Detection Of Mycoplasma Gallisepticum Research Articles

Detection of Mycoplasma gallisepticum, Isolation, and Determination of Tylosin Susceptibility of Isolates from Commercial Chickens.

Mycoplasma gallisepticum (MG) is a contagious avian pathogen that causes financial losses to the poultry industry. Isolation of the pathogen is difficult and time-consuming, and therefore, far from a routine method. Serological testing methods to detect antibodies resistant to MG are widely used in routine diagnosis. Tylosin is a class of macrolide antibiotics tremendously administered in veterinary medicine for the treatment of mycoplasmosis and prophylaxis. This study aimed to detect MG by immunoassay testing, culture, and polymerase chain reaction (PCR) in commercial poultry farms and to investigate the tylosin susceptibility of the isolates. To verify the presence of antibodies resistant to MG, 750 blood samples were randomly collected from 38 broiler farms from 2019 to 2022 in Mazandaran and Golestan provinces, Iran, and rapid slide agglutination (RSA) assay was performed. Positive results were analyzed by the enzyme-linked immunosorbent assay (ELISA) for further investigation. Here, 920 swab samples were collected from 38 non-vaccinated commercial farms for culture, and PCR tests were performed for the isolated strains. The activities of tylosin were tested in vitro against these isolates using the broth microdilution method. The lowest antibiotic concentration that resulted in a color change was considered the minimum inhibitory concentration (MIC) value. Twenty-four (63.1%) farms were positive in the RSA test, and 21 (55.2%) farms were positive in the ELISA test. Nine (23.68%) of the farms grew on culture media, and 8 (21.05%) were detected as Gallisepticum species by PCR. The geometric mean of MIC for tylosin was 5.75 µg/ml, MIC50 was 4 µg/ml, and MIC90 was 8 µg/ml. The results indicated that commercial farms were infected with MG. Considering the ability of MG to spread and the probable use of the RSA test as a rapid and cheap method, it can be argued that ELISA and RSA serological tests can be used to find MG in poultry flocks, and the positive result should be confirmed by standard microbiological tests or PCR. It was also found that the isolated parts of MG changed their sensitivity to tylosin, indicating the need for routine testing to optimize treatment dose and efficiency.

Molecular Detection of Mycoplasma gallisepticum in Broiler Chicken Farms of Satara and Pune Districts of Maharashtra

Molecular Detection of Mycoplasma gallisepticum in Broiler Chicken Farms of Satara and Pune Districts of Maharashtra

Detection of Mycoplasma gallisepticum in broiler chickens by PCR.

Background:Mycoplasma is a significant microorganism of poultry, which can cause respiratory infections and synovial inflammation, bringing about huge financial misfortunes to poultry workmanship worldwide.Aim:The goal of existing research was to determine the infection rate of Mycoplasma gallisepticum (MG) from chronic respiratory disease cases among broilers fields in Mosul/ Iraq using the polymerase chain reaction (PCR) technique.Methods:All 92 lungs samples were collected from broilers with classical respiratory signs in different regions of the Nineveh governorate for 3 months from February to April 2021.Results:PCR tests were performed using two couple primers, one for the qualitative amplification of 16S rRNA genes (285 base pairs) in Mycoplasma spp. and the second couple for the detection of M. gallisepticum (580 base pairs). Among the samples obtained from broilers, 87 (94.7%) were positive for Mycoplasma and 79 (85.9%) were positive for M. gallisepticum.Conclusion:Our results showed that MG infection in broiler chickens leads to serious clinical symptoms and severe lesions. The rate of Mycoplasma isolation in this study is high despite the short lifespan of broiler chickens.

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by using of cultural and PCR technique

Laboratory methods are essential for the diagnosis of Mycoplasmal infection. There are three laboratory approaches are essential for the diagnosis of Mycoplasmal infection in chicken including direct methods by culture method and polymerase chain reaction, and indirect methods by detection of Mycoplasmal antibodies by serological tests. This study aimed to detection of Mycoplasma by culture and PCR technique. Two hundred seventy-six samples were collected from infected adult boiler chicken in Salah Al-din province which suffering from respiratory signs and /or joint infection, 202 respiratory and 74 articular samples. According to the results of culture, Mycoplasma isolated in rate of 35.1% (36.6% from respiratory samples and 31.1% from articular samples). The sensitivity of culture was 100%, while the specificity of culture was 97.9% when comparing with PCR results. The current study concluded that the respiratory infection was more than articular infections, and Mycoplasma gallisepticum more distributed than Mycoplasma synoviae among chickens.

