Avian Mycoplasma Research Articles

Mycoplasma gallisepticum in vivo induced antigens expressed during infection in chickens

Until now only a few genes encoding virulence factors have been characterized in the avian pathogen Mycoplasma gallisepticum. In order to identify candidate targets associated with infection we applied an immunoscreening technique—in vivo induced antigen technology (IVIAT)—to detect immunogens of M. gallisepticum strain Rlow expressed preferentially during in vivo infection. We identified 13 in vivo-induced (IVI) proteins that correspond to different functional categories including: previously reported putative virulence factors (GapA, PlpA, Hlp3, VlhA 1.07 and VlhA 4.01), transport (PotE, MGA_0241 and 0654), translation (L2, L23, ValS), chaperone (GroEL) and a protein with unknown function (MGA_0042). To validate the in vivo antigenic reactivity, 10 IVI proteins were tested by Western blot analysis using serum samples collected from chickens experimentally (with strain Rlow) and naturally (outbreaks, N=3) infected with M. gallisepticum. All IVI proteins tested were immunogenic. To corroborate these results, we tested expression of IVI genes in chickens experimentally infected with M. gallisepticum Rlow, and in MRC-5 human lung fibroblasts cell culture by using relative real time reverse-transcription PCR (RT-PCR). With the exception of MGA_0338, all six genes tested (MGA_1199, 0042, 0654, 0712, 0928 and 0241) were upregulated at least at one time point during experimental infection (2–4 week post-infection). In contrast, the expression of seven out of eight IVI genes (MGA_1199, 0152, 0338, 0042, 0654, 0712, 0928) were downregulated in MRC-5 cell culture at both 2 and 4h PI; MGA_0241 was upregulated 2h PI. Our data suggest that the identified IVI antigens may have important roles in the pathogenesis of M. gallisepticum infection in vivo.

Transcriptome analysis reveals novel regulatory mechanisms in a genome-reduced bacterium

The avian bacterial pathogen Mycoplasma gallisepticum is a good model for systems studies due to small genome and simplicity of regulatory pathways. In this study, we used RNA-Seq and MS-based proteomics to accurately map coding sequences, transcription start sites (TSSs) and transcript 3′-ends (T3Es). We used obtained data to investigate roles of TSSs and T3Es in stress-induced transcriptional responses. We identified 1061 TSSs at a false discovery rate of 10% and showed that almost all transcription in M. gallisepticum is initiated from classic TATAAT promoters surrounded by A/T-rich sequences. Our analysis revealed the pronounced operon structure complexity: on average, each coding operon has one internal TSS and T3Es in addition to the primary ones. Our transcriptomic approach based on the intervals between the two nearest transcript ends allowed us to identify two classes of T3Es: strong, unregulated, hairpin-containing T3Es and weak, heat shock-regulated, hairpinless T3Es. Comparing gene expression levels under different conditions revealed widespread and divergent transcription regulation in M. gallisepticum. Modeling suggested that the core promoter structure plays an important role in gene expression regulation. We have shown that the heat stress activation of cryptic promoters combined with the hairpinless T3Es suppression leads to widespread, seemingly non-functional transcription.

Hydrogen peroxide production from glycerol metabolism is dispensable for virulence of Mycoplasma gallisepticum in the tracheas of chickens.

