Mycoplasma Gallisepticum Strains Research Articles

Mycoplasmosis in Poultry: An Evaluation of Diagnostic Schemes and Molecular Analysis of Egyptian Mycoplasma gallisepticum Strains.

Infections with Mycoplasma gallisepticum (MG) in poultry are associated with a wide range of disease conditions, including those affecting the respiratory and reproductive systems. The purpose of this study was to endorse the more sensitive diagnostic scheme for MG infection and identify the best molecular marker for MG phylogenetic analysis using six housekeeping genes: mgc2, mraW, atpG, ugpA, DUF31196, and lgT. For these purposes, 55 poultry flocks of different species were screened using either qRT-PCR or PCR techniques analogous to conventional culturing from non-cultured and cultured swabs on PPLO broth. The rate of MG positivity was the highest when using qRT-PCR from cultured broth (89.0%) and the lowest when using conventional culturing (34.5%). Compared to qRT-PCR from broth, statistical analysis using the Roc curve in MedCalc statistical software showed that the PCR schemes (qRT-PCR from swabs and PCR from swabs and broth) performed better than conventional culturing in terms of sensitivity, accuracy, and area under the curve (AUC), suggesting that they may be more reliable schemes. Further support was added by Cohen's kappa test, showing moderate agreement between the molecular approaches. Among the six screened genes, mgc2 and mraW had the highest detection rates (69% and 65.4%, respectively). The comparative phylogenetic analysis revealed that mgc2 or atpG gene sequences distinguished MG isolates into different clades with high discriminatory power.

Drug sensitivity and genome-wide analysis of two strains of Mycoplasma gallisepticum with different biofilm intensity.

Mycoplasma gallisepticum (MG) is one of the major causative agents of chronic respiratory diseases in poultry. The biofilms of MG are highly correlated to its chronic infection. However data on genes involved in biofilm formation ability are still scarse. MG strains with distinct biofilm intensity were screened by crystal violet staining morphotyped and characterized for the drug sensitivity. Two MG strains NX-01 and NX-02 showed contrasted ability to biofilm formation. The biofilm formation ability of NX-01 strain was significantly higher than that of NX-02 strain (p < 0.01). The drug sensitivity test showed that the stronger the ability of MG stain to form biofilms, the weaker its sensitivity to 17 antibiotic drugs. Moreover, putative key genes related to biofilm formation were screened by genome-wide analysis. A total of 13 genes and proteins related to biofilm formation, including ManB, oppA, oppD, PDH, eno, RelA, msbA, deoA, gapA, rpoS, Adhesin P1 precursor, S-adenosine methionine synthetase, and methionyl tRNA synthetase were identified. There were five major discrepancies between the two isolated MG strains and the five NCBI-published MG strains. These findings provide potential targets for inhibiting the formation of biofilm of MG, and lay a foundation for treating chronic infection.

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.

A Multiplex PCR Assay for Differential Identification of Wild-type and Vaccine Strains of Mycoplasma gallisepticum

Mycoplasma gallisepticum (MG) can cause respiratory disease in chickens and result in serious economic losses in the chicken industry. The use of live vaccines has been a favorable option for the control of MG infection in multi-age commercial layers and broiler breeders. There are three live vaccines, including ts-11, 6/85, and F strain, that have been commonly used in various parts of the world, including South Korea. The definitive diagnosis of the infection, therefore, requires the differentiation of wild-type field strains of MG from the vaccine strains used. Thus, we aimed to develop a novel multiplex PCR assay to discriminate between vaccine strains (ts-11, 6/85, and F strain) and wild-type field strains of MG isolated from infected chickens. We designed four novel primer sets that are each specific to MG species, ts-11, 6/85, and F strain. The multiplex PCR assay using the primer sets differentially identified wild-type and vaccine strains of MG but did not detect other avian bacteria. The detection limit of this assay was 250 fg/μL of genomic DNA of each strain tested. In addition, this assay was applied to 36 MG strains isolated from chickens over the past 20 years in South Korea. As a result, the assay identified 22 wild-type strains and 14 vaccine strains. Consequently, the novel multiplex PCR assay can discriminate between vaccine and wild-type field strains of MG and could be a valuable tool for the diagnosis of MG infection in MG-vaccinated chicken flocks.

