Development, validation and field evaluation of a quantitative real-time PCR able to differentiate between field Mycoplasma synoviae and the MS-H-live vaccine strain

Abstract

A quantitative PCR (qPCR) able to differentiate between field Mycoplasma synoviae and MS-H vaccine strain was developed, validated and evaluated. It was developed using nucleotide differences in the obg gene. Analytical specificity and sensitivity assessed using DNA from 194 M. synoviae field samples, three different batches of MS-H vaccine and from 43 samples representing four other avian Mycoplasma species proved to be 100%. The detection limit for field M. synoviae and MS-H vaccine strain was 10 and 10 colony-forming units PCR equivalents/g trachea mucus, respectively. The qPCR was able to detect both, field M. synoviae and MS-H vaccine strain in ratios of 1:100 determined both using spiked and field samples. One hundred and twenty samples from M. synoviae-infected non-vaccinated birds, 110 samples from M. synoviae-vaccinated birds from a bird experiment and 224 samples from M. synoviae negative (serology and PCR) birds were used to determine the relative sensitivity and specificity using a previously described M. synoviae PCR as reference. The relative sensitivity and specificity for field M. synoviae were 95.0% and 99.6%, respectively, and 94.6% and 100% for the MS-H-live vaccine, respectively. Field validation and confirmation by multi locus sequence typing revealed that the qPCR correctly distinguished between MS-H and field M. synoviae. Evaluation of the differentiating M. synoviae qPCR in three commercial flocks suggested transmission of MS-H-live vaccine from vaccinated to non-vaccinated flocks at the same farm. Furthermore, it showed evidence for the colonization with field M. synoviae in MS-H-vaccinated flocks.

Detection of Low Pathogenic Avian Influenza Virus Subtype H10N7 in Poultry and Environmental Water Samples During a Clinical Outbreak in Commercial Free-Range Layers, Netherlands 2017

“…In addition to the necropsy procedures, PCRs were executed to exclude infections with other pathogens or co-infections. Therefore, samples of the trachea were tested for the presence of Infectious bronchitis virus (34), Mycoplasma gallisepticum (35) and Mycoplasma synoviae (36). Standard PCR methods used by GD Animal Health excluded tracheal infections with Infectious laryngotracheitis virus and Avian metapneumovirus, and oviduct infections with Group I Aviadenovirus and Atadenovirus (Egg drop syndrome virus).…”

Diagnosing Infectious Diseases in Poultry Requires a Holistic Approach: A Review

“…Specific application of PCR is a DIVA (differentiating infected from vaccinated animals)-PCR, a method used for screening a vaccinated flock for infections with a filed/virulent strain, as for example in Marek's disease virus (MDV), Mycoplasma gallisepticum, Mycoplasma synoviae or turkey meningoencephalitis virus [223][224][225][226]. In order to accomplish functionality, DIVA tests should be able to distinguish between a vaccine and a field strain even in situations when multiple strains are present within the sample.…”

Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick

“…The rapid and accurate detection of MS is essential for the prevention and control of the disease. At present, common methods for MS detection include bacteriological culture, serological tests such as ELISA, and molecular approaches, including conventional PCR and real-time fluorescence quantitative PCR ( RFQ-PCR ) ( Moreira et al, 2015 ; Dijkman et al, 2017 ; Kuo et al, 2017 ). Although reliable and accurate, these methods are complex, time-consuming, and rely on specialized equipment and professional technicians, indicating that they are unsuitable for under-equipped laboratories or fields.…”