Mycoplasma Synoviae Fact Sheet - Veterinary Diagnostic Laboratory

The Minnesota Veterinary Diagnostic Laboratory has standardized and validated the use of a recentlypublished real-time TaqMan® PCR assay 3 for detection of Mycoplasma synoviae in clinical samples.This test is currently available for routine testing of respiratory and joint samples of avian origin.Test validationSpecificity: The analytical specificity of the real-time TaqMan® PCR assay was evaluated by testingDNA extracted from 10 field strains of M. synoviae and 17 additional avian Mycoplasma species, mostof which are nonpathogenic. Tracheal swabs from specific mycoplasma-free chickens and turkeys alsotested negative. 3Sensitivity: The real-time TaqMan® PCR detected 2 x 10 3 copies of M. synoviae per reaction. Testingwas performed by doing serial 10-fold dilutions of a pure culture (ATTC strain 25204) in PBS and inSP4 media4 (specific Mycoplasma culture broth). SP4 media is present in swabs frequently used byfield veterinarians to collect and submit samples for testing. Table 1 shows the effect of pooling, media,and storage time under refrigeration in the analytical sensitivity of the new M. synoviae real-timeTaqMan® test. Three concentrations of M. synoviae were tested (10 3 , 10 5 , 10 7 ), along with threedifferent diluents (phosphate-buffered saline [PBS] and SP4 broth) and two time points (2, 6, and 7days). All samples were maintained at 4°C until testing.Results from this experiment demonstrated that there was no significant effect of the diluents, storagetime, or pooling of up to 5 swabs in the sensitivity of the test. As expected, samples containing a higherinitial concentration (2 x 10 7 copies/ reaction) were detected earlier in the real-time reaction (lower CTvalue) compared with samples with a lower initial concentration (2 x 10 5 copies/ reaction). Samplescontaining the lowest initial concentration (2 x 10 3 copies/ reaction) were all negative in this experiment.ConclusionsThe new M. synoviae real-time TaqMan® PCR test has a detection limit of 2 x 10 3 copies/ reactionunder laboratory conditions. Pooling of up to 5 swabs in PBS or SP4 broth, or storage time of up to 7days under refrigeration does not affect the sensitivity of the test when samples contain an initialconcentration between 10 5 to 10 7 copies/ reaction.RecommendationsSamples: Appropriate swabs to use are synthetic (Dacron or Rayon) on plastic shafts (bacterialculturettes). The type of media can vary (most commonly used are liquid Stuarts and Hank's media),but be careful to avoid any agar gel-type media. One inexpensive, multi-purpose swab is BBLCultureSwab Liquid Stuart, Single Swab (pkg of 50), Product Number 220099. 5 should be applied totrachea, sinus/nasal cavity and swollen joints or tendon sheaths (hock tendon or foot pads). Theculturettes should be labeled according to anatomic site to facilitate pooling.Sample submission: Ideally, samples should be submitted immediately after collection, usingovernight delivery. Samples can be maintained under refrigeration until shipping, but should be testedwithin 7 days of collection.Pooling: Pooling of up to 5 swabs is acceptable and does not affect the sensitivity of the test. Swabscollected from the same anatomic site can be pooled at the VDL prior to testing. Swabs that arepooled in the field should be collected into 2ml of PBS or SP4 broth.SYS.WEBDOC.020 Rev 1 6/29/2012