Hydrogen peroxide (H2O2) is a by-product of glycerol metabolism in mycoplasmas and has been shown to cause cytotoxicity for cocultured eukaryotic cells. There appears to be selective pressure for mycoplasmas to retain the genes needed for glycerol metabolism. This has generated interest and speculation as to their function during infection. However, the actual effects of glycerol metabolism and H2O2 production on virulence in vivo have never been assessed in any Mycoplasma species. To this end, we determined that the wild-type (WT) R(low) strain of the avian pathogen Mycoplasma gallisepticum is capable of producing H2O2 when grown in glycerol and is cytotoxic to eukaryotic cells in culture. Transposon mutants with mutations in the genes present in the glycerol transport and utilization pathway, namely, glpO, glpK, and glpF, were identified. All mutants assessed were incapable of producing H2O2 and were not cytotoxic when grown in glycerol. We also determined that vaccine strains ts-11 and 6/85 produce little to no H2O2 when grown in glycerol, while the naturally attenuated F strain does produce H2O2. Chickens were infected with one of two glpO mutants, a glpK mutant, R(low), or growth medium, and tracheal mucosal thickness and lesion scores were assessed. Interestingly, all glp mutants were reproducibly virulent in the respiratory tracts of the chickens. Thus, there appears to be no link between glycerol metabolism/H2O2 production/cytotoxicity and virulence for this Mycoplasma species in its natural host. However, it is possible that glycerol metabolism is required by M. gallisepticum in a niche that we have yet to study.

Cytological evaluation of necropsy guided impression smears of chronic respiratory disease of chickens

The study was conducted to develop a cytology based diagnostic tool for the diagnosis of avian Mycoplasma and E. coli infections at post mortem in the field condition. A total of 38 culture and PCR confirmed Mycoplasma, E. coli or mixed infected samples were used for this study. Lung impression smears were prepared on glass slide from the samples at post mortem examination. Inflammatory cells were counted on microscope after Giemsa staining. Cell counts were analyzed with Bonferroni joint confidence interval and Mann-Whitney U test. The average cell percentages in healthy cases were 73.54- 81.66%, 9.63-13.37% and 7.42-14.38% for lymphocyte, heterophil, and macrophage, respectively. In case of Mycoplasma infection, average percentages of lymphocyte, heterophil and macrophages were 82.01-88.10%, 5.6 to 8.16% and 4.52 – 9.68%, respectively. In E. coli infection, average percentage of lymphocyte, heterophil and macrophages were found as 64.44-70.76%, 19.73-23.47% and 8.9- 12.7%, respectively. In mixed infection, lymphocyte, heterophil and macrophage were found as 76.08-80.50%, 13.47 –17.63% and 4.56 –7.66%, respectively. Statistical analyses revealed that in Mycoplasma infection number of lymphocyte and in E. coli infection number of heterophil increased significantly (p< 0.01). In MC complex, number of heterophil increased and macrophages decreased significantly (p < 0.01). These findings could help identification of Mycoplasma, E. coli or Mycoplasma- E. coli complex at post mortem examination in the field condition. DOI: http://dx.doi.org/10.3329/sja.v10i2.18331 SAARC J. Agri., 10(2): 95-105 (2012)

Molecular detection of infectious bronchitis virus and it is relation with avian influenza virus (H9) and Mycoplasma gallisepticum from different geographical regions in Iraq

Infectious bronchitis virus (IBV), Avian influenza virus (AIV) and Mycoplasma gallisepticum (MG) have been recognized as the most important pathogens in poultry cause acute respiratory infection and serous economic problems in Iraq and many other countries all over the world. This study was conducted to investigate the distribution of these diseases in commercial chicken flocks in different geographical region in middle part of Iraq by using qPCR. Tracheal swabs and tissue specimens from trachea, lung and kidney were taken from 38 different cases from commercial broiler chicken flocks in (Najaf, Hilla, Muthana and Theqaar governorates) in the period from November 2010 to June 2011, all these flocks were showed respiratory symptoms and mortality about 20-90%. The results showed that 92.1% of samples collected from these flocks were infected with IBV, 20% of samples were infected with IB alone and 45.71% of samples with IB combined with both GM and AIV subtype H9 and 25.71% of samples were positive to both IBV and AIV(H9). No samples were positive to AIV (H9) or MG alone. Because of importance of respiratory diseases as a most common conditions noted in commercial flocks in Iraq and no previous study detecting this pathogens by molecular techniques, this study come to detect and confirm the diagnosis of this pathogens by qPCR as new technique used in this field in Iraq.

