Validation of a previously developed quantitative polymerase chain reaction for the detection and quantification of Mycoplasma synoviae in chicken joint specimens

Figures & data

Table 1.  Primers and probes used for detection of M. synoviae and Synechococcus spp.

Species	Target gene	Oligo	Sequence
M. synoviae	16S-23S rRNA ISR	Forward	CTA AAT ACA ATA GCC CAA GGC AA
	(AY768810)	Reverse	CCT CCT TTC TTA CGG AGT ACA
		Probe	6FAM - AGC GAT ACA CAA CCG CTT TTA GAA T - BHQ1
Synechococcus spp.	16S-23S rRNA ISR	Forward	CAT TCT TGA CAA GTT AAC CAG TTA GCT G
	(AF317074)	Reverse	CAA GGT TCT GCT GAC ATT CAA ACA
		Probe	Cy5 - TCT CCA GGG CAG CAT TGA ATC CAG - BHQ2

Table 2.  Specificity of the quantitative PCR.

		Mycoplasma synoviae qPCR
Species	Strains and isolates	Number tested	Number positive	Number negative
Staphylococcus aureus	ATCC25923	1	—	1
Escherichia coli	ATCC25922	1	—	1
Enterococcus faecalis	ATCC29212	1	—	1
Mycoplasma gallisepticum	ATCC 15302, M1999.03a, M1999.03b, M1999.06, M2001.05, M2001.07, M2001.28, M2001.30, M2001.31, M2001.32, M2001.66, M2002.03, M2002.04, M2002.07, M2002.08b, M2002.14a, M2004.02, M2004.04c, M2004.07, M2004.07, M2004.07	21	—	21
Mycoplasma imitans	M1999.10a, M1999.10b	2	—	2
Mycoplasma gallinaceum	M1999.11	1	—	1
Mycoplasma meleagridis	KE^a, YH^a, ZR^a, RDJ^a, KVR^a, YST^a, BYD^a, ZYS^a, AJ^a, BSN^a, M1999.07, M1999.08, M1999.09, B46.04^b, B88.07^b, B2.08^b, B92.09^b	17	—	17
Mycoplasma synoviae	ATCC25204, M1995.02, M2000.05, M2001.27, M2001.29, M2002.02a, M2002.06, M2002.17, M2003.08, B45.04^b, M2004.08, M2005.09a, M2005.09b, M2005.09c, M2005.09d, M2005.26, M2005.04, M2006.07, B89.07^b, B119.07^b, M2007.20, M2007.23, M2007.07, M2007.17, M2007.21, B114.08^b, B86.08^b, M2008.01, M2008.02, M2008.03, M2008.17a, M2008.17b, M2008.17c, M2008.13, B60.09^b, B120.09^b, B130.09^b, M2009.03, M2009.05, B13.10^b, B14.10^b, B65.10^b, B152.10^b, B169.10^b, M2010.10, M2010.09a, M2010.09b, M2010.09c	48	48	—
^aCourtesy of Dr I. Lysnyansky, Mycoplasma Unit, Kimron Veterinary Institute, Bet-Dagan, Israel. ^bCourtesy of Prof J. Bradbury and Dr C. Dare, Department of Veterinary Pathology, Faculty of Veterinary Science, The University of Liverpool, UK.

Figure 1.  Percentage of birds with M. synoviae-induced macroscopic signs suggestive of arthritis with time.

Figure 2.  Ventral view of the distribution of macroscopic joint lesions found post-mortem in birds inoculated intravenously with M. synoviae. Each symbol represents an affected joint.

Table 3.  Quantitative PCR and mycoplasma culture results of M. synoviae infected birds (n=70) and FCA-injected controls (n=10) with time after the onset of clinical signs suggestive of arthritis.

		Birds with macroscopic joint lesions	qPCR				Culture
			Synovial fluid		Joint swab		Synovial fluid	Joint swab
Time after onset of clinical signs suggestive for arthritis (weeks)^a	n	Cumulative	Number positive/tested	CFU eq./ml (range)	Number positive/tested	CFU eq./ml (range)	Number positive/tested	Number positive/tested
Infected birds
<1	3	3	1/2	10^3.3	3/3	10^3.5 (10^2.7 ^to ^3.8)	0/2	0/1
1	17	20	12/12	10^3.1 (10^0.3 ^to ^4.2)	10/14	10^3.8 (10^1.9 ^to ^4.5)	6/12	0/5
2	4	24	3/4	10^3.2 (10^2.9 ^to ^3.8)	3/3	10^3.0 (10^2.8 ^to ^3.2)	2/4	0/0
3	13	37	7/7	10^3.0 (10^2.2 ^to ^3.6)	13/13	10^3.5 (10^1.6 ^to ^4.4)	1/7	1/6
4	5	42	1/1	10^3.2	3/5	10^2.5 (10^2.4 ^to ^2.6)	1/1	0/4
5	3	45	1/1	10^4.6	1/3	10^2.0	0/1	0/2
6	6	51	1/1	10^4.0	5/6	10^3.3 (10^2.1 ^to ^3.8)	0/1	0/5
7	3	54	0/0		2/3	10^3.9 (10^3.4 ^to ^4.3)	0/0	0/3
No arthritis^b	16	16	0/0		8/16	10^2.5 (10^2.0 ^to ^2.8)	0/0	0/16
FCA-injected controls^c
3	7		0/7		0/7		0/7	0/0
No arthritis	3		0/0		0/3		0/0	0/3
^aLameness and/or swollen hock and/or foot joints in individual birds. ^bAt the end of the experiment (12 weeks post inoculation of M. synoviae). ^cPost-mortem examination of all control birds was performed 3 weeks post injection of FCA.