Chronic Respiratory Disease In Chickens Research Articles

TLR2/MyD88/NF-κB signaling pathway regulates IL-1β production in DF-1 cells exposed to Mycoplasma gallisepticum LAMPs

Mycoplasma gallisepticum (M. gallisepticum) is one of the most important pathogens that cause chronic respiratory disease in chickens. M. gallisepticum-derived lipid-associated membrane proteins (LAMPs) are thought to be one of the major factors in mycoplasma pathogenesis and are potent inducers of the host innate immune response. However, the interaction of pathogenic M. gallisepticum-derived LAMPs with Toll-like receptors (TLRs) and the signaling pathways responsible for activating inflammation and NF-κB have not been fully elucidated. In this study, we found that IL-1β expression was induced in DF-1 cells stimulated with M. gallisepticum LAMPs. Subcellular localization experiments using immunofluorescence assays (IFAs) showed p65 translocation from the cytoplasm to the nucleus in DF-1 cells following stimulation with M. gallisepticum LAMPs. Phosphorylation of p65 was detected in LAMP-stimulated DF-1 cells. Treatment with an NF-κB-specific inhibitor showed that NF-κB is required for M. gallisepticum LAMP-induced IL-1β expression. In addition, the results indicated that TLR2 and myeloid differentiation primary-response protein 88 (MyD88)-dependent signaling pathways were involved in the activation of NF-κB by M. gallisepticum LAMPs. Together, these results provide evidence that M. gallisepticum LAMPs activate IL-1β production through the NF-κB pathway via TLR2 and MyD88.

Determination of the Mutant Selection Window and Evaluation of the Killing of Mycoplasma gallisepticum by Danofloxacin, Doxycycline, Tilmicosin, Tylvalosin and Valnemulin.

Mycoplasma gallisepticum is a common etiological cause of a chronic respiratory disease in chickens; its increasing antimicrobial resistance compromises the use of tetracyclines, macrolides and quinolones in the farm environment. Mutant selection window (MSW) determination was used to investigate the propensity for future resistance induction by danofloxacin, doxycycline, tilmicosin, tylvalosin and valnemulin. Killing of M. gallisepticum strain S6 by these antimicrobials was also studied by incubating M. gallisepticum into medium containing the compounds at the minimal concentration that inhibits colony formation by 99% (MIC99) and the mutant prevention concentration (MPC). Based on the morphology and colony numbers of M. gallisepticum on agar plates, the four kinds of sera in the order of the applicability for culturing M. gallisepticum were swine serum > horse serum > bovine serum > mixed serum. The MPC/MIC99 values for each agent were as follows: danofloxacin > tilmicosin > tylvalosin > doxycycline > valnemulin. MPC generated more rapid and greater magnitude killing than MIC99 against M. gallisepticum. Under exposure of 105–109 CFU/mL at MPC drug levels, valnemulin had the slowest rate of reduction in viable organisms and danofloxacin had the highest rate of reduction.

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.

Cloning, expression, and antigenic characterization of recombinant protein ofMycoplasma gallisepticum expressed inEscherichia coli

Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression inEscherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples ofMycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.

Preparation and Evaluation of Combined Polyvalent Inactivated Vaccine for The Protection Against Chronic Respiratory Disease in Chickens

Preparation and Evaluation of Combined Polyvalent Inactivated Vaccine for The Protection Against Chronic Respiratory Disease in Chickens

