Combination of differential growth at two different temperatures with a quantitative real-time polymerase chain reaction to determine temperature-sensitive phenotype of Mycoplasma synoviae

Figures & data

Table 1.  Origin of the M. synoviae strains/isolates used and their temperature-sensitivity (ts) phenotypes as determined by microtitration and vlhA Q-PCR.

			Viable count (CCU/ml)			Number of vlhA gene copies/reaction
Strain	Origin	Reference	33°C	39.5°C	ts phenotype	33°C	39.5°C	33/39.5°C vlhA gene copy ratio
MS-H	Vaccine strain derived from 86079/7NS, Australia	(Morrow et al., 1998)	3.11×10^10	1.02×10^2	ts ^+	3.58×10^7	6.01×10^5	59
86079/7NS	Parent strain of MS-H vaccine, palatine cleft, Australia	(Morrow et al., 1998)	1.11×10^7	1.70×10^6	ts ^−	1.35×10^7	1.98×10^7	0.68
MS-H^4	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.33×10^7	1.11×10^7	ts ^−	6.21×10^6	1.14×10^7	0.54
MS-H^5	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	4.92×10^7	5.46×10^9	ts ^−	4.55×10^6	1.90×10^7	0.24
WVU-1853	USA, type strain, joints	(Olson, 1956)	5.46×10^8	3.11×10^10	ts ^−	5.32×10^6	5.47×10^7	0.09
93205/1-2a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	2.42×10^7	1.02×10^2	ts ^+	8.49×10^6	7.28×10^5	11
93198/6-5b	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	7.05×10^5	0	ts ^+	1.05×10^7	9.87×10^4	106
94036/8-3a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.10×10^10	2.26×10^9	ts ^−	4.9×10^1	1.51×10^2	0.32
93198/6-1a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.00×10^8	2.73×10^7	ts ^−	4.25×10^7	5.86×10^9	0.01
94036/10-5a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	5.46×10^6	3.43×10^6	ts ^−	3.28×10^8	4.10×10^9	0.08
93198/5-10a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.54×10^6	1.02×10^2	ts ^+	4.43×10^10	1.52×10^8	290
93198/1-24b	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	7.23×10^8	2.26×10^8	ts ^−	2.45×10^5	1.70×10^7	0.01
93205/2-9a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	4.36×10^8	3.11×10^7	ts ^−	4.73×10^6	8.80×10^6	0.53
94036/2-1a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.97×10^3	0	ts ^+	2.35×10^7	1.03×10^5	227
93198/4-19a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.39×10^8	1.27×10^2	ts ^+	1.62×10^6	9.17×10^4	17
94036/2-2a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	2.26×10^6	1.02×10^2	ts ^+	1.26×10^7	2.63×10^3	4811
93198/3-13b	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.13×10^7	0.82×10^2	ts ^+	8.13×10^6	5.62×10^4	144
93198/3-15a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	9.23×10^6	0.68×10^2	ts ^+	5.37×10^6	1.84×10^5	29
93205/2-13a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	1.00×10^6	4.57×10^4	ts ^−	5.20×10^6	2.03×10^6	2.55
93205/9-3a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	8.48×10^4	2.48×10^1	ts ^+	2.64×10^6	1.49×10^5	17
93205/8-9c	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	2.03×10^7	1.50×10^8	ts ^−	1.16×10^6	7.19×10^6	0.16
93205/10-13a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	3.43×10^7	2.58×10^7	ts ^−	1.45×10^6	5.72×10^6	0.25
94036/9-2a	MS-H-vaccinated flock, Australia	(Markham et al., 1998b)	3.84×10^4	7.01×10^3	ts ^−	3.57×10^6	2.88×10^6	1.23

Figure 1.  Standard curve generated using pUC18 plasmid harbouring the vlhA3 gene. The mean and standard deviation of the cycle threshold (Ct) values for serially diluted plasmid are shown. The average coefficient of determination (R ²) was 0.983 and average efficiency was 85%.

Figure 2.  2a: vlhA gene copy numbers of 33°C and 39.5°C cultures of MS-H and 86079/7NS determined by vlhA Q-PCR at 24-h intervals. 2b: 33/39.5°C vlhA gene copy number ratios calculated at 24-h intervals. 2c: vlhA gene copy numbers plotted against pH at each time point. 2d: 33/39.5°C vlhA gene copy ratios plotted against pH at each time point. 2e: The vlhA gene copy number of MS-H cultures at 33°C and 39.5°C harvested at different pH irrespective of the time. 2f: 33/39.5°C vlhA gene copy number ratio of MS-H cultures at 33°C and 39.5°C harvested at different pH.

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