Detection and antibiotic resistance of Mycoplasma gallisepticum and Mycoplasma synoviae among chicken flocks in Egypt

Background and Aim: Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) are the most significant pathogens of avian mycoplasmosis. This study aimed to isolate and identify MG and MS from chickens and detect the various virulence genes in the isolates. Moreover, the efficacies of different antibiotics were tested to identify suitable treatment regimens. Materials and Methods: We isolated MG and MS from 487 chicken samples of different ages located in different Governorates in Egypt using conventional isolation methods. The isolates were characterized by polymerase chain reaction (PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method. Results: The prevalence of MG among the isolates was 9.85%, with the highest percentage isolated from air sacs, while the prevalence of MS among the isolates was 1.6%. Moreover, the highest levels of the prevalence of both MG and MS were during the winter and autumn sampling, while the lowest levels were in the summer and spring. Following the 16S rRNA-based detection of Mycoplasma isolates, 14 MG and 5 MS isolates were identified by different PCR-based detection methods for various virulence genes. Nine MG isolates contain the mgc2 gene, six MG isolates contain the gapA gene, and three MS isolates contain the vlhA gene. We validated a duplex PCR method for the simultaneous identification of MG and MS, based on 100% of the MG and MS isolates generating common bands at 55 and 17 kDa, respectively. The MIC method identified tiamulin and spiramycin as the antibiotics of choice for the treatment of MG and MS infections, respectively. Conclusion: For more precise diagnosis of Mycoplasma infections in chicken flocks, conventional isolation methods must be confirmed by PCR. SDS-PAGE analysis helps in epidemiological studies and vaccine preparation. The MIC method can be used to help develop therapies to control avian mycoplasmosis infections.


Introduction
Mycoplasmas are small-celled prokaryotes belonging to the class Mollicutes. They have a small genome and are unable to produce a cell wall, as well as many metabolic pathways [1]. Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) cause the most significant Mycoplasma infections among commercial poultry. MG infections usually lead to chronic respiratory disease (CRD) in chickens and infectious sinusitis in turkeys. MS causes subclinical upper respiratory infections, eggshell apex abnormalities, and tenosynovitis or bursitis in chickens and turkeys [2][3][4]. CRD caused by MG is prevalent in layer, broiler, and breeder poultry flocks. Infected birds display respiratory symptoms that include sneezing, rales, coughing, as well as an exudation from the nostrils and eyes [5]. The presence of MG and MS in infected tissues or swab samples is confirmed by isolating the organisms using a specific medium or by direct detection of their DNA.
There are several types of respiratory infections that impose a significant health and economic impacts. For example, viruses and bacteria are known to coinfect chickens, although the pathogenesis involved, and their connection with mucosal surfaces are poorly described [6]. MG and MS diseases in chickens may sometimes superficially appear like respiratory diseases caused by other pathogens such as those that cause mild Newcastle disease and avian infectious bronchitis. Ishfaq et al. [7] found that MG causes CRD in chickens, and infected chickens display abnormal morphology and cellular damage, including increases in inflammatory cell infiltration, cellular debris, and exudates, and mitochondrial and DNA damage in lungs. Infections with Avibacterium paragallinarum and Pasteurella multocida may present similarly and Available at www.veterinaryworld.org/Vol.13/July-2020/22.pdf should be excluded. MG in turkeys may be confused with avian pneumovirus infections, and the presence of sinusitis may also be suggestive of infections by P. multocida, Chlamydia, or MS. Moreover, care should be exercised in distinguishing between MS-related infectious synovitis, Staphylococcus aureus infections, and reovirus-related infectious tenosynovitis [8]. In general, MG and MS cause significant impacts on the poultry industry, thus detecting these pathogens is crucial for conducting epidemiological studies and control programs of avian mycoplasmosis.
This study aimed to characterize MG and MS isolates from chicken flocks using molecular methods and to test these isolates for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method, thus providing a guide to optimal therapy.

Ethical approval
The study protocol was approved by Animal Ethics Committee of the Faculty of Veterinary Medicine, Cairo University, Egypt.

Sampling
We collected 487 chicken samples from different flocks, breeds, and ages in Egypt (Giza, Fayoum, Benisuef, Menya, and Alexandria) from January 2017 to July 2019.
In acute cases, tracheal swabs, synovial fluid, trachea and lung samples were collected; while in chronic stages, air sac specimens and caseous material were collected (Table-1).