Effects of the In Ovo Vaccination of the ts-11 Strain of Mycoplasma gallisepticum in Layer Embryos and Posthatch Chicks.

Simple SummaryMycoplasma gallisepticum (MG) is responsible for reductions in egg production and other economic losses in the poultry industry. In this study, the potential application of in ovo vaccination of the ts-11of MG vaccine (ts-11MGV) in layer embryos for the subsequent early protection as well as live performance of pullets were investigated. The use of various dosages of live attenuated ts-11MGV ranging from 3.63 × 101 to 3.63 × 104 cfu that were delivered in ovo at 18 days of incubation were evaluated. The results of current study revealed that the in ovo injection of various dosage of ts-11MGV had no negative impacts on any hatch variables. Additionally, the higher dosage of ts-11MGV (3.63 × 104) resulted in a reduction in body weight gain in three-week-old pullets in comparison to all other treatments. Furthermore, MG DNA remained undetectable for hatchling and three-week-old pullets and no serological response was observed at 3 weeks posthatch. Total flock protection from field-strain MG infections is facilitated by the prior systemic establishment of vaccine strains in pullets. Therefore, it is concluded that the ts-11MGV may not be an appropriate candidate for in ovo injection due to the lack of its presence in hatchlings and posthatch chicks subsequent to its in ovo administration.The transmission of the ts-11 strain of Mycoplasma gallisepticum (MG) vaccine (ts-11MGV) between incubated eggs and between hatchlings that was administrated via in ovo injection, and its subsequent effects on their posthatch performance were evaluated. Marek’s disease diluent alone (sham-injected) or containing either 3.63 × 101, 102, 103, or 104 cfu of ts-11MGV was manually in ovo-injected into the amnion on 18 days of incubation. Egg residue analysis, percentage incubational egg weight loss, hatchability of viable injected eggs, and hatchling body weight (BW) were assessed. Selected hatchlings from each treatment replicate group were swabbed in the choanal cleft for MG DNA detection. Female chick live performance was also assessed through 21 days of posthatch age. Unexposed control sentinel chicks were allocated to each treatment replicate group to assess horizontal transmission. Birds were later swabbed and bled respectively, for detection of MG DNA and IgM production at 21 days posthatch. In all birds, no MG DNA was detected and SPA tests for IgM were negative. Among all variables, only 0 to 21 day BW gain was significantly affected by treatment and was lower in the 3.63 × 104 ts-11 MGV treatment in comparison to all the other treatments. Because ts-11MGV does not exhibit vertical or horizontal transmission capabilities under commercial conditions, it may not be a good candidate for in ovo injection.

In vitro behavior of Mycoplasmagallisepticum live-type nosode

As a step of a doctoral research project, in this study a live-type nosode was prepared from microorganism Mycoplasmagallisepticum strain R (ATCC 93-08/19610) according to Costa model and the rules by Brazilian Homeopathic Pharmacopoeia. Live nosode was tested in vitro to assess safety when used to immunize domestic fowl (Gallus gallus) against infection by this microorganism and to investigate its behavior under laboratory conditions. M. gallisepticum was not shown to grow in fluid (broth) and solid (plate) modified Frey medium with dilutions 11d, 12d, 20d and 30d. Inhibition halos about 2.0 mm were observed around paper disks impregnated with live-type nosode in microorganism-sown Petri dishes, whereas disks impregnated with conventional antibiotic oxytetracycline exhibited 8.0 mm inhibition halos. Protein assessment by Folin-Lowry method showed protein absence in dilutions 12d and 30d and neither microbial DNA traces were found in PCR assay in dilutions 12d, 20d and 30d.

