Antimicrobial susceptibility of pathogenic mycoplasmas in chickens in Asia.

Mycoplasma synoviae (n = 26) and M. gallisepticum (n = 11) isolates were gained from 164 clinical samples collected from China, India, Indonesia, Malaysia, Philippines, Republic of Korea and Thailand. Most isolates were from commercial chicken production systems. A method of filtering (0.45 µm) samples immediately after collection was convenient allowing over a week for transit to the laboratory. Minimum inhibitory concentrations (MICs) were characterized by a broth microdilution method to enrofloxacin, difloxacin, oxytetracycline, chlortetracycline, doxycycline, tylosin, tilmicosin, tylvalosin, tiamulin, florfenicol, lincomycin, spectinomycin and lincomycin and spectinomycin combination (1:2). Increased MICs to various antimicrobials were seen in different isolates but appeared largely unrelated to the antimicrobial treatment histories. Overall, the results were similar to other MIC surveys around the world. Generally, low MICs to tetracyclines, tiamulin and tylvalosin were observed. Increased tilmicosin MICs were observed in both M. synoviae and M. gallisepticum isolates (≥64 µg/ml MIC90 values) and this was seen in all isolates with high tylosin MICs. Increases in lincomycin MICs were mostly associated with increases in tilmicosin MICs. The results also suggested that antimicrobial use after mycoplasma vaccination may interfere with vaccine strain persistence and efficacy (field strains were more commonly observed in flocks that had treatments after vaccination) and this area warrants more investigation. The study shows that isolation and MIC determination can be done from remote locations and suggests that this may provide information that will allow more effective use of antimicrobials or other methods of control of avian mycoplasma in chickens (e.g. live vaccines) and therefore more responsible use of antimicrobials from a one health perspective.

Comparative genomic analyses of Mycoplasma synoviae vaccine strain MS-H and its wild-type parent strain 86079/7NS: implications for the identification of virulence factors and applications in diagnosis of M. synoviae.

Mycoplasma synoviae is an economically important avian pathogen worldwide, causing respiratory disease, infectious synovitis, airsacculitis and eggshell apex abnormalities in commercial chickens. Despite the widespread use of MS-H as a live attenuated vaccine over the past two decades, the precise molecular basis for loss of virulence in this vaccine is not yet fully understood. To address this, the whole genome sequence of the vaccine parent strain, 86079/7NS, was obtained and compared to that of the MS-H vaccine. Except for the vlhA expressed region, both genomes were nearly identical. Thirty-two single nucleotide polymorphisms (SNPs) were identified in MS-H, including 11 non-synonymous mutations that were predicted, by bioinformatics analysis, to have changed the secondary structure of the deduced proteins. One of these mutations caused truncation of the oppF-1 gene, which encodes the ATP-binding protein of an oligopeptide permease transporter. Overall, the attenuation of MS-H strain may be caused by the cumulative and complex effects of several mutations. The SNPs identified in MS-H were further analyzed by comparing the MS-H and 86079/7NS sequences with the strains WVU-1853 and MS53. In the genomic regions conserved between all strains, 30 SNPs were found to be unique to MS-H lineage. These results have provided a foundation for developing novel biomarkers for the detection of virulence in M. synoviae and also for designing new genotyping assays for discrimination of MS-H from field strains.

Development of Mycoplasma synoviae (MS) core genome multilocus sequence typing (cgMLST) scheme.

Mycoplasma synoviae (MS) is a poultry pathogen with reported increased prevalence and virulence in recent years. MS strain identification is essential for prevention, control efforts and epidemiological outbreak investigations. Multiple multilocus based sequence typing schemes have been developed for MS, yet the resolution of these schemes could be limited for outbreak investigation. The cost of whole genome sequencing became close to that of sequencing the seven MLST targets; however, there is no standardized method for typing MS strains based on whole genome sequences. In this paper, we propose a core genome multilocus sequence typing (cgMLST) scheme as a standardized and reproducible method for typing MS based whole genome sequences. A diverse set of 25 MS whole genome sequences were used to identify 302 core genome genes as cgMLST targets (35.5% of MS genome) and 44 whole genome sequences of MS isolates from six countries in four continents were used for typing applying this scheme. cgMLST based phylogenetic trees displayed a high degree of agreement with core genome SNP based analysis and available epidemiological information. cgMLST allowed evaluation of two conventional MLST schemes of MS. The high discriminatory power of cgMLST allowed differentiation between samples of the same conventional MLST type. cgMLST represents a standardized, accurate, highly discriminatory, and reproducible method for differentiation between MS isolates. Like conventional MLST, it provides stable and expandable nomenclature, allowing for comparing and sharing the typing results between different laboratories worldwide.

