Mycoplasma synoviae (MS) is the main Mycoplasma species in Mexican poultry production [1] . The route of MS infection may play a significant part in the resulting disease. Natural infection may occur vertically in ovo or horizontally by direct contact or airborne spread [2] . MS isolated from air sac lesions are more apt to cause airsacculitis, whereas those isolated from synovia are more apt to produce synovitis [3] . Vertical transmission plays a major role in spread of MS in chickens; however, several flocks hatched from infected breeders may remain free of infection (Vardaman). Vaccination and antibiotic treatments principally focus on leghorn breeder hens, have been used to prevent the vertical transmission of Mycoplasma synoviae [2] . In several countries, the live attenuated Mycoplasma synoviae strain MS-H, a temperature-sensitive (ts⁺) strain at <39.5˚C is used as a vaccination method to reduce virulent MS infection in commercial poultry. The vaccine is produced via chemical mutagenesis of the Australian field strain 86079/7NS [4] . Vaccination with the ts⁺ MS-H strain has the purpose of colonizing the extrapulmonary respiratory tract without causing systemic infection and transovarial transmission to the progeny. Nevertheless, the reversion from ts⁺ to temperature no-sensitive (ts⁻) (≥39.5˚C insulation) of live attenuated Mycoplasma spp. vaccines has been reported to revert virulence and vertical transmission to the progeny, produce infection of the air sacs of infected hens, and the ts⁻ isolates that lacked the ts⁺ characteristic of the original vaccine strain generated significantly higher tracheal mucosal thicknesses than MS-H-inoculated birds [5] . Moreover, recent studies have also shown reversion to virulence and vertical transmission of the ts-11, a live Mycoplasma gallisepticum vaccine [6] [7] . While the ts⁻ MS-H vaccine has received wide use in many countries over the past twenty five years, registration in the USA, the largest poultry producer country in the world has not been granted [2] . The authors of this paper asked 2022 the U.S. authorities why the MS-H vaccine has not been granted registration in the U.S. The authorities replied that the information is the company’s property that carried out the registration process in the U.S.A. The information related to this request depends on the final decision of the company to share sensitive information. However, the company still needs to send us ungranted registration information. Another alternative for the control of mycoplasmosis is antibiotic metaphylaxis. The antibiotics most widely used against Mycoplasma spp. are doxycycline, enrofloxacin, erythromycin, lincomycin, tiamulin, and tylosin [8] [9] . However, MS has been able to persist despite fluoroquinolone treatments in hens after two enrofloxacin treatments [10] .

The random amplification of polymorphic DNA (RAPD) is based on the analysis of bacterial samples of total genomic DNA subjected to PCR using short oligonucleotides of random sequences. This amplification protocol differs from standard PCR conditions because only a single random primer is used, and prior knowledge of the genome under analysis is not required [11] . This strategy provides an inherent advantage to de novo assays: not requiring prior sequencing studies and previous primers designed. Using a short (10-bp) primer, there is a high probability that the genome contains several amplification sites close to each other in an inverted orientation. The technique searches genomic material for small inverted sample repeats and amplifies intermediate segments of DNA of various lengths. The amplification product will depend on the template/primer combination and is reproducible for any combination. Amplification products are observed directly on agarose gels; Polymorphisms are considered dominant genetic markers inherited in a Mendelian genetic. The RAPD recognition technique has been demonstrated to be efficient, as well as especially effective for the epidemiological research of Mycoplasma gallisepticum (MG) strains and for the classification and distinction of vaccine strains and field isolates [1] [6] [12] [13] [14] [15] . However, the efficacy of RAPD in differentiating vaccine and field strains of MS has not been demonstrated. Marois et al. demonstrated that RAPD has a discriminatory indicator for MS from MG which is more significant than 0.95; therefore, RAPD was selected for the present work [16] .

Recently, our laboratories published a study showing the reverse of thermosensitivity of the ts⁺ MS-H strain [1] . However, as far as we are aware, there are no evidence on the reverse pathogenicity of the ts⁺ MS-H strain in poultry production, and the transmission to the progeny of hens vaccinated with ts⁺ MS-H, as has been previously reported with ts⁺ live attenuated MG vaccines [6] [7] . Therefore, the present study aimed to conduct a pilot study to lay the groundwork for evaluating the utility of the random amplification of polymorphic DNA (RAPD) method to analyze vertical transmission and possible reversal of thermosensitivity of the MS-H vaccine strain of Mycoplasma synoviae and evaluate the minimum inhibitory concentrations (MIC100) of the isolates (at 39.5˚C) in Hy-line® commercial breeder hens and progeny from the same parental batch.

In the present field report, isolation, and identification of the MS-H strains from tracheal samples of breeder hens at 48 and 72 weeks was demonstrated. Interestingly, the strains isolated from tracheal swabs grew at 37˚C and 39.5˚C. The ts⁻ MS-H strains isolated at 39.5˚C showed DNAbp equal to those of the ts⁺ MS-H vaccine strain. Moreover, the ts⁻ MS-H strain, with similar DNAbp as ts⁺ MS-H vaccine strain, was also isolated in progeny chickens in pullets showing clinical signs of synovitis or apparently healthy pullets. The laying breeder hens in the present study were positive for the MS-H strain, both by isolation and RAPD, until 48 and 70 weeks of age, indicating that the ts⁺ MS-H strain was slow to revert thermosensitivity. However, when the strain was transmitted to progeny, it produced synovitis at nine weeks of age. These findings suggest failures in vaccine immunity, mixed infection between the vaccine strain and the field strain, or the possibility that the ts⁺ strain can reverse its thermosensitivity at 39.5˚C, virulence, and replicate in laying breeder hens. In addition, the ts⁻ strain can be vertically transmitted to offspring from the same parent flock.