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Like humans, chickens can become sick from bacterial infections. Mycoplasma is a very small, slow-growing bacterium. There are multiple types of Mycoplasma that affect different animals, humans, and even plants and insects. In the current study, the whole genome of Mycoplasma synoviae was sequenced through the joint sequencing method of the third-generation PacBio and second-generation Illumina of the high-throughput sequencing platform, and the complete genome map of the Mycoplasma synoviae was successfully obtained. SMRT sequencing and assembly of whole genome: Canu v1.5/ WTDBG v2.2 software was used for preliminary assembly of filtered subreads. Pilon V1.22 software was used to further correct the assembled genome with high-quality second-generation data, and the genome with higher accuracy was finally obtained. Sequencing results showed that the Mycoplasma was composed of a 0.83Mb chromosome with a G+C percentage of 28.42%. Bioinformatics was used to predict the encoding protein genes, and it was found that Mycoplasma synoviae genome encoded a total of 1415 ORF open reading frames, including 1.73% of the repeats, 7 ribosomal RNAs, 34 transport RNAs, 4 non-coding RNAs and 12 pseudogenes. Mycoplasma synoviae genomes also contain 2 CRISPR, 1 gene island and 2 prophages. Using multiple strains of Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma anatis as reference genomes, we constructed phylogenetic trees of 12 species of Mycoplasma. The genetic and taxonomic status of Mycoplasma synoviae strains were evaluated. The results showed that the sequencing strains of Mycoplasma synoviae and the model strain Mycoplasma synoviae WVU1853 were closely clustered together, suggesting the genetic relationship was relatively close and the sequencing strains were relatively conservative, and represent that they share the same genetic origin.

Effects of immunosuppression on the efficacy of vaccination against Mycoplasma gallisepticum infection in chickens

Immunosuppression can increase the susceptibility of chickens to other disease-causing pathogens and interfere with the efficacy of vaccination against those pathogens. Chicken anaemia virus (CAV) and infectious bursal disease virus (IBDV) are common causes of immunosuppression in chickens. Immunosuppression was induced by experimental infection with either CAV or IBDV to assess the effect of immunosuppression on the efficacy of vaccination with Mycoplasma gallisepticum strain ts-304 against infection with virulent M. gallisepticum, a common bacterial pathogen of chickens worldwide. Birds were experimentally infected with either CAV or IBDV at 1 week of age, before vaccination and challenge with M. gallisepticum to examine the effect of immunosuppression at the time of vaccination, or at 6 weeks of age, after vaccination against M. gallisepticum but before challenge with virulent M. gallisepticum, to investigate the effect of immunosuppression at the time of challenge. All birds were vaccinated with a single dose of the ts-304 vaccine at 3 weeks of age and experimentally challenged with the virulent M. gallisepticum strain Ap3AS at 8 weeks of age. In immunosuppressed chickens there was a reduction in protection offered by the ts-304 vaccine at two weeks after challenge, as measured by tracheal mucosal thicknesses, serum antibody levels against M. gallisepticum, air sac lesion scores and virulent M. gallisepticum load in the trachea. Immunosuppressed birds with detectable serum antibodies against M. gallisepticum were less likely to have tracheal lesions. This study has shown that immunosuppression caused by infection with CAV or IBDV can interfere with vaccination against mycoplasmosis in chickens.

Transcriptomic Analysis of Long-Term Protective Immunity Induced by Vaccination With Mycoplasma gallisepticum Strain ts-304.