Phylogenetic and pathogenic analysis of Mycoplasma Synoviae isolated from native chicken breeds in China.

Mycoplasma synoviae (M. synoviae) infection leads to serious economic losses in the world every year. Between 2013 and 2014, the infectious synovitis, caused by M. synoviae infection, occurred in native chickens in China and resulted in the loss of millions of chickens in Chinese poultry farms. However, there has been no data about phylogenetic and pathogenic analysis of Chinese M. synoviae isolates. In this study, a total of 110 M. synoviae strains were isolated from M. synoviae infected chickens. The isolates identified in the present study were classified into a new distinct subgroup based on analysis of the 5'-end vlhA sequences, tentatively termed the K group. In addition, though the pathogenicity was significantly different among isolates, there was no close relationship between pathogenicity and genotype for Chinese M. synoviae based on a pathogenic analysis of the 5'-end of the vlhA gene.

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro.

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5+/-1.5%) and CEC-32 (RIF 7.0+/-0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R(low). Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2+/-0.3%) similar to that of R(low) (1.1+/-0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.

Mycoplasma synoviae infection on Newcastle disease vaccination of chickens / Infecção por Mycoplasma synoviae na vacinação da doença de Newcastle em galinhas

Newcastle disease is characterized by respiratory manifestations in association with nervous and/or digestive symptoms. Its prevention is done by vaccination with live attenuated (lentogenic strains) and/or killed vaccines. The lentogenic strains can lead to strong post-vaccination reaction, principally due to the presence of other pathogenic agents. Among them, Mycoplasma synoviae is worldwide important, mainly in Brazil. The dissemination of this agent in poultry flocks has been achieved due to difficulties in diagnosis and disease reproduction, virulence variations among different M.synoviae strains, and attribution of typical M.synoviae disease manifestation to other disease agents. This experimental study in SPF chicks (Gallus gallus), previously infected by M.synoviae and thereafter vaccinated against Newcastle disease, was done with the objective of evaluating M.synoviae pathogenicity through assessment of post-vaccinal respiratory reactions and serologic responses to Newcastle disease virus vaccine in the absence of environmental factors. A total of 86 three days old chicks were used, being 57 infected by eye and nostril drop, with chicken activated M. synoviae strain WVU 1853. Seven days later, 21 mycoplasma infected birds plus 29 not mycoplasma infected ones were vaccinated against Newcastle disease. As results, the not infected and vaccinated birds yielded, significantly, higher and longer lasting serologic responses to Newcastle disease vaccine virus than those infected and vaccinated. Similarly, the infected and vaccinated birds yielded lower serologic reactions to M.synoviae than those only mycoplasma infected. No post-vaccinal respiratory reaction was observed in the vaccinated birds.

A doença de Newcastle é caracterizada por manifestações respiratórias associadas a sintomas nervosos e/ou digestivos. Sua prevenção é feita pela vacinação com vacinas vivas atenuadas (cepas lentogênicas) e/ou inativadas. As cepas lentogênicas podem determinar acentuada reação pós-vacinal, principalmente na presença de outros patógenos. Entre eles, o Mycoplasma synoviae tem importância mundial, principalmente no Brasil. A disseminação deste agente nos planteís avícolas tem sido facilitada, devido a dificuldades de reprodução e diagnóstico da doença em aves, variação de virulência entre as diferentes cepas de M.synoviae e atribuição a outros patógenos de manifestação típica da micoplasmose por M.synoviae. Este estudo experimental em aves (Gallus gallus) SPF, previamente infectadas por M.synoviae e depois vacinadas contra Newcastle, foi realizado com objetivo de avaliar a patogenicidade do M.synoviae pela obtenção dareação respiratória pós-vacinal e a resposta sorológica para o vírus vacinal da doença de Newcastle, na ausência de fatores ambientais. Um total de 86 aves, com três dias de idade foram utilizadas, sendo 57 infectadas via ocular e intranasal, com cepa MS WVU 1853, ativada em galinhas. Sete dias depois, 21 aves infectadas por micoplasma e 29 não infectadas foram vacinadas contra a doença de Newcastle. Como resultados, aves não infectadas e vacinadas produziram resposta sorológica para o vírus vacinal da doença de Newcastle, significativamente mais elevada e mais duradora que aquelas infectadas e vacinadas. Igualmente, aves infectadas e vacinadas produziram reações sorológicas para M.synoviae mais baixa, que aquelas apenas infectadas. Não foram observadas reações respiratórias pós-vacinal nas aves vacinadas.