Sinusitis Of Turkeys Research Articles

Респираторные заболевания у кур и индеек, вызванные Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) is a prokaryote, intermediate between bacteria and viruses, and lacks a cell wall. MG mainly causes chronic respiratory disease in chickens and other poultry and infectious sinusitis in turkeys. In addition, MG infection can cause poor carcass sorting and feed processing efficiency in broiler chickens. Egg production and hatching rate in laying hens tends to decrease with increasing numbers of weak broods. Several methods are available for isolation of the microorganism: culturing, PCR diagnosis, etc. There is no definite treatment against this disease, but there are a number of attenuated vaccines produced by different countries as prophylaxis. At this point in time, the question remains both the need for further identification of a more sensitive and specific diagnostic system for the invention, as well as more in-depth studies of mycoplasma at the molecular level, which will allow further production of vaccines with a large number of strains.

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.

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.

Detection of some Pathogenic Bacteria Causing Sinusitis in Turkeys by Using Multiplex PCR

Detection of some Pathogenic Bacteria Causing Sinusitis in Turkeys by Using Multiplex PCR

A Mycoplasma gallisepticum Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen Mycoplasma gallisepticum To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in malF (MalF-deficient ST mutant 04-1; ΔmalF) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the ΔmalF mutant, compared to the wild type. Stable isotope labeling using [U-13C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into M. gallisepticum and that, in the absence of MalF, the transcription of gtsA, which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF. The loss of MalF appeared to have a global effect on glycerol metabolism, suggesting that it may also play a regulatory role, and cellular morphology was also affected, indicating that the change to glycerol metabolism may have a broader effect on cellular organization. Overall, this study suggests that the reduced virulence of the ΔmalF mutant is due to perturbed glycerol uptake and metabolism and that the operon including malF should be reannotated as golABC to reflect its function in glycerol transport.IMPORTANCE Many mycoplasmas are pathogenic and cause disease in humans and animals. M. gallisepticum causes chronic respiratory disease in chickens and infectious sinusitis in turkeys, resulting in economic losses in poultry industries throughout the world. Expanding our knowledge about the pathogenesis of mycoplasma infections requires better understanding of the specific gene functions of these bacteria. In this study, we have characterized the metabolic function of a protein involved in the pathogenicity of M. gallisepticum, as well as its effect on expression of selected genes, cell phenotype, and H2O2 production. This study is a key step forward in elucidating why this protein plays a key role in virulence in chickens. This study also emphasizes the importance of functional characterization of mycoplasma proteins, using tools such as metabolomics, since prediction of function based on homology to other bacterial proteins is not always accurate.

Complete Sequence-Based Genotyping of mgc2/pvpA Genes in Chicken-Derived Mycoplasma gallisepticum Isolates of Iran.

Mycoplasma gallisepticum (MG) is a major pathogen of the poultry industry throughout the world. MG causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite constant improvements in the biosecurity of the poultry industry in Iran, MG infection still occurs and causes significant economic issues. To evaluate genetic variability, 10 Iranian MG isolates along with 17 available sequences were characterized by gene-targeted sequencing (GTS) analysis of complete mgc2/pvpA genes. According to the findings, 21 different sequence types within the sample set of 27 strains were typed by this method. The discriminatory power of this typing assay was established to be 0.97. Although no insertions and deletions of nucleotides were observed in the mgc2 gene among the Iranian strains, different lengths of pvpA genes with 1086, 1095, and 1101 nucleotides were detected within direct repeats (DRs) 1 and 2. Generally, eight tetrapeptides Pro-Arg-Pro-Met/Gln/Asn were found in the DRs of PvpA. Analysis of the carboxyl ends of PvpA proteins exhibited various repeats of prolines. In the phylogenetic tree of partial and complete mgc2/pvpA genes, all Iranian MG isolates were clustered into two distinct groups. Because this typing assay could provide a higher discriminatory power than the previously reported GTS scheme of partial mgc2/ pvpA genes, these results can be considered a blueprint for future national control and diagnostic strategies. Furthermore, consistent surveillance with larger datasets will be needed to clarify the epidemiologic characteristics of MG outbreaks in different poultry hosts.