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae among Commercial Poultry in Khouzestan Province, Iran.

Mycoplasmas are important avian pathogens, which can cause both respiratory disease and synovitis in poultry that result in considerable economic losses to the poultry industry all over the world. The aim of this study was to determine the prevalence of Mycoplasma gallisepticum and Mycoplasma synoviae infections among commercial poultry flocks in Khouzestan province, Iran, using the polymerase chain reaction (PCR) technique. Totally, 290 tracheal swab samples were collected from 19 broiler flocks and 4 layer-breeder flocks, with or without respiratory signs, in different areas of Khouzestan province within six months. The PCR tests were applied for the specific amplification of 16S rRNA (185 bp) and vlhA (392 bp) genes. Out of 100 swab samples obtained from the layer-breeder flocks, 1 and 72 specimens were positive for M. gallisepticum and M. synoviae, respectively. In this regard, out of the 4 layer-breeder flocks, 1 (25%) and 4 (100%) flocks were positive for M. gallisepticum and M. synoviae, respectively. However, none of the studied broiler flocks were M. gallisepticum- or M. synoviae-positive. According to the results, the PCR technique could be concluded as a rapid method for the accurate identification of M. gallisepticum and M. synoviae infections in commercial poultry flocks. The results were indicative of the low prevalence of M. gallisepticum in the studied flocks in Khouzestan province. On the other hand, M. synoviae was widely distributed among layer-breeder flocks in this province.

Duplex PCR for Direct Detection of Mycoplasma gallisepticum and Mycoplasma synoviae from Tissues of Poultry Affected with Respiratory Infections

In the present study, 92 pooled tissue samples including trachea, lungs and air sacs were collected from 92 different poultry flocks affected with respiratory infections and were subjected to mycoplasmological examination by duplex PCR. Result of duplex PCR revealed, 25/92 (27%) MG, 2/92 (2.1%) MS and 1 (1.08%) sample was positive for both MG and MS. In conclusion, duplex PCR was found to be a better assay to detect two most pathogenic avian mycoplasmas (M. gallisepticum and M. synoviae) simultaneously directly from poultry tissue sample and also could serve as a sensitive, specific and rapid tool in monitoring and surveillance programmes on poultry farms in Haryana. It seems to be the first study of this type in India.

전북지역 육용종계에서 Mycoplasma gallisepticum과 M. synoviae의 항체 및 유전자 양성률 조사

전북지역 육용종계에서 Mycoplasma gallisepticum과 M. synoviae의 항체 및 유전자 양성률 조사

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by Real-Time PCRs and Mycoplasma gallisepticum-antibody Detection by an ELISA in Chicken Breeder Flocks

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by Real-Time PCRs and Mycoplasma gallisepticum-antibody Detection by an ELISA in Chicken Breeder Flocks

A Multiplex real-time PCR for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from Brazilian commercial poultry flocks

Mycoplasma gallisepticum (MS) and Mycoplasma synoviae (MS) are important avian pathogens and cause economic losses to the poultry industry. Molecular biology techniques are currently used for a rapid detection of these pathogens and the adoption of control measures of the diseases. The aim of this study was to develop and validate a technique for simultaneous detection of MG and MS by multiplex real time polymerase chain reaction (PCR). The complete assay (Multiplex MGMS) was designed with primers and probes specific for each pathogen and developed to be carried out in a single tube reaction. Vaccines, MG and MS isolates and DNA from other Mycoplasma species were used for the development and validation of the method. Further, 78 pooled clinical samples from different poultry flocks in Brazil were obtained and used to determine the sensitivity and specificity of the technique in comparison to 2 real time PCR assays specific for MG (MG PCR) and MS (MS PCR). The results demonstrated an agreement of 100% (23 positive and 44 negative samples) between Multiplex MGMS and MG PCR in the analysis of 67 samples from MG positive and negative poultry flocks, and an agreement of 96.9% between Multiplex MGMS and MS PCR in the analysis of 64 samples from MS positive and negative poultry flocks. Considering the single amplification tests as the gold standard, the Multiplex MGMS showed 100% of specificity and sensitivity in the MG analysis and 94.7% sensitivity and 100% specificity in the MS analysis. This new assay could be used for rapid analysis of MG and MS in the poultry industry laboratories.

