The development and application of a Mycoplasma gallisepticum sequence database

Figures & data

Table 1. Genetic regions selected for analysis, with expected PCR product sizes and discrimination (D) indices for targeted genetic sequencing.

Genetic region analysed	PCR product size (bp)	D index^Footnotea
IGSR	812	0.950
mgc2	824	0.915
MGA_0319	590	0.874
gapA	332	0.713

^a As described in Ferguson et al. (2005) and Raviv et al. (2007).

Table 2. M. gallisepticum reference and vaccine strains and DNA samples from other countries used to generate the IGSR/mgc2 dendrogram for comparison with South African wild-type genotypes.

Isolate name^Footnotea	GenBank numbers (IGSR; mgc2)	Country of origin	Strain type
COL/68938/CK08	KC247840; KC247873	Colombia	Field strain
COL/79663/CK09	KC247841; KC247874	Colombia	Field strain
ECU/77025/CK09	KC247842; KC247875	Ecuador	Field strain
ECU/77029/CK09	KC247843; KC247876	Ecuador	Field strain
GUA/79672/CK09	KC247844; KC247877	Guatemala	Field strain
PAN/68630/CK08	KC247845; KC247878	Panama	Field strain
PAN/69016/CK08	KC247846; KC247879	Panama	Field strain
VEN/69993/CK08	KC247847; KC247880	Venezuela	Field strain
VEN/69994/CK08	KC247848; KC247881	Venezuela	Field strain
USA/K6001B/CK07	KC247849; KC247882	USA (Georgia)	Field strain
USA/65099/PF08	KC247850; KC247883	USA (Georgia)	Field strain
USA/68614/CK08	KC247851; KC247884	USA (Alabama)	Field strain
USA/75695/CK09	KC247852; KC247885	USA (Georgia)	Field strain
USA/82065/CK10	KC247853; KC247886	USA (Georgia)	Field strain
USA/87387/CK11	KC247854; KC247887	USA (Florida)	Field strain
USA/92439/CK12	KC247855; KC247888	USA (Georgia)	Field strain
EGY/67240/CK08	HQ143380^Footnoteb; HQ143372^Footnoteb	Egypt	Field strain
EGY/75651/CK09	HQ143381^Footnoteb; HQ143373^Footnoteb	Egypt	Field strain
EGY/82133/CK10	KC247856; KC247889	Egypt	Field strain
ISR/K3868/CK95	HQ143384^Footnoteb; HQ143377^Footnoteb	Israel	Field strain
JOR/111/CK09	KC247857; KC247890	Jordan	Field strain
SPA/5742-3/CK09	KC247858; KC247891	Spain	Field strain
IND/38825/CK05	KC247859; KC247892	India	Field strain
THA/3/CK08	KC247860; KC247893	Thailand	Field strain
THA/5/CK08	KC247861; KC247894	Thailand	Field strain
USA/A5969/CK55	KC247862; KC247895	USA	Reference strain
USA/R/CK60	KC247863; KC247896	USA (Georgia)	Reference strain
USA/F/CK58	HQ143383^Footnoteb; KC247897	USA (California)	Reference F strain
ts-11	KC247864; KC247898	Australia	Vaccine strain
6/85	KC247865; KC247899	USA	Vaccine strain

^a CK = chicken; PF = peafowl; last two digits in field strain names indicates year of isolation. ^bSequences published by Gharaibeh et al. (2011).

Table 3. South African M. gallisepticum genotypes identified, with corresponding sequence types and genotype prevalence.

Genotype	IGSR (R)	mgc2 (C)	MGA_0319 (P)	gapA (G)	Number of isolates	% of wild-types
SA-WT#1	R4 (KC247830)^Footnotea	C0^Footnoteb (KC247866)	P5 (KC247834)	G0/1 (KC247838)	2	5.7
SA-WT#2	R4	C3 (KC247867)	P5	G3 (KC247839)	3	8.6
SA-WT#3	R3 (KC247831)	C4 (KC247868)	P4 (KC247835)	G0/1	1	2.9
SA-WT#4	R0 (KC247832)	C5^Footnotec	P0 (KC247836)	G0/1	2	5.7
SA-WT#5	–	C6 (KC247869)	P5	G3	1	2.9
SA-WT#6	R4	C7 (KC247870)	P5	G0/1	3	8.6
SA-WT#7	R4	C8 (KC247871)	P5	G0/1	15	42.9
SA-WT#8	R5 (KC247833)	C8	P5	G0/1	3	8.6
SA-WT#9	R0	C8	P3 (KC247837)	G0/1	1	2.9
SA-WT#10	R3	C8	P5	G0/1	1	2.9
SA-WT#11	–	C9 (KC247872)	P5	G0/1	2	5.7
SA-WT#12	R4	–	P4	G0/1	1	2.9
ts-11	R0	C0	P0	G0/1	1	N/A

^a GenBank accession number of the sequence of a representative sample. ^bSequences identical to those of the live vaccine strains were designated as follows: ts-11, “0”; 6/85, “1”; F strain, “2”. Sequences not identical to the vaccine sequences were assigned numbers greater than 2. ^cNote that this sequence differed from that of ts-11 by a SNP. Repeated amplification and sequencing to determine whether this sequence was in fact wild-type (sequence type C5) or ts-11-like (sequence type C0) yielded inconclusive results.

Table 4. Comparison of targeted genetic sequencing methods for discrimination of South African M. gallisepticum genotypes.

Genotyping target(s)	Number of sequence types identified (s)	Total number of genotypes (N)^Footnotea	D index^Footnoteb
IGSR	4	11	0.745
mgc2	8	12	0.894
MGA_0319	4	13	0.615
gapA	2	13	0.295
IGSR/mgc2	10	10	1.000
mgc2/MGA_0319	10	12	0.955
IGSR/MGA_0319	7	11	0.927
IGSR/mgc2/MGA_0319	10	10	1.000

^a N, total number of genotypes considered for each genetic target (excluding genotypes for which target sequences were not available). ^b; N=total number of genotypes described, s=number of sequence types identified, n=number of genotypes belonging to jth sequence type.

Figure 1. Dendrogram of South African M. gallisepticum wild-types and live vaccine strains, constructed by Clustal-W alignment of adjoined IGSR, mgc2, MGA_0319 and gapA sequences by the neighbour-joining method with 1000-bootstrap replicates using MEGA 5.05 (http://www.megasoftware.net).

Figure 2. Dendrogram of M. gallisepticum wild-types from South Africa and 12 other countries, with vaccine and reference strains, constructed by Clustal-W alignment of adjoined IGSR and mgc2 (fragment) sequences by the neighbour-joining method with 1000-bootstrap replicates using MEGA 5.05 (http://www.megasoftware.net). Origin of foreign M. gallisepticum DNA is listed in Table 2.

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