Culturing, isolation, and preliminary identification
The isolation and preliminary identification of Mycoplasma isolates followed the standard procedures defined by Kleven [9]. The samples were cultured on sterile PPLO broth and agar media, as described by Frey [10]. Mycoplasma colonies were identified based on their characteristic fried egg appearance [11].
The digitonin sensitivity test was performed to differentiate between Mycoplasma and Acholeplasma isolates [12]. The glucose fermentation test [13] and the arginine hydrolysis test [14] were performed to differentiate Avian Mycoplasma species. Finally, the film and spot formation test used to identify Mycoplasma synoviae [15] and the growth inhibition test [16] were performed to identify a Mycoplasma isolate depending on serology.
Purification and maintenance of isolates [17] A single fried egg-shaped colony was picked, including the agar block, and transplanted into a broth medium to obtain a pure culture. The purified isolates were kept at −20°C in the form of agar blocks.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
The isolates were characterized to determine strain variability. SDS-PAGE followed the methods described by Laemmli [18]. The samples were run alongside preserved molecular weight markers ladder from Cleaver Scientific Ltd (Blue Wide Range Prestained Protein Ladder, UK) and low molecular weight protein ladder (Pharmacia Biotech, USA). 200 µl of activated MG or MS Broth culture was mixed with equal volume of denaturation mixture containing 2-mercaptoethanol. The mixture was boiled for 5 min then 20 µl was applied to each well of the SDS-PAGE gel. The gel was run at a constant power of 25 mA for approximately 4-5 h using a horizontal electrophoresis connected with power supply (Biometra, USA). Low molecular weight and wide range protein ladders were included in each run. The gel was stained overnight (50% methanol, 7% glacial acetic acid, and 0.2% Coomassie Brilliant Blue) and de-stained using a solution containing 40% methanol and 7% glacial acetic acid. The bands were visualized, photographed, and analyzed using Alpha View-AlphaImager HP (ProteinSimple, CA, USA).

PCR
In PCR analysis of 16 Mycoplasma isolates was analyzed by PCR to specifically amplify the 16S rRNA gene. Only 14 isolates were identified as MG, while the mgc2 and gapA virulence gene was detected in only nine and six of the 14 MG isolates, respectively.
Five Mycoplasma isolates were analyzed by PCR. Based on their 16S rRNA gene, all five isolates are classified as MS. The vlhA virulence gene was detected in only three out of five MS isolates.
Duplex PCR analysis was designed to simultaneously detect MG and MS by targeting the 16S-23S rRNA gene region of the isolates. MG positive results were 14/16 (87.5%). 14 MG OIE positive isolates were tested for virulence using mgc2 virulent gene and gapA virulent gene and we found that 9/14 were mgc2 positive isolates (64.3%) and 6/14 were gapA positive isolates (42.9%) (Figure-2). MS positive results were 5/5 (100%). MS OIE positive isolates were tested for virulence using vlhA virulent and we found 3/5 were vlhA gene-positive isolates (60%) (Figure-3). Results indicated a difference in virulence between the isolates. Finally, we adopted and validated duplex PCR for simultaneous identification of MG and MS as it was more rapid and cheaper method in comparison to single PCR for identification of MG and MS.

Microbroth antibiotic sensitivity test
Antimicrobial sensitivity tests were conducted on 5 MG and 3 MS isolates using the MIC method (Table-4). The MIC values among the isolates ranged from 0.0625 to 0.5 ug/ml.

Discussion
MG and MS are important pathogens that affect poultry worldwide, causing enormous economic losses in the poultry industry [25,26].
MG is a major poultry pathogen [27] that causes CRD in chickens and turkeys and also negatively impacts egg hatchability and development [28]. MG infections also affect virus titers and the efficacy of some poultry viral vaccines [29,30]. In our study, analysis of tracheal swabs, trachea, lungs, and air sac samples showed that MG is most prevalent in the air sacs (15.7%), a result that is similar to that reported by Reda and El-Samie [31] and and Mohamed [32], who isolated MG from air sacs at the highest rates of 23.3% and 21.5% respectively. Meanwhile, EI-Jakee et al. [33] isolated and cultured 14 Mycoplasma isolate from tracheal swabs collected from 12 broiler breeder flocks.
Necropsy of the diseased birds revealed chronic macroscopic lesions accumulating as caseous material in the lungs [34]. This material is sticky, attaching to the respiratory surface of the air sacs. All of the MG isolates in our study were obtained from caseous material.
MS is an important poultry pathogen that causes air sacculitis, synovitis, and reduced egg production, resulting in great economic losses [4,35]. The prevalence of MS in the synovial fluid was only 7.1%. This low percentage is attributed to the difficulty in isolating MS due to its fastidious growth.
MG and MS were digitonin positive, glucose fermentation positive, and arginine hydrolysis negative. Meanwhile, only suspected MS isolates gave positive results in the film and spot assay. In the growth inhibition test, 85% of suspected MG isolates were inhibited by specific antiserum against MG, while all MS isolates were inhibited by anti-MS antiserum. These results agree well with the results of biochemical identification.
The present study shows that MG and MS infections are more predominant during winter and autumn compared to summer and spring. This seasonal difference is probably due to poor temperature control measures, resulting in low-temperature stress [36].
SDS-PAGE has been used to demonstrate minor but distinct and reproducible differences in protein patterns among strains of MG and MS. Therefore, SDS-PAGE is a useful procedure in epidemiological and other studies, where it is important to be able to distinguish between bacterial strains based on protein patterns. The molecular detection of pathogenic isolates of MG by SDS-PAGE was indicated by the presence of specific 72 and 73 kDa bands in 60% Available at www.veterinaryworld.org/Vol.13/July-2020/22.pdf