Preparation of ELISA and Lateral Flow Kits for rapid Diagnosis of Mycoplasma gallisepticum in Poultry

Avian mycoplasmas were mainly the cause of poultry industry economic losses; reduced meat and egg production and increases the antibiotic treatment cost. Mycoplasma gallisepticum (MG) infection is designated as infectious sinusitis of turkeys and chronic respiratory disease of chickens (gasping, depression, semi closed eyes, infraorbital sinuses edema and decrease in egg production). This study aimed to prepare, evaluate and Compare in-house ELISA kits and lateral flow assay (LFA) from a local strain of MG with commercial ELISA kits and PCR consequently. A total of 54 samples (27 tracheal swabs, 10 trachea and 17 lung) and 50 serum samples collected from birds suffering from chronic respiratory disease were tested by prepared in-house ELISA, commercial ELISA kits, PCR and LFA; a high correlation coefficient between in-house ELISA using whole antigen or sonicated antigen and commercial kit was recorded. Lateral Flow assay (LFA) performance indicate a low sensitivity (77.5%) but maintain a high specificity (92%) compared to PCR. The in-house ELISA kits and LFA prepared could be used as a fast diagnostic technique for detection of MG in Egypt. According to the available knowledge the prepared LFA for diagnosis of MG infection in chickens was developed for the first time in Egypt.

Evaluation of Mycoplasma gallisepticum (MG) ts-304 vaccine as a live attenuated vaccine in turkeys

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide that causes chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys. Vaxsafe MG (strain ts-11) is a live attenuated temperature sensitive vaccine that has been proven to be effective in controlling CRD in chickens, but it is not efficacious in turkeys. The gapA gene, which encodes a mature cytadhesin protein with a molecular weight of approximately 105 kDa, is not expressed in strain ts-11 because a 20 base pair reiterated sequence introduces a frame shift and causes premature truncation of the translated peptide. A GapA positive clone, MG ts-304, isolated from strain ts-11 has been shown to have enhanced efficacy in chickens. Here we describe studies we conducted to assess the safety and efficacy of the MG ts-304 vaccine candidate in turkeys. We found that MG ts-304 was able to colonise the trachea of 3-week-old turkeys and was safe, even at a tenfold overdose, inducing no adverse clinical signs of respiratory disease or significant gross lesions in the respiratory tract (air sacs or trachea), and was poorly transmissible to in-contact birds. We also showed that it was efficacious when administered to 3-week-old turkeys, inducing protective immunity against challenge with the M.gallisepticum wild-type strain Ap3AS. MG ts-304 is therefore a promising live attenuated vaccine candidate for use in turkeys.

Identification of genes involved in Mycoplasma gallisepticum biofilm formation using mini-Tn4001-SGM transposon mutagenesis

Mycoplasma gallisepticum (MG) is an important pathogen that can cause chronic respiratory disease in chickens and infectious sinusitis in turkeys. MG has the ability to form biofilms. The molecular mechanisms underlying MG biofilm formation are complex and poorly understood. To better understand the mechanisms involved in biofilm formation, mini-Tn4001-SGM, a novel transposon vector containing the gentamicin gene was constructed and electroporated into MG strain Rlow. Of the 738 mutants obtained, 12 had significantly reduced capacity to form biofilms in a polystyrene microtiter-plate biofilm assay. Ten different genes were identified as disrupted in these mutants using genomic walking from the transposon insertion sites and Southern bolt hybridization with a transposon-based probe. Four genes were associated with cellular processes, especially synthesis of extracellular polysaccharide and several lipoproteins encoded. Other genes were associated with translation, metabolism and gene regulation, and one had unknown function. Seven genes identified in this study have been previously associated with biofilm formation in MG or other bacterial species. The other three have not been previously reported to play a role in biofilm formation in MG. In conclusion, a new transposon vector was shown to be a powerful tool for future studies of MG pathogenesis. This study adds to our understanding of the molecular mechanisms involved in MG biofilm formation and may shed light on the persistence of MG infections.

Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance.

Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

Evaluation of a PCR multiplex for detection and differentiation of Mycoplasma synoviae, M. gallisepticum, and M. gallisepticum strain F-vaccine

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the mycoplasma infections of most concern for commercial poultry industry. MG infection is commonly designated as chronic respiratory disease (CRD) of chickens and infections sinusitis of turkeys. MS causes sub clinical upper respiratory infection and tenosynovitis or bursitis in chickens and turkeys. The multiplex PCR was standardized to detect simultaneously the MS, MG field strains and MG F-vaccine strain specific. The generic PCR for detection of any species of Mollicutes Class was performed and compared to the multiplex PCR and to PCR using species-specific primers. A total of 129 avian tracheal swabs were collected from broiler-breeders, layer hens and broilers in seven different farms and were examined by multiplex PCR methods. The system (multiplex PCR) demonstrated to be very rapid, sensitive, and specific. Therefore, the results showed a high prevalence of MS in the flocks examined (27.9%), and indicate that the MS is a recurrent pathogen in Brazilian commercial poultry flocks.

Factors influencing the cell adhesion and invasion capacity of Mycoplasma gallisepticum

BackgroundThe cell invasiveness of Mycoplasma gallisepticum, the causative agent of respiratory disease in chickens and infectious sinusitis in turkeys, may be a substantial factor in the well-known chronicity of these diseases and in the systemic spread of infection. To date, not much is known about the host factors and mechanisms involved in promotion or obstruction of M. gallisepticum adherence and/or cell invasion.In the current study, the influence of extracellular matrix (ECM) proteins such as fibronectin, collagen type IV and heparin, as well as plasminogen/plasmin, on the adhesion and cell invasion levels of M. gallisepticum to chicken erythrocytes and HeLa cells was investigated in vitro. Two strains, Rhigh and Rlow, which differ in their adhesion and invasion capacity, were analyzed by applying a modified gentamicin invasion assay. Binding of selected ECM molecules to M. gallisepticum was proven by Western blot analysis.ResultsCollagen type IV, fibronectin, and plasminogen exerted positive effects on adhesion and cell invasion of M. gallisepticum, with varying degrees, depending on the strain used. Especially strain Rhigh, with its highly reduced cell adhesion and invasion capabilities seemed to profit from the addition of plasminogen. Western and dot blot analyses showed that Rhigh as well as Rlow are able to adsorb horse fibronectin and plasminogen present in the growth medium. Depletion of HeLa cell membranes from cholesterol resulted in increased adhesion, but decreased cell invasion.ConclusionECM molecules seem to play a supportive role in the adhesion/cell invasion process of M. gallisepticum. Cholesterol depletion known to affect lipid rafts on the host cell surface had contrary effects on cell adherence and cell invasion of M. gallisepticum.

Infecção experimental por Mycoplasma gallisepticum e Escherichia coli em perus

Mycoplasma gallisepticum (MG) é responsável por provocar sinusite infecciosa em perus. A infecção por Mycoplasma spp. torna a ave susceptível a infecção por Escherichia coli. O objetivo deste estudo foi desenvolver em perus, um modelo experimental para a sinusite infecciosa. Utilizou-se 250 peru,s machos da linhagem Nicholas (Aviagen®) divididos em grupo não infectado (T1) e grupo desafiado (T2) que recebeu por via ocular, com um dia de idade, Mycoplasma gallisepticum cepa F e aos 21 dias de idade E. coli por via saco aéreo. Analisou-se a mortalidade, os sinais clínicos e lesões em sacos aéreos, fígado e coração. Concluiu-se que o delineamento experimental utilizado foi eficaz para simular a infecção natural por MG e E. coli, sendo que a vacina contra MG-F utilizada para poedeiras é patogênica para perus.