Development of a Mycoplasma gallisepticum infection model in turkeys

Mycoplasma gallisepticum causes chronic respiratory disease in chickens and is also highly pathogenic in turkeys. Several live attenuated M. gallisepticum vaccines are available for prevention of disease in chickens but they are considered to be either not safe or not efficacious in turkeys. The studies presented here aimed to develop a suitable infection model in turkeys, a prerequisite for development of a vaccine against M. gallisepticum for turkeys. Two wild-type Australian M. gallisepticum strains, Ap3AS and 100809/31, were used and their capacity to induce lesions was evaluated in 5-week-old to 6-week-old turkeys exposed to aerosols of these strains. Gross air sac lesion scores in the group exposed to Ap3AS were significantly greater than those in the group exposed to 100809/31 (P < 0.05). Histological tracheal lesion scores and tracheal mucosal thicknesses were significantly greater in birds exposed to either strain than in the unexposed birds (P < 0.05), but no significant differences were observed between the two infected groups. In a subsequent experiment, 6-week-old to 7-week-old turkeys were exposed to different doses of M. gallisepticum Ap3AS. Serology and M. gallisepticum re-isolation performed 14 days after infection showed that all birds exposed to Ap3AS were positive by rapid serum agglutination and by culture. Gross air sac lesion scores in the groups exposed to the highest dose, 8.17 × 108 colour-changing units Ap3AS/ml, as well as a 10-fold lower dose were significantly more severe than in the uninfected control group. Lesion scores and tracheal mucosal thicknesses were significantly greater in birds exposed to Ap3AS than in the unexposed birds (P < 0.05). However, no significant differences were seen in tracheal mucosal thicknesses or lesion scores between the groups exposed to the different doses of Ap3AS. This study has established a reliable challenge model for M. gallisepticum infection in turkeys, which will be useful for evaluation of potential M. gallisepticum vaccine candidates for this species.

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&lt; 0.01). In MC complex, number of heterophil increased and macrophages decreased significantly (p &lt; 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)

Mycoplasma gallisepticum and Escherichia coli mixed infection model in broiler chickens for studying valnemulin pharmacokinetics

A Mycoplasma gallisepticum-Escherichia coli mixed infection model was developed in broiler chickens, which was applied to pharmacokinetics of valnemulin in the present experiment. The velogenic M. gallisepticum standard strain S6 was rejuvenated to establish the animal model, and the wild E. coli strain O78 was injected as supplementary inoculum to induce chronic respiratory disease in chickens. The disease model was evaluated based on its clinical signs, histopathological examination, bacteriological assay, and serum plate agglutination test. The pharmacokinetics of valnemulin in infected chickens was determined by intramuscular (i.m.) injection and oral administration (per os, p.o.) of a single dose of 10 mg/kg body weight (BW). Plasma samples were analyzed by liquid chromatography-tandem mass spectrometry. The plasma concentration-time curve of valnemulin was analyzed using the noncompartmental method. After the i.m. administration, the mean values of Cmax , Tmax , AUClast , MRT, CLβ /F, Vz /F, and t1⁄2β , were 27.94 μg/mL, 1.57 h, 171.63 μg·h/mL, 4.51 h, 0.06 L/h/kg, 0.56 L/kg, and 6.50 h, respectively. By contrast, the corresponding values after p.o. administration were 5.93 μg/mL, 7.14 h, 47.60 μg·h/mL, 9.80 h, 0.22 L/h/kg, 3.35 L/kg, and 10.60 h. The disposition of valnemulin was retarded in infected chickens after both modes of extravascular administration as compared to the healthy controls. More attention should be given to monitoring the therapeutic efficacy and adverse effects of mixed infection because of higher required plasma drug concentration and enlarged AUC with valnemulin treatment.

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 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.

Differentiation of Mycoplasma gallisepticum strains using PCR and high-resolution melting curve analysis

Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines. In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vlhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG strains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respiratory swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11-vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs.

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.

The Mycoplasma gallisepticum OsmC-like protein MG1142 resides on the cell surface and binds heparin