Antimicrobial
Isolates ( of the isolates, which was also observed by Hassan et al. [36]. Strategies to control MG and MS infections are based primarily on serological and/or bacteriological screening. However, culture-based methods are laborious, time-consuming and may fail to detect Mycoplasma species from medicated birds. They are also less sensitive than PCR-based methods. PCR provides a rapid, sensitive, and precise method for diagnosing MG from suspected cases [5].  culture-based method. Periodic in vitro testing of MICs of antibiotics against Mycoplasma field isolates is required to monitor the impact of mass medication programs and to assist in developing effective therapies.

Authors' Contributions
ME collected the data, participated in the design of the work protocol, performed the laboratory work, and was involved in the preparation of the manuscript. ME and YMH found the research idea, shared in the performed data and designed the work protocol. JE participated in the design of the work protocol, contributed to the manuscript review and interpreted the results. All authors were involved in the preparation of the manuscript and had read and approved the final manuscript.
MG and MS isolated from chicken flocks were biochemically tested and then identified using PCR. Targeting using the 16S rRNA gene, PCR positively identified 87.5% and 100% of the MG and MS isolates, respectively. This finding agrees with those of Atique et al. [37] who confirmed that PCR provides more reliable, specific, and rapid results compared to culture.
The isolates that were positive for the mgc2 and gapA genes accounted for 64.3% and 42.9% of the MG OIE-positive isolates. The isolates that were positive for the vlh gene accounted for 60% of the MS OIE-positive isolates. These results suggest that the isolates may differ in their respective virulence levels. Therefore, Norouzian et al. [38] highlighted the importance of understanding the relationship between changes in MG antigenicity and pathogenicity to genetic variations.
Duplex PCR is cheaper and faster than single PCR for the identification of MG and MS. It is, therefore a good alternative method. The duplex technique was able to detect MG and MS simultaneously, although the MG bands are more intense than those of MS [39]. In our study, we adopted and validated duplex PCR for the simultaneous identification of MG and MS.
The PCR technique not only has several advantages, but it is also limited by the development of severe contamination due to the improper handling of samples, thus resulting in false-positive results [40,41]. Therefore, conventional cultural methods should be carried out in parallel with PCR.
Abd El-Hamid et al. [42] demonstrated that MG and MS infections can be controlled by proper treatment procedures. To develop an effective antibiotic treatment for MG and MS infections, we performed MIC experiments using eight different antimicrobials against five and three different MG and MS field isolates, respectively. On the one hand, tiamulin was the most effective antimicrobial for inhibiting MG growth in vitro. Thus, we recommend treating MG-infected birds using this antimicrobial, which agrees with Xiao et al. [43], who concluded that tiamulin is highly active against MG. On the other hand, spiramycin was the most effective antimicrobial for inhibiting the growth of MS in vitro. The use of this antibiotic has not yet resulted in any case of mycoplasma resistance; thus, we recommend it for treating MS infection. Hong et al. [44] suggested treating MS infections with antibiotics that do not cause the rapid development of MS resistance.

MG and MS are the predominant species of
Mycoplasma that causes respiratory and joint problems in chickens in the different Egyptian governorates (Giza, Fayoum, Benisuef, Menya, and Alexandria). Although time-consuming, the traditional cultural approach remains the gold standard for MG and MS diagnosis. PCR-based methods are more specific and faster than traditional culture-based methods; however, they should be performed in parallel with the traditional