Identification and serotyping
Of the 117 G. parasuis isolates, 105 were assigned to ten distinct serovars, and twelve of the isolates tested were NT. Serovar 10 (17.9%) was the most prevalent, followed by serovars 15 (14.5%), 6 (12.0%), 8 (11.1%), 4 (8.5%), 9 (7.7%), 1 (7.7%), 7 (6.0%), 5/12 (4.3%), and 2 (0.9%)(Fig. 1A). Serovars 3, 11, 13, and 14 were not identified. Serovars 4, 6, 15, and NT were observed in all three provinces. However, serovar 2 was observed only in Shanghai and serovar 7 was observed only in Jiangxi(Fig. 1B).
VG analysis of the G. parasuis isolates
The VGs vacJ and clpP (95.7%) were the most prevalent, followed by cheY (93.2%), rfaE(92.3%), hsdR(91.5%), capD(88.9%), fhuA(40.2%), vta1(35.9%), hhdA(33.3%), hhdB(26.5%), HPM1372 (22.2%),nhaC(21.4%), lsgB(19.7%), H0254(10.3%), fimB(10.3%), wbgY(7.7%), HPM1373(6.8%), HPM1371(5.3%), HPM1370(1.7%)(Fig. 2). All G. parasuis isolates were clustered according to the presence of VGs. Four clusters were obtained (clusters A, B, C, and D) (Fig. 3). Cluster A includes serovars 1, 2, 4, 6, 7, 8, 9, 10, 15, and NT isolates, harboring 4 to 11 VGs; Cluster B includes serovars 4, 5,6, and NT isolates, harboring 9 to 17 VGs; Cluster C includes serovars 1, 7, and 10, harboring 5 to 8 VGs; and Cluster D includes only NT isolates, harboring 0 to 4 VGs. Interestingly, some serovars were distributed in 2 or 3 clusters. For example, serovars 4 and 6 were found in clusters A and B, serovars 1, 7, and 10 were found in clusters A and C, and NT isolates were found in clusters A, B, and D (Fig. 3).
Association between serovars and VGs
The distribution of VGs in the isolates allocated to different serovars varied greatly, and a significant correlation was found between serovars and some VGs. A significant positive correlation was found between the following: serovar 1 and vta1; serovar 4 and hhdB, H0254, nhaC, and vta1; serovar 5 and fhuA, wbgY, hhdA, hhdB, lsgB, H0254, nhaC, vta1, and HPM1373; serovar 6 and both HPM1371, and HPM1372; serovar 7 and both hhdA and vta1; serovar 8 and hhdA, hhdB, and HPM1371; serovar 10 and fimB, HPM1371, and HPM1372; serovar 15 and hsdR. However, a significant negative correlation was found between serovar 1 and capD, serovar 4 and HPM1371, serovar 6 and vta1, serovar 8 and vta1, serovar 9 and both fhuA and HPM1371, and the following: serovar 10 and fhuA, hhdA, hhdB, nhaC, and vta1, serovar 15 and hhdA, lsgB, nhaC, and HPM1371, and NT and rfaE, vacJ, cheY, clpP, and hsdR (p< 0.05, Table 2).