Identification of biofilm formation by Mycoplasma gallisepticum

Mycoplasma gallisepticum is the causative agent of chronic respiratory disease in chickens and of infectious sinusitis in turkeys, chickens, game birds, pigeons, and passerine birds of all ages. This study investigated the biofilm-producing ability of M. gallisepticum strains in an attempt to explain its intriguing persistence in commercial flocks. Eleven strains of M. gallisepticum were investigated for their biofilm formation, which varied considerably. Strains Nobilis MG 6/85, S6 (P5 and P20), D9604, and SU15 were strong biofilm producers. Strains Rlow (P10 and P100), NCL, CG5, YL4, and F were weak biofilm producers. Strains Vaxsafe MG ts-11 and F36 did not produce biofilm as verified using a crystal violet staining assay. In addition, highly differentiated biofilm structures of strain Nobilis MG 6/85 with characteristic stacks and channels were observed under confocal scanning laser microscopy and scanning electron microscopy. The carbohydrates (sucrose, glucose), disodium ethylenediaminetetraacetic acid (EDTA), antibiotics (tetracycline, gentamicin), or detergent (Triton X-100) were further used to determine their effects on biofilm formation. Biofilm formation was significantly inhibited by 5% sucrose and 5mmol/L EDTA. Compared with the planktonic mycoplasma, these biofilm-grown cultures were more resistant to tetracycline, gentamicin, and Triton X-100 treatments. Furthermore, real-time reverse transcriptase-polymerase chain reaction was performed to investigate the transcription of several genes that may be associated with biofilm formation. The results indicated that the transcriptions of some genes in the biofilm-grown cells were markedly decreased, including vlhA3.03, csmC, hatA, gapA, neuraminidase, and mgc2. Our results will benefit further research on the persistence of M. gallisepticum infections.

Development and immunogenicity of recombinant GapA+Mycoplasma gallisepticum vaccine strain ts-11 expressing infectious bronchitis virus-S1 glycoprotein and chicken interleukin-6

Mycoplasma gallisepticum (MG) is a major pathogen of poultry that causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. The efficacy of this vaccine is highly dose dependent and the flock antibody response is weak. To improve the functionality of the vaccine and investigate its potential as a delivery vector for foreign antigens and immunomodulatory proteins, we developed a derivative of ts-11 expressing infectious bronchitis virus-S1 glycoprotein (IBV-S1) and releasing chicken interleukin-6 into the extracellular milieu (MG ts-11 C3 (+CS)) using a transposon-based delivery vector. Following administration of MG ts-11 C3 (+CS) to chickens by eye-drop, an antibody response to MG and IBV-S1, as determined by the rapid serum agglutination test (RSA) and Western blotting, respectively, could be detected. Birds inoculated with the recombinant vaccine had significantly enhanced weight gain and were partially protected against damage by pathogenic IBV. These results indicate that the ChIL-6 released by MG ts-11 C3 (+CS) may have had a non-specific effect on growth rate. They also suggest that ts-11 is a promising vaccine vector, capable of delivering heterologous protective antigens, and may also provide non-specific benefits when engineered to express immunomodulatory proteins. With some improvements in the expression system, it could be used to induce a targeted immune response against specific mucosal pathogens, and co-expression of several antigens would allow development of a novel multivalent vaccine.

Mutations in 23S rRNA gene associated with decreased susceptibility to tiamulin and valnemulin in Mycoplasma gallisepticum