Mycoplasma gallisepticum is an avian pathogen that causes a chronic respiratory disease of chickens and results in significant economic losses to the poultry industry worldwide. Colonization of the host and the establishment of chronic disease are initiated by the cytadherence of M. gallisepticum to the host respiratory epithelium. While several proteins involved in cytadhesion have been characterized, molecules that interact with components of the host extracellular matrix, a process that is central to pathogenesis, are only now being identified. In this study, M. gallisepticum whole cells were shown to bind heparin in a specific and saturable manner. Heparin also significantly inhibited the binding of M. gallisepticum to the human lung fibroblast cell line MRC-5, suggesting a potential role for glycosaminoglycans (GAGs) in cytadherence. M. gallisepticum protein MG1142 (encoded by mga 1142), which displays homology to the osmotically induced (OsmC) family of proteins, binds strongly to heparin, is highly expressed during in vitro culture, and is surface accessible. Recombinant MG1142 bound heparin in a dose-dependent and saturable manner with a dissociation constant (K(d)) of 10+/-1.8 nM, which is within a physiologically significant range, compared to that of other heparin-binding proteins. Binding to heparin was inhibited by the heavily sulfated polysaccharide fucoidan, but not by mucin or chondroitin sulfate A or B, suggesting that electrostatic interactions between the sulfate groups of heparin and the positively charged basic residues of the MG1142 protein are important in binding. The ability of M. gallisepticum to bind GAGs may contribute to host adherence and colonization.

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.

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.

Mycoplasmoses in poultry

The most important mycoplasmas isolated from domestic avian species include Mycoplasma gallisepticum (MG), M. synoviae (MS), M. meleagridis (MM) and M. iowae (MI). MG causes chronic respiratory disease of chickens and infectious sinusitis in turkeys, resulting in economic losses. MS causes infectious synovitis or mild upper respiratory disease. MM infects only turkeys, causing airsacculitis and sub-optimal production and hatchability. MI is associated with reduced hatchability in turkey flocks. Transmission is either direct, from bird to bird or through the egg, or indirect. Diagnosis is based on isolation and identification of mycoplasmas, according to biochemical, serological or molecular biology tests, or serological examination of host sera by slide agglutination, haemagglutination inhibition or enzyme-linked immunosorbent assay (ELISA) tests. Antibiotics (i.e. tetracyclines, macrolides, quinolones and tiamulin) may be used for therapeutic treatment or prophylactic medication. The eradication of mycoplasma infection can be achieved through improvements in hygiene and management practices, therapeutic treatment of breeder layers and/or of hatching eggs and better monitoring procedures.

Aerosol Studies with Avian Mycoplasma. I. Survival in the Air

Grumbles et al. (4) and Fahey and Crawley (2) presented data indicating airborne spread of agents responsible for chronic respiratory disease in chickens. This method of transmission requires that the Mycoplasma microorganisms be discharged from birds in a manner to become airborne, survive in the airborne state, and be infective upon inhalation by susceptible birds. This study was made to determine whether Mycoplasma gallisepticum and M. meleagridis could be recovered, and for what period, from artificially created areosols.

Neuraminidase-like Enzyme present in Mycoplasma gallisepticum

Mycoplasma gallisepticum is the agent responsible for chronic respiratory disease in chickens. It will agglutinate mammalian and avian erythrocytes, and this phenomenon has been used to detect M. gallisepticum haemagglutinating inhibition antibodies. Two antigens have been used in the haemagglutination-inhibition (HI) test. The first will here be referred to as the “prepared antigen”. This was obtained by growing the organism in broth, centrifuging and resuspending the organism in equal parts of glycerol and phosphate buffered saline (pH 7.0), freezing and storing until required for use, when it is further diluted in phosphate buffered saline. The other antigen is a rapidly growing 24 h broth culture grown in PPLO broth, 10 per cent horse serum, 1 per cent yeast autolysate, and 0.1 per cent dextrose, with the pH. adjusted to 7.8 (ref. 1).

A Report on Broilers from Parents Free of Mycoplasma gallisepticum

ELAPLANE and Stuartl first described a chronic respiratory disease in chickens in 1943. Since that time much information has been published on this subject. The name, changed several times, is now most commonly which is quite descriptive. Research has revealed that chicks infected with Mycoplasma gallisepticum are more susceptible to aerosacculitis than uninfected chicks. Chicks infected with this organism, when complicated with other agents, develop serious pathological changes resulting in deaths and, in those that survive, high condemnations when processed. Flocks grown free of this organism apparently do not develop this condition. This experiment was designed to secure some data on a group of chicks hatched from parents free of M. gallisepticum. A Department of Agriculture news release indicated that in May, 1961, 43% of the condemnations in broiler chickens at slaughter were due to aerosacculitis, representing a tremendous loss of chicken meat.