Live attenuated vaccines are commonly used to control Mycoplasma gallisepticum infections in chickens. M. gallisepticum ts-304 is a novel live attenuated vaccine strain that has been shown to be safe and effective. In this study, the transcriptional profiles of genes in the tracheal mucosa in chickens challenged with the M. gallisepticum wild-type strain Ap3AS at 57 weeks after vaccination with ts-304 were explored and compared with the profiles of unvaccinated chickens that had been challenged with strain Ap3AS, unvaccinated and unchallenged chickens, and vaccinated but unchallenged chickens. At two weeks after challenge, pair-wise comparisons of transcription in vaccinated-only, vaccinated-and-challenged and unvaccinated and unchallenged birds detected no differences. However, the challenged-only birds had significant up-regulation in the transcription of genes and enrichment of gene ontologies, pathways and protein classes involved in infiltration and proliferation of inflammatory cells and immune responses mediated through enhanced cytokine and chemokine production and signaling, while those predicted to be involved in formation and motor movement of cilia and formation of the cellular cytoskeleton were significantly down-regulated. The transcriptional changes associated with the inflammatory response were less severe in these mature birds than in the relatively young birds examined in a previous study. The findings of this study demonstrated that vaccination with the attenuated M. gallisepticum strain ts-304 protects against the transcriptional changes associated with the inflammatory response and pathological changes in the tracheal mucosa caused by infection with M. gallisepticum in chickens for at least 57 weeks after vaccination.

Isolation and Antimicrobial Sensitivity of Mycoplasma synoviae and Mycoplasma gallisepticum from Vaccinated Hens in Mexico.

Mycoplasma synoviae (MS) and Mycoplasma gallisepticum (MG) strains were isolated at 39.5 °C to rule out temperature-sensitive strains (ts+) and identified using random amplification of polymorphic DNA. Then, their minimum inhibitory concentrations (MIC100) were calculated in isolated strains from broiler breeders and laying hens vaccinated with ts+ MS-H and ts+ MG TS-11 vaccines in Mexico. We sampled 631 lots of hens. A total of 28 of the 123 MS isolates and 12 of the 23 MG isolates were analyzed using random amplification of polymorphic DNA, of which 24 and 3 matched the DNA banding patterns of the MS-H and MG-F strains, respectively. The isolated MS and MG strains were sensitive to tiamulin and tylosin and showed intermediate sensitivity or resistance to lincomycin, florfenicol, erythromycin, enrofloxacin, and curcumin. Although both the MS and MG strains were sensitive to the same antibiotics (MIC100 lower than 1 mg mL−1), the MG strains were 5 to 10 times more sensitive than the MS strains. MS is the most frequently isolated mycoplasma in Mexican poultry production. The MS vaccine used (ts+ MS-H) could reverse its thermosensitivity and therefore could regain its virulence. MS was less sensitive to tiamulin and tylosin compared to MG.

Duration of protective immunity induced by Mycoplasma gallisepticum strain ts-304 vaccine in chickens

Mycoplasma gallisepticum (MG) is an important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. MG ts-304 is a GapA positive clone recovered from Vaxsafe MG (strain ts-11) that has been shown to be safe in chickens when delivered by the eye drop route to 3-week-old specific-pathogen-free chickens and to confer protection against challenge at 4 weeks after vaccination, as measured by tracheal mucosal thickness and air sac lesion scores. In this study, specific pathogen-free chickens (SPF) were vaccinated with a single dose of the MG ts-304 vaccine (106.0 colour changing units) at 3 weeks of age and experimentally challenged by aerosol with the virulent M. gallisepticum strain Ap3AS at 40, 48 and 57 weeks after vaccination. There were no significant differences in tracheal mucosal thickness 2 weeks after challenge between chickens challenged at the three time points, or between the vaccinated birds after challenge and unvaccinated/unchallenged control birds. Thus there was clear evidence that the immunity conferred by vaccination with the MG ts-304 vaccine resulted in significant protection against tracheitis in chickens that extended to, but was highly likely to exceed, 57 weeks after vaccination and that similar long term protective immunity could be expected to be conferred by a vaccine dose lower than that used in this study.