Detection of Mycoplasma gallisepticum in experimentally infected broiler chickens using Culture, SPA, ELISA, and PCR methods

Avian mycoplasmosis is caused by several pathogenic mycoplasmas of which Mycoplasma gallisepticum and Mycoplasma synoviae are the most important. This experiment was designed to study the diagnosis of M. gallisepticum infection in broiler chicken receiving M. gallisepticum. One hundred and twenty one-day-old chickens were raised in separate isolated rooms and equally allocated into two groups and renamed groups A and B were 121-day-old chickens. At 3 weeks old, group A was considered as the control group while group B was challenged with M. gallisepticum containing 1 × 106 CFU/bird via an eye drop. In this study, culture, serum plate agglutination (SPA) test, the commercially available enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) tests were compared for the detection of M. gallisepticum infection during the 1–7 weeks of age. Results revealed that M. gallisepticum was found in group B using culture and PCR during the 4–7-week-old period. The SPA results showed positive reactors in group B during the 5–7-week-old period. The positive reactors were detected by commercial ELISA test kit from 6 to 7 weeks of age. In this study, sensitivity of the PCR method was higher as compared to culture and serological tests. Its high sensitivity and fast turnaround time is making it the most frequently used test to monitor M. gallisepticum infection beside other tests.

Influence of Swab Material on the Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by Real-Time PCR

Recent reports have shown an increased recovery of cells from flocked nylon swabs which may improve the specimen quality and the real sensitivity of diagnostic tests in a clinical setting. In this study, the detection of Mycoplasma gallisepticum (MG) and M. synoviae (MS), using dry swabs of different materials (nylon flocked, cotton, and polyester), was investigated using real-time TaqMan PCR protocols. Different types of samples, including dilutions of pure broth cultures of MG and MS as well as swabs from tracheas of experimentally infected chickens and field cases of infection, were analyzed. There were no statistical differences in real-time PCR results among the different swab types (P < 0.05), indicating that this is not likely to be a significant factor in MG and MS detection by this method.

Influence of Swab Material on the Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by Real-Time PCR

Influence of Swab Material on the Detection of Mycoplasma gallisepticum and Mycoplasma synoviae by Real-Time PCR

Development of a duplex real-time TaqMan PCR assay with an internal control for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae in clinical samples from commercial and backyard poultry

In this study, we report the development and validation of a duplex real-time polymerase chain reaction (PCR) assay with an internal control using TaqMan-labelled probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae (duplex MGMS PCR). The MGMS PCR was highly specific with a sensitivity of 7 and 1 colony-forming units/ml for M. gallisepticum and M. synoviae, respectively, using dilution of pure culture that corresponds to 34 and 29 DNA copies per reaction. Validation of the assay was completed with 260 and 27 pooled samples (tracheal swabs) from commercial chickens and turkeys, respectively, with potential M. gallisepticum and M. synoviae involvement and 42 samples (palatine cleft swabs) from backyard geese and ducks. Using isolation as the gold standard, the MGMS PCR was more sensitive than isolation and the analytical sensitivity was 0.944 and 0.958 for M. gallisepticum and M. synoviae, respectively. In comparison with a gapA-based assay (gapA PCR) and a 16S rRNA-based assay (16S PCR) for M. gallisepticum and M. synoviae, respectively, the results agreed for 94.5% and 96.6%, respectively. The use of the internal control allowed monitoring of proper extraction and inhibition of amplification that was detected in 12 samples. The duplex MGMS PCR was shown to be superior to the presently reported real-time PCR assays in terms of combination of sensitivity, specificity and capacity of detection of more than one target in a single tube. In conclusion, the duplex MGMS PCR was highly specific, sensitive, and reproducible and could be used on clinical samples from commercial chickens, turkeys and backyard poultry including ducks and geese.

Real-time polymerase chain reaction for the qualitative and quantitative detection of Mycoplasma gallisepticum