Diacylated lipopeptide from Mycoplasma synoviae mediates TLR15 induced innate immune responses

Avian-specific toll like receptor 15 (TLR15) is functionally equivalent to a group of TLR2 family proteins that the mammalian innate immune system utilizes to recognize a broad spectrum of microbe-associated molecular patterns, including bacterial lipoproteins. In this study we examined the role of chicken TLR2 family members in the innate immune response to the avian pathogenic bacterium, Mycoplasma synoviae. We found that Mycoplasma synoviae, and specifically the N-terminal diacylated lipopeptide (MDLP) representing the amino-terminal portion of its mature haemagglutinin protein, significantly induces the expression of TLR15, but not TLR1 and TLR2 in chicken macrophages and chondrocytes. TLR15 activation is specific and depends on diacylation of the lipopeptide. Activation of TLR15 after stimulation with Mycoplasma synoviae and MDLP triggers an increase in the expression of transcription factor nuclear factor kappa B and nitric oxide production. Moreover, transfection of avian macrophage cells with small interfering RNA reduces the expression of TLR15 after stimulation with MDLP. This leads to decreased activation of the innate immune response, as measured by nitric oxide production. Additionally, pretreatment of cells with neutralizing anti-TLR15 antibody results in a notable attenuation of MDLP-driven release of nitric oxide. This positive correlation may constitute a mechanism for stimulating the innate immune response against avian mycoplasmas in chicken cells via TLR15.

Development of Duplex PCR Assay for the Detection of Mycoplasma Gallisepticum and Mycoplasma Synoviae Prevalence in Pakistan

Mycoplasma infections are of great concern in avian medicine, because they cause economic losses in commercial poultry production of Pakistan. Timely and efficient diagnosis of the mycoplasma is needed in order to practice prevention and control strategies. A duplex Polymerase Chain Reaction (PCR) was optimized for simultaneous detection of two pathogenic species of avian mycoplasmas. Two sets of oligonucleotide primers specific for Mycoplasma gallisepticum and Mycoplasma synoviae, were used in the test. The developed assay exhibited high sensitivity and 100% specificity for the simultaneous detection of mycoplasma species prevalence in Pakistan.

MalF is essential for persistence of Mycoplasma gallisepticum in vivo

There is limited understanding of the molecular basis of virulence in the important avian pathogen Mycoplasma gallisepticum. To define genes that may be involved in colonization of chickens, a collection of mutants of the virulent Ap3AS strain of M. gallisepticum were generated by signature-tagged transposon mutagenesis. The collection included mutants with single insertions in the genes encoding the adhesin GapA and the cytadherence-related protein CrmA, and Western blotting confirmed that these mutants did not express these proteins. In two separate in vivo screenings, two GapA-deficient mutants (ST mutants 02-1 and 06-1) were occasionally recovered from birds, suggesting that GapA expression may not always be essential for persistence of strain Ap3AS. CrmA-deficient ST mutant 33-1 colonized birds poorly and had reduced virulence, indicating that CrmA was a significant virulence factor, but was not absolutely essential for colonization. ST mutant 04-1 contained a single transposon insertion in malF, a predicted ABC sugar transport permease, and could not be reisolated even when inoculated by itself into a group of birds, suggesting that expression of MalF was essential for persistence of M. galliseptium strain Ap3AS in infected birds.

Comparison of three diagnostics methods of Mycoplasma gallisepticum in Batna governorate (Algeria)

Mycoplasma gallisepticum infection is commonly designated as chronic respiratory disease in chickens. It is the most pathogenic avian Mycoplasma; however, strains may differ markedly in virulence. In this study, the technical performance of culture, a commercially available polymerase chain reaction (PCR) test and rapid plate agglutination (SPA) test were compared for the detection of Mycoplasma gallisepticum infections from 18 birds. Results showed a high percentage of positive samples of both culture and PCR tests (72.22% and 63.63% respectively). SPA showed a less positive rate (61.11%). the utilization of SPA towards MG diagnosis is limited by its reduced specificity and the high incidence of false positives Contradictory to other studies, bacteriology was more sensitive than PCR. Several studies support strongly the use of the PCR as a technique for the diagnosis of Mycoplasma infections since this technique is more sensitive than serology and culture. This study showed that it is not advisable to rely completely on one test (system) only.

