applied by diagnostic laboratories without access to expensive equipment.
Project description:Mycoplasma gallisepticum (MG) is economically important pathogen of poultry causes airsacculitis and frequently infraorbital sinusitis in turkeys. Infections may remain without clinical signs, but they can make birds susceptible to secondary infections. This study was carried out for molecular detection and phylogenetic analysis of MG infections in commercial and backyard turkey flocks in some parts of Iran. A total number of 600 swab samples were collected from 18 commercial and 31 backyard turkey flocks. The PCR technique was performed for detecting 16S rRNA gene in the samples. Positive sample were subjected for sequencing of mgc2 gene. The results showed that 48.38% of backyard and 16.66% of commercial farms were positive for MG. These findings suggested the presence of MG in the commercial and backyard turkeys' farms of Iran. The molecular analysis indicated high sequence similarity between some Iranian turkeys isolates with Indian and Pakistanian MG isolates. Furthermore, substitutions of MG nucleic acids and correlated amino acids sequences may lead to some antigenic modifications.
Project description:Mycoplasma gallisepticum is an avian respiratory and reproductive tract pathogen that has a significant economic impact on the poultry industry worldwide. Although membrane proteins of Mycoplasma spp. are thought to play crucial roles in host interactions, very few have had their biochemical function defined. In this study, we found that the GroEL protein (heat shock protein 60) of Mycoplasma gallisepticum could induce apoptosis in peripheral blood mononuclear cells, and the underlying molecular mechanism was further determined. The GroEL gene from Mycoplasma gallisepticum was cloned and expressed in Escherichia coli to facilitate the functional analysis of recombinant protein. The purified GroEL protein was shown to adhere to peripheral blood mononuclear cells (PBMCs) and DF-1 cells and cause apoptosis in PBMCs. A protein pulldown assay coupled with mass spectrometry identified that annexin A2 possibly interacted with GroEL protein. Coimmunoprecipitation assays confirmed that GroEL proteins could bind to annexin A2, and confocal analysis further demonstrated that GroEL colocolized with annexin A2 in HEK293T cells and PBMCs. Moreover, annexin A2 expression was significantly induced by a recombinant GroEL protein in PBMCs, and knocking down annexin A2 expression resulted in significantly reduced apoptosis. Taken together, these data suggest that GroEL induces apoptosis in host cells by interacting with annexin A2, a novel virulence mechanism in Mycoplasma gallisepticum Our findings lead to a better understanding of molecular pathogenesis in Mycoplasma gallisepticum.
Project description:A putative cytadhesin-related protein (PvpA) undergoing variation in its expression was identified in the avian pathogen Mycoplasma gallisepticum. The pvpA gene was cloned, expressed in Escherichia coli, and sequenced. It exhibits 54 and 52% homology with the P30 and P32 cytadhesin proteins of the human pathogens Mycoplasma pneumoniae and Mycoplasma genitalium, respectively. In addition, 50% homology was found with the MGC2 cytadhesin of M. gallisepticum and 49% homology was found with a stretch of 205 amino acids of the cytadherence accessory protein HMW3 of M. pneumoniae. The PvpA molecule possesses a proline-rich carboxy-terminal region (28%) containing two identical directly repeated sequences of 52 amino acids and a tetrapeptide motif (Pro-Arg-Pro-X) which is repeated 14 times. Genetic analysis of several clonal isolates representing different expression states of the PvpA product ruled out chromosomal rearrangement as the mechanism for PvpA phase variation. The molecular basis of PvpA variation was revealed in a short tract of repeated GAA codons, encoding five successive glutamate resides, located in the N-terminal region and subject to frequent mutation generating an in-frame UAA stop codon. Size variation of the PvpA protein was observed among M. gallisepticum strains, ranging from 48 to 55 kDa and caused by several types of deletions occurring at the PvpA C-terminal end and within the two directly repeated sequences. By immunoelectron microscopy, the PvpA protein was localized on the mycoplasma cell surface, in particular on the terminal tip structure. Collectively, these findings suggest that PvpA is a newly identified variable surface cytadhesin protein of M. gallisepticum.
Project description:<i>Mycoplasma gallisepticum</i> causes chronic respiratory diseases in poultry and economic losses to the chicken and turkey industry. We report the complete genome sequence of the field isolate strain KUVMG001 of <i>Mycoplasma gallisepticum</i> from South Korea.