The aim of this study was to evaluate the sensitivity and the detection limit of a real-time polymerase chain reaction (Q-PCR) developed for the qualitative and quantitative detection of Mycoplasma gallisepticum. No cross-reactivity was observed with DNA from other important avian mycoplasmas, including Mycoplasma synoviae and Mycoplasma meleagridis. However, the Q-PCR could not distinguish between M. gallisepticum and Mycoplasma imitans. The Q-PCR had detection limits 10 to 1000 times lower than a conventional commercial PCR method and than culture. The Q-PCR was used quantitatively by incorporating a set of external M. gallisepticum DNA standards, derived from a M. gallisepticum log-phase culture of a known concentration. The number of colony-forming unit equivalents per millilitre in tracheal swabs from experimentally infected birds could be determined from a single sample. The method had good reproducibility and correlated well with standard counting techniques using culture. It can be concluded that the Q-PCR described is suitable for qualitative and quantitative detection of M. gallisepticum in clinical samples. PCR en temps réel pour la détection qualitative et quantitative de Mycoplasma gallisepticum Le but de cette étude a été d'évaluer la sensibilité et les limites de détection du test d'amplification en chaîne par polymérase en temps réel (Q-PCR) développé pour la détection qualitative et quantitative de Mycoplasma gallisepticum (Mg). Aucune réaction croisée n'a été observée avec l'ADN d'autres mycoplasmes aviaires importants, incluant M. synoviae et M. meleagridis . Cependant, le test Q-PCR n'a pas pu différencier Mg de M. imitans. Les limites de détection ont été de 10 à 1000 fois inférieures à celles d'un test PCR du commerce (cPCR) et à la culture. Le test Q-PCR a été utilisé quantitativement en incorporant un set de standards externes d'ADN de Mg dérivé d'une culture de Mg, en phase logarithmique, de concentration connue. Le nombre d'équivalents unité formant colonie/ml dans les écouvillons trachéaux des animaux infectés expérimentalement a pu être déterminé à partir d'un seul échantillon. La reproductibilité de la méthode a été bonne et une bonne corrélation a été observée avec les techniques standard de comptage après culture. Il peut être conclu que le test Q-PCR décrit est approprié à la détection qualitative et quantitative de Mg à partir d'échantillons cliniques. Real-Time-PCR für den qualitativen und quantitativen Nachweis von Mycoplasma gallisepticum Ziel dieser Studie war es, die Sensibilität und die Nachweisgrenze einer für die qualitative und quantitative Bestimmung von Mycoplasma gallisepticum (MG) entwickelten Real-Time-Polymerasekettenreaktion (Q-PCR) zu untersuchen. Mit der DNS anderer wichtiger aviärer Mykoplasmen einschließlich M. synoviae und M. meleagridis wurden keine Kreuzreaktionen beobachtet. Die Q-PCR konnte jedoch nicht zwischen MG und M. imitans unterscheiden. Die Q-PCR hatte eine 10-1000-mal niedrigere Nachweisgrenze als die konventionelle kommerzielle PCR (cPCR) und die kulturelle Nachweismethode. Die Q-PCR wurde zum quantitativen Nachweis genutzt, indem ein Satz externer MG-DNS-Standards aus MG-log-Phasen-Kulturen mit bekannter Konzentration inkorporiert wurde. Die Zahl der Kolonie bildenden Einheiten/ml in Trachealtupfern von experimentell infizierten Tieren konnte in einer einzigen Probe bestimmt werden. Die Methode weist eine gute Reproduzierbarkeit auf und korrelierte mit Standard-Zählverfahren bei der Kultivierungsmethode. Daraus kann geschlossen werden, dass die beschriebene Q-PCR für den qualitativen und quantitativen Nachweis von MG in Untersuchungsproben geeignet ist. PCR a tiempo real par la detección cuantitativa y cualitativa de Mycoplasma gallisepticum El objetivo de este estudio fue el de evaluar la sensibilidad y el límite de detección de una reacción en cadena de la polimerasa a tiempo real (Q-PCR) desarrollado para la detección cualitativa y cuantitativa de Mycoplasma gallisepticum (Mg). No se observó reactividad cruzada con el ADN de otros importantes micoplasmas aviares, incluidos M. synoviae y M. meleagridis. Aún así, la Q-PCR no pudo distinguir entre Mg y M. imitans. LA Q-PCR presentó límites de detección de 10 a 1,000 veces menores que la PCR comercial convencional (cPCR) y que el cultivo. La Q-PCR se utilizó como método cuantitativo mediante la incorporación de un grupo de ADN de Mg estándares externos, derivados de un cultivo de Mg en fase log, de concentración conocida. El número de de equivalentes de unidades formadoras de colonias/ml en hisopos traqueales de aves infectadas experimentalmente pudo ser determinado a partir de una sola muestra. El método presentó buena reproducibilidad y se correlacionó bien con las técnicas estándares de contaje usadas en cultivos. Se puede concluir que la Q-PCR descrita es útil para la detección cualitativa y cuantitativa de Mg en muestras clínicas.