Identification of avian Mycoplasma species in commercial broilers and layers with respiratory symptoms in Balochistan

Among many avian mycoplasmas, only the Mycoplasmas gallisepticum (MG) andMycoplasmas synoviae (MS) are responsible for causing respiratory disease in commercial poultry. This study reported for the very first time the serological occurrence of M. gallisepticum and M. synoviae in blood samples (n = 600) from sixty flocks (n = 42 broiler and n = 18 layer flocks) with respiratory symptoms in Quetta, Pishin and Kuchlak districts of Balochistan, Pakistan. Sera were tested for MG and MS antibody responses by serum plate agglutination (SPA) test and by enzyme-linked immunosorbant assay (ELISA) Synbiotics kit. It was found that M. gallisepticumantibodies in broiler flocks detected by SPA and by ELISA tests were 10.47 and 19.76%, whereas M. synoviae were 7.86 and 11.19%, respectively. In layer flocks the MG and MS antibodies detected by SPA and ELISA were 19.44, 31.66 and 8.8, 25%, respectively. The overall antibodies of MG and MS in both broiler and layer flocks tested by SPA and ELISA was found to be 13.16, 23.33 and 8.16, 15.33%, respectively. In broiler and layer flocks the presence of antibodies against both M. gallisepticum and M. synoviae were found in tested flocks with respiratory symptoms. Further studies on prevalence and diagnosis of both the M. gallisepticum and M. synoviae in causing respiratory disease in commercial broilers and layers in Balochistan are required. Key words: Mycoplasma, broilers, serum plate agglutination, enzyme-linked immunosorbent assay.

A diagnostic polymerase chain reaction for Mycoplasma iowae using primers located in the intergenic spacer region and the 23S rRNA gene

Mycoplasma iowae is primarily a pathogen of turkeys and, although uncommon, it still persists in some areas of the world, where it may cause embryo mortality and leg lesions. A species-specific diagnostic polymerase chain reaction was developed using a forward primer based in the intergenic spacer region between the 16S rRNA and the 23S rRNA ribosomal genes and a reverse primer located within the 23S rRNA gene. The polymerase chain reaction proved to be both sensitive and specific. It detected M. iowae DNA in the six reference strains of serotypes I, J, K, N, Q and R and in 28 field isolates. With the six serotypes the test detected between 1 and 5 pg of M. iowae DNA. There were no non-specific reactions with the other avian Mycoplasma species. When the closest phylogenetically related species were checked, a weak reaction with Mycoplasma muris was observed that disappeared when the annealing temperature was increased by 2°C.

Detection and Differentiation of Avian Mycoplasmas by Surface-Enhanced Raman Spectroscopy Based on a Silver Nanorod Array

Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array-surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.

A Comparison of Serological and Bacteriological Methods for Detection of Mycloplasma gallisepticum in Experimentally-Infected Chickens

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to Mycoplasma gallisepticum. Three antigens were used in this experiment. Antigen 1 was prepared from whole cell of M. gallisepticum, antigen 2 was a sodium dodecyl sulfate-solubilized preparation from whole cells, and antigen 3 was prepared by sonication of the whole cell antigen. The assay was then used to detect (anti)-M. gallisepticum antibodies in experimentally-infected chickens compared with serum-plate-agglutination (SPA), haemagglutination-inhibition (HI) tests, and tracheal culture. Data obtained in this experiment showed that there was a correlation between seropositivity and rate of isolation of M. gallisepticum. ELISA was found to be less sensitive, but more specific than SPA, and more sensitive than the HI test. The whole cell antigen gave the highest optical densities but was less specific than the other two antigens. The ELISA using all three antigens successfully identified the M. gallisepticum-infected chickens uniformly and positively through 14-35 days post infection, and correctly identified the control group as negative through the 35 day experimental period. The ELISA obviously has a place in the serodiagnosis of avian mycoplasma. Improved-specificity and -sensitivity of the M. gallisepticum antigen is desirable.