Table 2 Association between serovars and VGs of G. parasuis isolates
serovar
|
VGs
|
VG +
|
VG−
|
−VG +
|
−VG−
|
OR
|
95% CI
|
P
|
1
|
capD
|
5
|
4
|
99
|
9
|
0.11
|
0.03-0.48
|
0.009
|
|
vta1
|
8
|
1
|
34
|
74
|
17.41
|
2.09-144.78
|
0.001
|
5
|
fhuA
|
5
|
0
|
42
|
70
|
∞
|
/
|
0.009
|
|
wbgY
|
5
|
0
|
4
|
108
|
∞
|
/
|
0.000
|
|
hhdA
|
5
|
0
|
34
|
78
|
∞
|
/
|
0.003
|
|
hhdB
|
5
|
0
|
26
|
86
|
∞
|
/
|
0.001
|
|
lsgB
|
5
|
0
|
18
|
94
|
∞
|
/
|
0.000201
|
|
H0254
|
5
|
0
|
7
|
105
|
∞
|
/
|
0.000005
|
|
nhaC
|
5
|
0
|
20
|
92
|
∞
|
/
|
0.000317
|
|
vta1
|
5
|
0
|
37
|
75
|
∞
|
/
|
0.005
|
|
HPM 1373
|
5
|
0
|
3
|
109
|
∞
|
/
|
0.000
|
10
|
fhuA
|
4
|
17
|
43
|
53
|
0.29
|
0.09-0.93
|
0.047
|
|
hhdA
|
1
|
20
|
38
|
58
|
0.08
|
0.01-0.62
|
0.002
|
|
hhdB
|
0
|
21
|
31
|
65
|
0
|
/
|
0.001
|
|
nhaC
|
0
|
21
|
25
|
71
|
0
|
/
|
0.006
|
|
fimB
|
8
|
13
|
4
|
92
|
14.15
|
3.73-53.68
|
0.000094
|
|
vta1
|
0
|
21
|
42
|
54
|
0
|
/
|
0.000031
|
|
HPM1371
|
20
|
1
|
42
|
54
|
25.71
|
3.31-199.41
|
0.000007
|
|
HPM 1372
|
9
|
12
|
17
|
79
|
3.49
|
1.27-9.59
|
0.019
|
4
|
hhdB
|
7
|
3
|
24
|
83
|
8.07
|
1.94-33.61
|
0.003
|
|
H0254
|
5
|
5
|
7
|
100
|
14.29
|
3.33-61.37
|
0.001
|
|
nhaC
|
8
|
2
|
17
|
90
|
21.18
|
4.13-108.52
|
0.000053
|
|
vta1
|
9
|
1
|
33
|
74
|
20.18
|
2.46-165.85
|
0.000391
|
|
HPM 1371
|
2
|
8
|
60
|
47
|
0.2
|
0.04-0.99
|
0.044
|
15
|
hhdA
|
1
|
16
|
38
|
62
|
0.1
|
0.01-0.78
|
0.011
|
|
lsgB
|
0
|
17
|
23
|
77
|
0
|
/
|
0.023
|
|
hsdR
|
12
|
5
|
95
|
5
|
0.13
|
0.03-0.52
|
0.006
|
|
nhaC
|
0
|
17
|
25
|
75
|
0
|
/
|
0.022
|
|
HPM 1371
|
2
|
15
|
60
|
40
|
0.09
|
0.02-0.42
|
0.000336
|
8
|
hhdA
|
9
|
4
|
30
|
74
|
5.55
|
1.59-19.41
|
0.009
|
|
hhdB
|
8
|
5
|
23
|
81
|
5.63
|
1.68-18.87
|
0.005
|
|
vta1
|
0
|
13
|
42
|
62
|
0
|
/
|
0.004
|
|
HPM 1371
|
13
|
0
|
49
|
55
|
∞
|
/
|
0.000158
|
6
|
vta1
|
1
|
13
|
41
|
62
|
0.12
|
0.02-0.95
|
0.017
|
|
HPM 1371
|
13
|
1
|
49
|
54
|
14.33
|
1.81-113.61
|
0.001
|
|
HPM 1372
|
7
|
7
|
19
|
84
|
4.42
|
1.39-14.10
|
0.014
|
7
|
hhdA
|
5
|
2
|
34
|
76
|
5.59
|
1.03-30.26
|
0.040
|
|
vta1
|
7
|
0
|
35
|
75
|
∞
|
/
|
0.001
|
9
|
fhuA
|
0
|
9
|
47
|
61
|
0
|
/
|
0.011
|
|
HPM 1371
|
1
|
8
|
61
|
47
|
0.1
|
0.01-0.83
|
0.012
|
NT
|
rfaE
|
6
|
5
|
102
|
4
|
0.05
|
0.01-0.24
|
0.000289
|
|
vacJ
|
6
|
5
|
106
|
0
|
0
|
/
|
0.000003
|
|
cheY
|
7
|
4
|
102
|
4
|
0.07
|
0.01-0.34
|
0.003
|
|
clpP
|
7
|
4
|
105
|
1
|
0.02
|
0-0.20
|
0.000212
|
|
hsdR
|
7
|
4
|
100
|
6
|
0.11
|
0/03-0.48
|
0.007
|
VG +: Number of isolates in the corresponding serovar but carrying the VG
VG−: Number of isolates in the corresponding serovar but no carrying the VG
−VG +: Number of isolates no in the corresponding serovar but carrying VG
−VG −: Number of isolates no in the corresponding serovar but no carrying VG.
