Detection and quantification of Mycoplasma gallisepticum genome load in conjunctival samples of experimentally infected house finches (Carpodacus mexicanus) using real-time polymerase chain reaction

Figures & data

Table 1.  Real-time primer and probe sequences utilized in this study

Gene	Primer/probe	Sequence	Reference
mgc2	Forward	5′-GGTCCTAATCCCCAACAAAGAAT	Designed using Primer Express
	Reverse	5′-CTTGGTTGGTTCATATTAGGCATTT
	Probe	5′-6-FAM-CCACAGGGCTTTGGTGGCCCA-BHQ-1
18S rRNA	Forward	5′-AGCCTGCGGCTTAATTTGAC	Lopez-Andreo et al. (2005)
	Reverse	5′-CAACTAAGAACGGCCATGCA
	Probe	5′-6-FAM-AGGATTGACAGATTGAG-BHQ-1
nos2	Forward	5′-GAGTGGTTTAAGGAGTTGGATCTGA	Jarosinski et al. (2002)
	Reverse	5′-TTCCAGACCTCCCACCTCAA
	Probe	5′-TET–CTCTGCCTGCTGTTGCCAACATGC-TAMRA
rag1	Forward	5′-TCAGGACTGCCACTCTCAATTG	Designed using Primer Express
	Reverse	5′-CAAACCAAGGCTGCATCGTA
	Probe	5′-6-FAM-ACCCAGTAGACACAATTGCAAAGAGATTCCG-BHQ-1

Figure 1.  Linearity of standard curve. 1a: Standard curve based on mgc2 plasmid. 1b: Standard curve based on M. gallisepticum genomic DNA.

Table 2.  Reproducibility and limit of detection of the standard curves used for the mgc2 assay

Mgc2 plasmid standard						Genomic standard
Inter-assay reproducibility			Intra-assay reproducibility			Inter-assay reproducibility
Copies	Mean Ct	Standard deviation	Copies	Mean Ct	Standard deviation	Copies	Mean Ct	Standard deviation
1.40 x 10^8	12.52	0.452	1.40 x 10^8	12.92	0.03	1.00 x 10^6	18.39	1.025
1.40 x 10^7	15.70	0.352	1.40 x 10^7	15.99	0.05	1.00 x 10^5	21.87	1.049
1.40 x 10^6	19.18	0.354	1.40 x 10^6	19.59	0.05	1.00 x 10^4	25.58	1.097
1.40 x 10^5	22.85	0.344	1.40 x 10^5	23.01	0.15	1.00 x 10^3	29.18	1.143
1.40 x 10^4	26.36	0.290	1.40 x 10^4	26.5	0.16	1.00 x 10^2	33.05	1.393
1.40 x 10^3	29.72	0.431	1.40 x 10^3	30.04	0.21	1.00 x 10^1	36.61	0.795
1.40 x 10^2	32.86	0.313	1.40 x 10^2	33.12	0.2	1.00 x 10^0	40.00	–
1.40 x 10^1	34.91	0.653	1.40 x 10^1	35.5	1.17
1.40 x 10^0	40.00	–

Figure 2.  2a: Mean M. gallisepticum (MG) load present in total swab samples of the conjunctiva of four inoculated house finches that developed clinical conjunctivitis, as a function of time p.i., with standard error bars. 2b: Mean M. gallisepticum (MG) load shown as the log₁₀ value of mgc2 copy number per host cell, with standard error bars.

Figure 3.  Data from individual birds showing M. gallisepticum (MG) load over time and occurrence of conjunctivitis. Solid circles, an eye score of 1 on the day of conjunctival sampling; dashed circle, a more severe eye score of 2. R, right eye; L, left eye. Eye score code: 0 = no visible inflammation; 1 = pink conjunctival discolouration and slight periorbital oedema, 2 = pink conjunctival discolouration, slight to moderate periorbital oedema, and epiphora with mucoid discharge.

Table 3.  Summary of maximal mycoplasmal load and eye score at day 7 p.i.

Bird	Day 7 eye score	Maximum M. gallisepticum load (M. gallisepticum/host cell)	Day of maximum M. gallisepticum load
1	0	16	7
2	1	2079	5
3	1	400	7
4	1	1328	7
5	2	3359	5