Adaptation and cytopathic effects of avian mycoplasma in chicken embryo cell cultures.

Summary An investigation was carried out to adapt six avian Mycoplasma namely M. gallisepticum (S6), M. gallinarum (PG16) and four local Mycoplasma isolates in chicken embryo cell cultures. Four of the six Mycoplasma cultures produced cytopathic effects in chicken embryo cell cultures. The time of appearance of cytopathic effects decreases with the increase of serial passages. Zusammenfassung Adaptation und cytopathische Effekte von aviären Mykoplasmen in Hühnerembryozellkulturen Untersuchungen zur Adaption von sechs aviären Mykoplasmastämmen M. gallisepticum (S6), M. gallinarum (PG16) und vier lokalen Mykoplasmaisolaten an Hühnerembryofibroblasten wurden durchgeführt. Vier der sechs Stämme in duzierten cytopathische Effekte in Hühnerembryozellkulturen. Die Zeit des Auftretens cytopathischer Effekte verkürzte sich mit laufender Passagezahl.

Differences of Seropositivity against Selected Infectious Agents in Relation to Farm Management Characteristics to Broiler Production in Uruguay

The objective of this study was to investigate risk factors, especially for management characteristics associated with the seropositivity of respiratory infectious agents such as avian pneumovirus, Ornithobacterium rhinotracheale, Mycoplasma gallisepticum and Mycoplasma synoviae in broiler chickens on farms in Uruguay. Seventeen farms of broiler chickens (>35 days old) were studied between October 2008 and April 2009, comprised data collection through questionnaire interviews for each study farm, in combination with blood sample collections for each chicken (n = 1861). Of all the 17 study farms, 13, 13, five and nine farms were classified as seropositive against avian pneumovirus, Ornithobacterium rhinotracheale, Mycoplasma gallisepticum and Mycoplasma synoviae, respectively. The seropositivity against Ornithobacterium rhinotracheale in relation to the use of vaccination programmes indicated statistical significance. The farms with the vaccination programmes would be more likely to practise the same or similar sanitary measures, which could contribute to prevent the chickens from various infection. Practice of a sanitary measure would be indicative of a poultry farm to distinguish potential risks of diseases.

Phytopathogenicity of avian mycoplasma Mycoplasma gallisepticum S6: Morphologic and ultracytostructural changes in plants infected with the vegetative forms and the viable but nonculturable forms of the bacterium

The data obtained in this study proved that Mycoplasma gallisepticum S6 known as avian pathogen had a phytopathogenic potential. The vegetative forms and the viable but nonculturable (VBNC) forms of this mycoplasma could infect the plants via an assemblage of rootlets, invade different tissues, persist there and cause destructive events characteristic to phytomycoplasmoses. In comparison with the vegetative forms, the VBNC forms induced more prominent destructive changes. This phenomenon might be connected to increasing expression of proteins responsible for virulence in the bacterial cells. The fact that M. gallisepticum S6 could demonstrate virulent features (infectivity, invasiveness, persistence and toxigenicity) in regard to plants seems to require a development of new ways for controlling phytomycoplasmoses taking into account the probable presence of asymptomatic carriers of this bacterium.