Oliveira and Pijoan (2004) reported that G. parasuis was divided into three groups based on different serovars: highly pathogenic serovars (1, 5, 10, 12, 13, and 14), moderately pathogenic serovars (2,4, and 15), and non-pathogenic serovars (3, 6, 7, 8, 9, and 11). The current study identified a significant correlation between different pathogenic serovar groups and several VGs. The highly pathogenic serovars had a significant positive association with wbgY, fimB, 1371, and 1373, and a significant negative association with hhdB. The moderately pathogenic serovars had a significant positive association with hsdR and vta1, and a significant negative association with HPM1371. The non-pathogenic serovars had a significant negative association with H0254, fimB, and vta1 (p< 0.05, Table 3).
Table 3 Association between pathogenic serovar group and VGs of G. parasuis isolates
pathogenic serovar group
|
VGs
|
VG +
|
VG−
|
−VG +
|
−VG−
|
OR
|
95% CI
|
P
|
highly pathogenic
group
|
wbgY
|
7
|
28
|
1
|
70
|
17.5
|
2.06-148.84
|
0.002
|
hhdB
|
5
|
30
|
25
|
46
|
0.31
|
0.11-0.90
|
0.038
|
fimB
|
9
|
26
|
3
|
68
|
7.85
|
1.97-31.28
|
0.002
|
HPM 1371
|
27
|
8
|
32
|
39
|
4.11
|
1.64-10.28
|
0.002
|
HPM 1373
|
6
|
29
|
1
|
70
|
14.48
|
1.67-125.66
|
0.005
|
Moderately pathogenic
group
|
hsdR
|
22
|
6
|
78
|
0
|
0
|
/
|
0.0002
|
vta1
|
15
|
13
|
22
|
56
|
2.94
|
1.21-7.17
|
0.021
|
HPM 1371
|
4
|
24
|
55
|
23
|
0.07
|
0.02-0.22
|
0.000
|
non-pathogenic
group
|
H0254
|
1
|
42
|
10
|
53
|
0.13
|
0.02-1.06
|
0.026
|
fimB
|
0
|
43
|
12
|
51
|
0
|
/
|
0.001
|
vta1
|
9
|
34
|
28
|
35
|
0.33
|
0.14-0.80
|
0.014
|
VG +: Number of isolates in the corresponding serovar but carrying the VG
VG−: Number of isolates in the corresponding serovar but no carrying the VG
−VG +: Number of isolates no in the corresponding serovar but carrying VG
−VG −: Number of isolates no in the corresponding serovar but no carrying VG.
Association between MLST and VGs
The MLST analysis revealed two major clades (clade 1 and clade 2) based on the MLST target sequences of 43 G. parasuis isolates. Clade 1 includes 37 isolates of serovars 1, 2, 4, 6, 7, 8, 9, 10, 15, and NT, harboring 1 to 11 VGs each. Clade 2 includes 6 isolates of serovars 1, 4 and 5, harboring 8 to 16 VGs each(Fig. 4). Interestingly, isolates in the second clade had a significantly increased probability of containing the VGs vta1, nhaC, hhdA, hhdB, lsgB, H0254, wbgY, fimB, and 1373 (p< 0.05, Table 4).
Table 4 Association between MLST clade and VGs of G. parasuis isolates
VG
|
Clade1+
|
Clade1−
|
Clade2+
|
Clade2−
|
OR
|
95% CI
|
P
|
vta1
|
13
|
24
|
6
|
0
|
0
|
/
|
0.004
|
nhaC
|
8
|
29
|
5
|
1
|
0.06
|
0.01-0.59
|
0.007
|
hhdA
|
8
|
29
|
5
|
1
|
0.06
|
0.01-0.59
|
0.007
|
hhdB
|
7
|
30
|
5
|
1
|
0.05
|
0.01-0.50
|
0.004
|
lsgB
|
5
|
32
|
4
|
2
|
0.08
|
0.01-0.56
|
0.01
|
H0254
|
0
|
37
|
5
|
1
|
0
|
/
|
0.000006
|
wbgY
|
1
|
36
|
4
|
2
|
0.01
|
0-0.14
|
0.001
|
fimB
|
1
|
36
|
3
|
3
|
0.03
|
0-0.38
|
0.006
|
HPM 1373
|
1
|
36
|
3
|
3
|
0.03
|
0-0.38
|
0.006
|
+: Number of isolates in the corresponding clade but carrying the VG
−: Number of isolates in the corresponding clade but no carrying the VG