Effects of dietary Original XPC on selected blood variables in layer pullets challenged with Mycoplasma gallisepticum , ,

Effects of dietary Original XPC (XPC) on 17 selected blood variables in commercial layer pullets challenged with the virulent, low-passage R strain of Mycoplasma gallisepticum (RlowMG) were investigated. Hy-Line W-36 pullets sourced from M. gallisepticum–clean layer breeders were fed a basal diet with XPC (1.25 kg/metric ton) or without from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with RlowMG. Blood samples were taken immediately before the initial RlowMG challenge at 8 woa and again at 12 woa (4 wk after challenge). At 8 woa, blood pH was lower and glucose concentration was higher in the preassigned challenge treatment groups. At 12 woa, the concentration of oxygen dissolved in the blood was significantly lower in the RlowMG-challenged group than the unchallenged group of birds regardless of dietary treatment. The RlowMG challenge significantly increased blood carbon dioxide partial pressure, calcium, sodium, anion gap, osmolality, glucose, and corticosterone levels but significantly decreased blood oxygen partial pressure, oxyhemoglobin concentration, concentration of oxygen dissolved in the blood, chloride, and pH levels. Because blood pH and glucose concentration at 8 woa were examined before challenge, their baseline values were biased with respect to challenge treatment before treatment was applied. However, the lack of a significant main effect due to diet at 8 woa for blood pH and glucose concentration, along with the other 15 blood variables, indicate that the baseline data with respect to dietary treatment were unbiased, allowing for real dietary effects to be accurately assessed. In conclusion, layer pullets challenged with RlowMG undergo a stress response associated with changes in various physiological blood variables, and a decrease in pH and increase in carbon dioxide partial pressure, in association with a lack of change in bicarbonate, indicates that the stress response caused by the RlowMG challenge was associated with respiratory acidosis. Nevertheless, feeding XPC did not influence the effects of challenge treatment on these postchallenge physiological blood values.

Differential Response of the Chicken Trachea to Chronic Infection with Virulent Mycoplasma gallisepticum Strain Ap3AS and Vaxsafe MG (Strain ts-304): a Transcriptional Profile.

Mycoplasma gallisepticum is the primary etiological agent of chronic respiratory disease in chickens. Live attenuated vaccines are most commonly used in the field to control the disease, but current vaccines have some limitations. Vaxsafe MG (strain ts-304) is a new vaccine candidate that is efficacious at a lower dose than the current commercial vaccine strain ts-11, from which it is derived. In this study, the transcriptional profiles of the trachea of unvaccinated chickens and chickens vaccinated with strain ts-304 were compared 2 weeks after challenge with M. gallisepticum strain Ap3AS during the chronic stage of infection. After challenge, genes, gene ontologies, pathways, and protein classes involved in inflammation, cytokine production and signaling, and cell proliferation were upregulated, while those involved in formation and motor movement of cilia, formation of intercellular junctional complexes, and formation of the cytoskeleton were downregulated in the unvaccinated birds compared to the vaccinated birds, reflecting immune dysregulation and the pathological changes induced in the trachea by infection with M. gallisepticum Vaccination appears to protect the structural and functional integrity of the tracheal mucosa 2 weeks after infection with M. gallisepticum.

Mycoplasma gallisepticum strain ts-304 is a safe and effective live attenuated vaccine for use in chickens

Vaxsafe MG (strain ts-11) is a live attenuated vaccine against the important poultry pathogen Mycoplasma gallisepticum that has been used globally to improve poultry health. However, the majority of the bacterial cells in Vaxsafe MG do not express the GapA cytadhesin, reducing their capacity to colonise the respiratory tract. Vaxsafe MG (strain ts-304) is a GapA positive clone recovered from Vaxsafe MG (strain ts-11) that has been shown to be safe and efficacious in turkeys, and preliminary studies have suggested that Vaxsafe MG (strain ts-304) may have greater efficacy in chickens than Vaxsafe MG (ts-11). The studies described here aimed to meet the international regulatory requirements for safety and efficacy in chickens. The vaccine colonised the trachea of 3-week-old chickens without inducing signs of respiratory disease or significant lesions in the respiratory tract, and was safe at a tenfold overdose and after repeated administration. It was transmissible from vaccinated to naïve chickens with no evidence of reversion to virulence following multiple in vivo passages. Finally, the superiority of Vaxsafe MG (strain ts-304) was demonstrated by its capacity to induce similar protection against infection with wild type M. gallisepticum at a 40 fold lower dose than the end of shelf life titre dose of Vaxsafe MG (ts-11). The lower effective dose of Vaxsafe MG (strain ts-304) allows it to be freeze-dried, enhancing its stability, making it easier to transport and store the vaccine and increasing its shelf life. Vaxsafe MG (strain ts-304) is, therefore, a highly efficacious and promising live attenuated vaccine candidate suitable for use in chickens.