The Development of Diagnostic Real-Time Taqman PCRs for The Four Pathogenic Avian Mycoplasmas

Four avian mycoplasmas are commonly recognized as poultry pathogens: Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Mycoplasma meleagridis (MM), and Mycoplasma iowae (MI). The avian mycoplasmas are associated with respiratory disease, synovitis and arthritis, poor performance, skeletal deformities, and embryo mortality. Three main approaches are used for the diagnosis of avian mycoplasmosis: isolation and identification, detection of antibodies, and molecular detection of the organism's nucleic acid by PCR. In recent years real-time PCR technology has revolutionized the way clinical microbiology laboratories diagnose infectious diseases, but so far only a limited number of diagnostic real-time PCRs have been proposed for avian mycoplasma diagnostics. We developed a complete set of reliable diagnostic real-time TaqMan PCR assays for the four pathogenic avian mycoplasmas. The selected genomic targets of the developed assays were species specific and intraspecifically conserved and included the 16S-23S intergenic spacer region of MS and MM, the upstream region to the 16S ribosomal DNA of MI, and highly conserved foci of the mgc2 gene of MG. The four assays were demonstrated to be highly specific and sensitive to their target avian mycoplasma, with detection limits of one copy per reaction mix for the MG assay and 10 copies per reaction mix for the MS, MM, and MI assays. We believe that the incorporation of the developed assays in avian mycoplasma diagnostics will contribute to the accuracy, efficiency, and feasibility of diagnosis of these pathogens.

Development and evaluation of an improved diagnostic PCR for Mycoplasma synoviae using primers located in the haemagglutinin encoding gene vlhA and its value for strain typing

Using published primers, detection of Mycoplasma synoviae and strain identification using the vlhA gene sequence was attempted. However, of 21 M. synoviae strains examined, three could not be amplified, so a new reverse primer was designed with a target in the conserved region of the vlhA gene. This allowed all 21 M. synoviae strains, a further nine strains and also material from 11 swab samples from M. synoviae-positive birds, to produce a PCR product, suggesting that the method could also be suitable for clinical specimens. The protocol was then tested on the type strains of M. synoviae and the other 22 recognised avian Mycoplasma species, with amplification of M. synoviae only. Further testing demonstrated that this PCR was equally or more sensitive than other PCR tests used to detect M. synoviae. Subsequent DNA sequence analysis of the PCR product based on percent similarity and evolutionary relationship appeared to be a useful tool for strain differentiation.

Use of polymerase chain reactions to detect Mycoplasma gallisepticum, Mycoplasma imitans, Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae in birds of prey

Certain Mycoplasma spp. are pathogens of poultry, but little is known of the role of mycoplasmas in disease of birds of prey. Species-specific polymerase chain reactions (PCRs) for the detection of the poultry pathogens Mycoplasma gallisepticum, Mycoplasma imitans, Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae were therefore evaluated for use in birds of prey. The specificities of the PCR methods were established using avian and other mycoplasmas and also selected walled bacteria. The sensitivities of the different PCR assays varied between 100 fg and 10 pg DNA. Fifty-three tracheal swabs from healthy captive and free-ranging birds of prey were then investigated using these PCRs, and in no case was an amplicon obtained for M. gallisepticum/M. imitans, M. iowae or M. synoviae. Species-specific primers for M. meleagridis amplified a product from eight birds of prey but restriction enzyme analysis as well as sequencing of PCR products demonstrated these results to be false positives. Alignment studies of the sequenced products with the 16S rRNA gene sequence of various Mycoplasma species in GenBank demonstrated an identity of 91% to M. meleagridis but of 98% to Mycoplasma buteonis or Mycoplasma gallopavonis. Isolation and attempted identification of these mycoplasmas suggested it may be a previously unrecognized species.

Control of Avian Mycoplasma Infections in Commercial Poultry

Control of pathogenic avian mycoplasmas can consist of one of three general approaches: Maintaining flocks free of infection, medication, or vaccination. Maintaining flocks free of pathogenic mycoplasmas consists of maintaining replacements from mycoplasma-free sources in a single-age, all-in all-out management system. Good biosecurity and an effective monitoring system are necessary aspects of this program. Medication can be very useful in preventing clinical signs and lesions, as well as economic losses, but cannot be used to eliminate infection from a flock and is therefore not a satisfactory long-term solution. Vaccination against Mycoplasma gallisepticum (MG) or M. synoviae (MS) can be a useful long-term solution in situations where maintaining flocks free of infection is not feasible, especially on multi-age commercial egg production sites.