Polymerase chain reaction for detection of Mycoplasma gallisepticum in environmental samples

The polymerase chain reaction (PCR) was used to detect Mycoplasma gallisepticum in samples collected from the environment of experimentally or naturally infected poultry. Culture was also used in the experimental infections. Of 160 samples of food, drinking water, feathers, droppings or dust collected during experimental infection, 103 were positive using a M. gallisepticum -specific PCR (MG-PCR) and 68 were positive using a PCR (mycoplasma-PCR) that detects all species of the genera Mycoplasma , Spiroplasma, Acholeplasma and Ureaplasma . Six of these samples were also positive by culture. In environmental samples collected on a depopulated M. gallisepticum -positive turkey farm, three and two out of a total of 12 were positive by mycoplasma-PCR and MG-PCR, respectively. These results indicate the disseminating capacity of this mycoplasma and the possible use of PCR methods for epidemiological analyses and control of farm decontamination before the introduction of new birds.

Experiences with multispecies polymerase chain reaction and specific oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae

Amplified fragments of the rDNA coding for 16S rRNA of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were blotted on nylon membranes, followed by dot-blot detection with two species-specific digoxigenin-(DIG)-labeled oligonucleotide probes. The sensitivity and specifity of the tests were determined in titration studies with purified homologous and heterologous DNA. With the detection protocol used, the MSYV8/31 probe showed 100% specifity for MS, while both MG and the related species Mycoplasma imitans were recognized by the MGAV8/31 probe. Both DIG-labeled oligonucleotides gave positive results in the colorimetric assay with 10 to 100 ng homologous non-amplified DNA and polymerase chain reaction (PCR) amplificates of 100 fg homologous template DNA. There was no reaction with heterologous strains when amplificates starting with a 106-fold amount of template DNA (100 ng) were tested in dot-blots. The suitability for field samples was demonstrated with tracheal swabs from turkeys and chickens, and the results were compared with mycoplasma growth in cultures of the same swabs. Both tests had an accuracy of over 95%, a high sensitivity and specificity, and high predictive values of positive or negative results. There was no significant difference between the results obtained by the two methods. PCR in combination with dot-blotting is a relatively simple method for the detection of mycoplasma infections, and a valuable extension of current diagnostic tools.

A comparison of a commercial PCR‐based test to culture methods for detection of Mycoplasma gallisepticum and Mycoplasma synoviae in concurrently infected chickens

The suitability of commercial PCR‐based test kits for the detection of either Mycoplasma gallisepticum (MG) or M. synoviae (MS) was compared to detection by culture. The MG and MS kit detected six and five homologous strains respectively in broth cultures and there were no reactions with thirteen het‐erologous species including M. imitans, a species phylogenetically closely related to MG. Tracheal and lung/air‐sac swabs were collected from twenty 17‐week‐old commercial pullets which were seropositive for MS and were compared for detection of MS by kit and by culture. The results were in agreement for 13 positive and 22 negative swabs, while the remaining five swabs were either PCR‐positive only (two) or culture‐positive only (three). Tracheal swabs taken from seventy‐six 31‐week‐old layers from a MS seropositive commercial flock which had been experimentally infected with MG were used to compare the MG DNA probe kit and culture. Of 76 swabs 39 were MG positive and 12 were negative by both methods. MG was detected by PCR test only in 23 other specimens, while only two swabs were negative by PCR but positive by culture. The difference between the detection methods was significant (McNemar test, P < 0.001). Concurrent MS infection was detected by the PCR‐based kit in 45 of these hens.

Detection ofMycoplasma Gallisepticum antibodiesin the sera of village chickens by the enzyme-linked immunosorbent assay

Detection ofMycoplasma Gallisepticum antibodiesin the sera of village chickens by the enzyme-linked immunosorbent assay

Detection ofMycoplasma gallisepticum antibodiesin the sera of village chickens by the enzyme-linked immunosorbent assay

Detection ofMycoplasma gallisepticum antibodiesin the sera of village chickens by the enzyme-linked immunosorbent assay

Detection of Mycoplasma gallisepticum, M. synoviae, and M. iowae by Multi-Species Polymerase Chain Reaction and Restriction Fragment Length Polymorphism

A single set of oligonucleotide primers was designed from known 16S ribosomal RNA (rRNA) sequences of Mycoplasma gallisepticum (MG), M. synoviae (MS), and M. iowae (MI). This set of primers selectively amplifies a 780-base-pair DNA fragment within the 16S rRNA gene of MG, MS, and MI but does not amplify other avian mycoplasmas or other bacteria. The detection limit of the multi-species polymerase chain reaction (PCR) was approximately 100 mycoplasma (MG, MS, MI) colony-forming units per PCR reaction. The PCR product was differentiated by restriction fragment length polymorphism with the restriction enzymes HpaI, HpaII, and MboI. Preliminary results from field samples suggest that this technique could be a useful and rapid diagnostic test for the detection of these three pathogenic poultry mycoplasmas.