Growth and humoral immune effects of dietary Original XPC in layer pullets challenged with Mycoplasma gallisepticum

Effects of dietary Original XPC (XPC) in commercial layer pullets challenged with the virulent, low passage R strain of Mycoplasma gallisepticum (Rlow MG) were investigated. Hy-Line W-36 pullets sourced from MG-clean breeders were fed a basal diet with or without (CON) XPC (1.25 kg/metric ton) from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with Rlow MG. Body weight was recorded at 3, 8, and 12 woa, and ovary, ceca, and bursa weights were recorded at 3 and 12 woa. Blood samples were taken immediately before the initial Rlow MG challenge at 8 woa and again at 12 woa to test for IgM and IgG antibody production against MG. All birds were evaluated for MG lesion scores at 12 woa. Regardless of challenge, inclusion of XPC in the diet did not significantly alter BW at 3 or 8 woa or relative organ weights at 3 or 12 woa. However, at 12 woa, BW of XPC-fed birds, regardless of challenge was significantly (P = 0.0038) heavier than CON by 25.7 g. All birds tested negative for MG antibodies before the 8 woa challenge. Respective percentage serum plate agglutination and ELISA positive birds at 12 woa were 0 and 0% (CON, nonchallenged), 1.4 and 0% (XPC, nonchallenged), 100 and 47.2% (CON, challenged), and 100 and 50.0% (XPC, challenged). Diet did not significantly affect ELISA titers, but they were significantly (P < 0.0001) increased due to challenge. Furthermore, lesion scores were significantly higher for Rlow MG-challenged birds (P = 0.0012), and dietary treatment with XPC in challenged birds numerically reduced MG lesion scores from 0.278 to 0.194. In conclusion, although dietary XPC did not significantly alter the humoral immune response, antibody titer levels, or severity of MG lesions in layer pullets that were or were not challenged with Rlow MG, it led to an increase in their rate of growth through 12 woa.

Molecular detection and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae strains in backyard poultry in Italy

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) represent the most important avian Mycoplasma species in the poultry industry, causing considerable economic losses. In Italy, the presence of MG or MS has been investigated especially in commercial poultry farms. To our knowledge, no systematic investigations on MG or MS presence using highly specific diagnostic assays have been performed in backyard poultry. The aim of this study was to detect and molecularly characterize MG and MS strains in 11 backyard poultry flocks located in different regions of Italy. Tracheal swabs were collected and DNA was extracted. For MS, a PCR targeting a vlhA gene fragment was performed, and typing and subtyping was attempted. The presence of MG was investigated by a screening PCR, then MG typing by gene-targeted sequencing (GTS). All the amplicons were sequenced, then MG and MS dendrograms were constructed. All the flocks examined resulted Mycoplasma positive: 5 out of 11 (45.45%) were MG and MS positive, 3 (27.27%) were MG positive, and the remaining 3 (27.27%) were MS positive. The MS detections were assigned to types C, D, and F. All strains of type D belonged to subtype D1 and 2 unknown subtypes were identified. A MS sequence showed peculiar characteristics, which did not allow assignment to a known MS type or subtype. MG GTS analysis identified 6 MG strains belonging to 5 subclusters circulating in Italian backyards chicken flocks. The results of this study provide evidence of a risk for commercial poultry farms, especially in areas where backyard and commercial farms are close, suggesting the implementation of biosecurity measures.