In a study published by Noormohammadi et al., 2003, 9/50 isolates from flocks vaccinated with the ts⁺ MS-H strain were found to have lost the ts⁺ phenotype of the original vaccine strain. In 2/9 ts⁻ MS clones were recovered from air sacs and 3/9 isolates produced increased tracheal mucosal thicknesses compared to MS-H inoculated birds, and 1/9 induced increased tracheal mucosal thicknesses [5] .

Data expressed as mean (median). ^†IS = infectious synovitis. Dissimilar letters within the same column show significant differences (p < 0.05).

The reverse of thermosensitivity has been also demonstrated with other commercial live attenuated Mycoplasma vaccines. In a study published by El Gazzar et al., 2011 from commercial broiler flocks in northeastern Georgia, USA that descended from the same parent flock were found to be Mycoplasma gallisepticum (MG) positive using serology, culture, and PCR, indicating reversion to virulence and vertical transmission of the ts-11 live MG vaccine [7] . Other scientists have also provided conclusive evidence of transovarian transmission and reverse virulence of the isolate genotyped as MG ts-11 [6] .

In the present, no differences were observed in MIC100s between laying breeder hens and progeny for doxycycline, erythromycin, lincomycin, and tylosin. However, a significant increase in MIC100 for enrofloxacin was observed in the progeny, following the standard therapeutic treatment of the poultry company. The ability of MS to persist in spite of fluoroquinolone treatments has been previously demonstrated by Le Carrou et al., 2006 who found a significant increase in the resistance level to enrofloxacin of five reisolated Mycoplasma clones, which was observed after the second treatment [10] . Furthermore, we found that the antimycoplasmic agent with the highest sensitivity for MS was tylosin, and this result is in agreement with a previous study [1] . Remarkably, in a study published by Pakpinyo and Sasipreeyajan [13] , the molecular characterization and determination of MIC tested on MG isolated from commercial chickens had similar RAPD patterns and properties of antimicrobial resistance, providing useful information to plan for prophylactic and therapeutic impacts on the poultry industry.

The results of this field report suggest that vaccination failed or that the strain of the live attenuated ts⁺ MS-H vaccine can revert its temperature sensitivity to perform vertical transmission to the progeny, recover virulence, and induce synovitis. The RAPD technique could easily differentiate between the different avian Mycoplasma species and differentiate between MG vaccine and field strains. However, for MS strains, there are no reports showing differences between strains. The underreporting may be since the RAPD method for interpreting MS isolates does not distinguish between genetic variation among MS strains during an outbreak and the presence of different but closely related strains. Hence, it is important to contemplate the potential danger when using ts⁺ MS-H is a reversion of ts⁺ with a possible reversion of pathogenicity to the original Australian field strain 86079/7NS and vertical transmission to progeny when using the ts⁺ MS-H vaccine strain or other life ts⁺ vaccines against Mycoplasma spp. This work should be taken as a pilot study for further studies confirming the findings of this field report under experimental and laboratory conditions and ruling out or confirming the use of the RAPD method to differentiate MS strains.

Conceptualization, V.M.P-G.; methodology, V.M.P.-G.; software, V.M.P.-G.; validation, G.T.-I., R.L.-A, S. E.-A., E.O.; formal analysis, G.T.-I.; investigation, V.M.P.-G.; resources, V.M.P.-G. data curation, I.C.-H. and D.G.; writing— original draft preparation, V.M.P-G.; writing—review and editing, V.M.P-G, G.T.-I., R.L.-A., and F.A.-H; visualization, V.M.P.-G; project administration, G.T.-I., and R.L.-A.; funding acquisition, G.T.-I., R.L.-A., S. E.-A., E.O. and V.M.P.-G. All authors have read and agreed to the published version of the manuscript.

This research obtained funding from the PAPIIT IG200923 project of DGAPA-UNAM, Universidad Nacional Autónoma de México. This research was also supported in part by funds provided by USDA-NIFA, Sustainable Agriculture Systems, Grant No. 2019-69012-29905. Title of project: Empowering U.S. Broiler Production for Transformation and Sustainability USDA-NIFA (Sustainable Agriculture Systems): no. 2019-69012-29905. The authors thank CONACyT for the doctoral grant, number 494367. Institutional Review Board Statement: This study was carried out in accordance with the recommendations for the management of chickens provided by the Internal Committee for Care and Use of Experimental Animals (CICUAE) of the National Autonomous University of Mexico (UNAM). Ethical approval code: CICUAE-FESC C20_06. And by the Researchers Supporting Project (RSP2023R111), King Saud University, Riyadh, Saudi Arabia.

The authors declare no conflict of interest.

Petrone-Garcia, V.M., López-Arellano, R., Castellanos-Huerta, I., El-Ashram, S., Al-Olayan, E., Graham, D. and Tellez-Isaias G. (2023) Isolation and Progeny Transmission of Non-Temperature- Sensitive MS-H Vaccine Strains of Mycoplasma synoviae from Temperature-Sensitive MS-H-Vaccinated Laying Breeder Hens. Food and Nutrition Sciences, 14, 579-588. https://doi.org/10.4236/fns.2023.147038