Molecular assay for detecting MS-H vaccine strain and immune response mechanisms in chickens receiving one or two doses of live MS-H vaccine.

The MS-H vaccine, containing a live strain of Mycoplasma synoviae, is a feasible option for controlling M. synoviae infection in poultry flocks. A comprehensive understanding of vaccinated chickens, including strain differentiation and immune response mechanisms, is required to optimize vaccination strategy. This study aimed to verify the PCR-RFLP molecular assay as a convenient technique for detecting the MS-H vaccine strain and to characterize the immune response mechanisms in experimental layer-type chickens receiving one of three different vaccination programmes; a single dose at either 9 or 12 weeks of age or two doses at both 9 and 12 weeks of age. The PCR-RFLP assay, using restriction enzyme TasI to digest vlhA gene-targeted PCR amplicons, was performed to evaluate vaccine administration by detecting the MS-H vaccine strain in vaccinated chickens and differentiating it from non-vaccine strains such as WVU1853 reference strain and Thai M. synoviae field strains. Results demonstrated that vaccination in layer-type chickens, whether as one or two doses, stimulated immune response mechanisms with no significant advantages of two administrations over a single administration. Serological responses in vaccinated chickens, examined by RPA test and ELISA, were initially detected at 2 weeks post-vaccination, continuously increased, and then remained at the baseline levels from 6 to 9 weeks post-vaccination. Cellular immune responses against both homologous and heterologous antigens, examined by the MTS tetrazolium assay, were similar in the early period post-vaccination, whereas cellular immune response against the homologous MS-H antigen was improved in the late period post-vaccination.

Molecular characterization and antimicrobial susceptibility profiles of Thai Mycoplasma synoviae isolates.

Mycoplasma synoviae (MS) infection is mainly controlled by pathogen-free flocks' maintenance, medication in infected flocks, and vaccination in high-risk flocks. The effective control strategy requires convenient approach for detecting and differentiating MS strains and reliable drug susceptible evidence for deciding on reasonable antimicrobial usage. This study aimed to characterize the partial vlhA gene of nine Thai MS isolates circulated in chickens in 2020, to verify the PCR-RFLP assay for strain differentiation, and to determine the eight antimicrobial susceptibility profiles using microbroth dilution method. Based on sequence analysis of the partial vlhA gene, Thai MS isolates in 2020 were classified as types E and L with 19 and 35 amino acid lengths, respectively. The developed PCR-RFLP assay could detect and differentiate vaccine and Thai field strains. Most Thai MS isolates in this study were susceptible to tylosin, tylvalosin, tiamulin, doxycycline, oxytetracycline, tilmicosin, and lincomycin-spectinomycin at MIC50 values of 0.0391, 0.0098, 0.0781, 0.1563, 0.1563, 0.625 and 0.625 µg/mL, respectively; and resistance to enrofloxacin at MIC50 value of 10 µg/mL. In conclusion, this study revealed diagnostic assays for differentiating MS strains and the antimicrobial susceptibility profiles of Thai MS, which are necessary to design suitable MS control procedures for poultry flocks.

Targeted sequencing analysis of Mycoplasma gallisepticum isolates in chicken layer and breeder flocks in Thailand.

Mycoplasma gallisepticum (MG) is one of the most economically important pathogens worldwide. MG affects the respiratory system and impairs growth performance in poultry. In developing countries, the most widely used technique to identify MG is the conventional PCR assay. In this study, 24 MG isolates collected from Thailand farms with unvaccinated chickens during 2002-2020 were characterized by gene-targeted sequencing (GTS), followed by phylogenetic analysis using unweighted pair group method with arithmetic mean. These 24 Thai MG isolates differed from vaccine strains, including the F, ts-11 and 6/85 strains. One isolate showed 99.5-100% genetic similarity to the F strain with 4 partial gene analyses. This result may have been due to contamination from vaccinated flocks because the F strain is the most commonly used vaccine strain in Thailand. However, the GTS analysis using the partial MG genes in this study showed that the isolates could be grouped into different patterns based on individual gene sequences. The phylogenetic analysis of partial mgc2, gapA, pvpA and lp gene sequences classified the Thai MG isolates into 7, 11, 7 and 2 groups, respectively. In conclusion, at least 2 partial MG genes, especially partial gapA and mgc2 genes, are needed to differentiate MG isolates.

Characterization of Thai Mycoplasma synoviae Isolates by Sequence Analysis of Partial vlhA Gene.

Mycoplasma synoviae (MS), a remarkable pathogen in poultry, causes subclinical infection of the upper respiratory tract and an infectious synovitis, especially in the tendon sheaths and synovial membranes of joints. Because the specific detection of MS 16S rRNA gene-based PCR was unsuitable for strain differentiation, vlhA gene-based PCR was designed to differentiate the MS strains. The vlhA gene of MS encodes for hemagglutinin and other immunodominant membrane proteins involved in colonization, antigenic variations, and virulence. Sequence analysis of the vlhA gene based on the nucleotide insertion/deletion of the proline-rich repeat (PRR) region and the nucleotide polymorphisms of the RIII region in vlhA gene fragments was useful for typing and subtyping of MS strains. This study aimed to characterize the Thai MS field isolates and to differentiate the field and vaccine strains in Thailand by using sequence analysis of the partial vlhA gene. In total, 20 MS field isolates submitted from registered chicken farms in Thailand during 2015 were identified as Type C1 (n = 1), C2 (n = 4), E1 (n = 9), E2 (n = 1), and L (n = 5). The results revealed that six of the nine isolates resulting in respiratory signs were Type E1. In addition, four isolates from lame chickens showing joint swelling were identified as Type L, with a length of 105 nucleotides. This study provides the first molecular data of Thai MS isolates and the first evidence of Type L for being an arthropathic strain that differs from a previous study demonstrating that only MS Type B, with a longer PRR of 135 nucleotides, could be highly invasive strains and associated with infectious synovitis in chickens. Furthermore, one farm showed coinfection of MS Types E and L, but most of the farms were affected by only one type of MS. The results indicated that sequence analysis of the partial vlhA gene can be used as a tool for tracing MS characterization.