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Comparison of GTS and RAPD assays to characterize Thai Mycoplasma gallisepticum strains

Comparison of GTS and RAPD assays to characterize Thai Mycoplasma gallisepticum strains

Application of high-resolution melting-curve analysis on pvpA gene for detection and classification of Mycoplasma gallisepticum strains

Mycoplasma gallisepticum (MG) is an avian species pathogen which causes heavy economic losses in the poultry industry. The purpose of this study was to determine genomic diversity of 14 MG field strains from chicken, Chuker partridge and peacock collected during 2009–2012 in Iran by polymerase chain reaction and partial sequencing of the pvpA gene. A High-Resolution Melting (HRM) technique was also developed and applied to differentiate between field and vaccine strains. Sequencing of the pvpA gene revealed a 51 nucleotide deletion, within DR-1 and DR-2, among MG strains from chicken and partridge whilst 63 nucleotides were deleted in MG strain from peacock. One nucleotide substitution was also observed among chicken MG strains. Phylogenetic analysis of the sequences clustered all of the Iranian MG strains into two clades or phylogeny groups; the strains from chicken and partridge in one group (group 1) and the strain from peacock into another group (group 4). HRM analysis has also produced comparable outcome to those of sequencing; four distinct melting curves which correspond to the three MG strains from chicken, Chukar partridge and peacock and ts-11 vaccine strain. Overall, findings of this study point towards a single source of infection for the chicken and partridge MG strains and likelihood of the strains being native and endemic in Iran. Peacock considered as an exotic species in Iran, hence the genetic distance for the pvpA gene. MG can be transmitted easily among different avian species and this distinct peacock strain may pose a threat to poultry industry. Our findings also show that molecular variation among pvpA gene of MG strains could be revealed using the relatively rapid and affordable HRM technique.

Performance, serologic and tracheal responses of laying hens exposed to F strain of Mycoplasma gallisepticum

ABSTRACT: The F strain of Mycoplasma gallisepticum (MG-F) protects chickens against mycoplasma infections, in which monitoring is made by serology and histopathology of trachea. This trial used 90 chickens, being 30 unvaccinated (G1 group), 30 eye-drop vaccinated at 8 weeks of age with MG-F (Ceva Animal Health, São Paulo, SP, Brazil) (G2), and 30 immunized at 8 and 11 weeks of age (G3). Samples were obtained from chickens on the 8, 12, 15, 18, 20 and 24th weeks of age for the enzyme-linked immunosorbent assay (ELISA) test. Tracheal fragments were collected after necropsies on the 15 and 24th weeks of age. Up to 12 weeks, the ELISA reactions in optical density (OD) were 0.165 (G1), 0.151 (G2) and 0.151(G3), all below 0.20 and with no significant difference among groups (p &gt; 0.05). After the 15th week, the ELISA reactions rose, yielding the following group averages by collecting dates: G1 (0.18, 0.19, 0.18, and 0.16), G2 (0.36, 0.49, 0.47, and 0.44) and G3 (0.41, 0.52, 0.59, 0.60), being the means in G2 and G3 not significantly different between than, but significantly different from G1. The initial weight (592.71, 621.33, and 594.40), the final weight (1,932.58, 1,987.59, and 1,875.20) and the weekly weight gain (11.65, 11.90, and 11.14) were not significantly different among groups. At necropsy the gross tracheal score means by group and dates were: 15th week (0.25, 0.61, and 0.54) and 24th week (0.54, 0.58, and 0.67), being these difference not significantly (p &gt; 0.05). On microscopy, the tracheal score averages by groups G1, G2 and G3, respectively, were: 15th week (0.25, 0.32, and 0.47) and 24th week (0.07, 0.75, and 0.08). G2 yielded higher score average than G1 and G3 on the 24th week. Higher tracheal changes for G2 and G3 as compared to G1 could be ascribed to MG-F infection. There were no evident prejudicial effects on live weight, weight gain and tissue changes by applying one or two vaccination doses.