Mycoplasma gallisepticum is a major etiological agent of chronic respiratory disease (CRD) in chickens and sinusitis in turkeys. The pleuromutilin antibiotics tiamulin and valnemulin are currently used in the treatment of M. gallisepticum infection. We studied the in vitro development of pleuromutilin resistance in M. gallisepticum and investigated the molecular mechanisms involved in this process. Pleuromutilin-resistant mutants were selected by serial passages of M. gallisepticum strains PG31 and S6 in broth medium containing subinhibitory concentrations of tiamulin or valnemulin. A portion of the gene encoding 23S rRNA gene (domain V) and the gene encoding ribosome protein L3 were amplified and sequenced. No mutation could be detected in ribosome protein L3. Mutations were found at nucleotide positions 2058, 2059, 2061, 2447 and 2503 of 23S rRNA gene (Escherichia coli numbering). Although a single mutation could cause elevation of tiamulin and valnemulin MICs, combinations of two or three mutations were necessary to produce high-level resistance. All the mutants were cross-resistant to lincomycin, chloramphenicol and florfenicol. Mutants with the A2058G or the A2059G mutation exhibited cross-resistance to macrolide antibiotics erythromycin, tilmicosin and tylosin.

Transcriptional Responses of Mycoplasma gallisepticum Strain R in Association with Eukaryotic Cells

Mycoplasma gallisepticum is an etiologic agent of chronic respiratory disease in chickens and infectious sinusitis in turkeys. Other than proteins important for cytadherence, few M. gallisepticum factors or pathways contributing to host cell interactions have been identified. In this study, an oligonucleotide-based microarray was utilized to investigate transcriptional changes in M. gallisepticum strain R(low) upon exposure to eukaryotic cells. Fifty-eight genes were either up- or downregulated upon exposure to MRC-5 lung fibroblasts grown in vitro, including genes encoding transport-, metabolism-, and translation-associated proteins. Twenty of the 58 regulated genes have no assigned function. These results indicate that M. gallisepticum regulates gene expression upon exposure to eukaryotic cells, revealing genes and pathways likely to be important for host-bacterium interaction.

Phenotypic switching in Mycoplasma gallisepticum hemadsorption is governed by a high-frequency, reversible point mutation.

Mycoplasma gallisepticum is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in M. gallisepticum hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the gapA gene as part of the same transcription unit. In clones derived from strain R(low), detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the gapA structural gene. In HA(-) variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA(-) mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the crmA gene by using transposition mutagenesis. Despite a fully functional gapA gene, the amount of GapA detected in this mutant was considerably lower than in HA(+) clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover.

Multiplex PCR of avian pathogenic mycoplasmas.

AbstractMore than 20 mycoplasma species have been isolated and characterized from avian sources (1). Only four avian mycoplasmas species are known to cause economic losses in commercial poultry production. Mycoplasma gallisepticum (Mg) infection commonly causes chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys (2), M. synoviae (Ms) infection most frequently occurs as a subclinical upper respiratory infection and synovitis in chickens and turkeys (3), M. iowae (Mi) causes decrease in hatchability and high embryo mortality in turkeys (4), and M. meleagridis (Mm) is the cause of an egg-transmitted disease of turkeys in which the primary lesion is an airsacculitis in the progeny, which leads to lower hatchability and skeletal abnormalities in young turkeys (5).KeywordsMultiplex Polymerase Chain ReactionSwab SampleMicrofuge TubeMycoplasma SpeciesMycoplasma GallisepticumThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Lymphoproliferative responses of specific‐pathogen‐free chickens to Mycoplasma gallisepticum strain PG31

Mycoplasma gallisepticum (MG) is one of the aetiologic agents of chronic respiratory disease in chickens and infectious sinusitis in turkeys. We investigated humoral and cellular immune mechanisms following experimental infection with four different strains of MG. Peripheral blood leukocytes (PBL) obtained from chickens were examined for proliferation using antigen preparations of whole cell MG as stimuli in vitro. A consistent lymphoproliferative response was observed against the homologous whole cell antigens in the group of chickens infected with strain PG31. Significant lymphoproliferation was detected as early as 1 week post‐infection. We further characterized antigen‐specific proliferation by measuring the production of interferon and nitric oxide by the PBL of infected chickens. Consistent with lymphoproliferation, we also detected the presence of interferon and nitric oxide in vitro in antigen‐stimulated cultures. These results indicate a possible role of cell‐mediated immune responses in the development of immunity following MG infection in chickens.