MLST subtypes and sequence polymorphism
Forty-one of the 46 isolates were successfully amplified at all eight loci. Among them, dz-hrgp, rpgr, and mucin1 had relatively higher sequence polymorphism, with 5, 4, and 4 subtypes being identified, respectively. In contrast, the 44 isolates generated the same sequence at the normally polymorphic chom3t locus (Supplementary Table 1). Altogether, 17 MLGs were obtained from these isolates of C. parvum. Among them, the IIdA19G1 isolates from Guangdong, Jiangsu and Shanghai consisted of 12 MLGs. In addition, the IIdA20G1 isolates from Hebei and Heilongjiang had two geographically segregated MLGs. The IIdA15G1 isolates from Xinjiang had 3 different MLGs (Supplementary Table 1).
Sequence data of all eight loci were concatenated to make a multilocus contig of 4,740 bp in length. There was a high genetic diversity (Hd = 0.89) within C. parvum IId population in China (Table 2). Among the IIdA19G1 isolates, the genetic diversity of isolates from Shanghai (Hd = 0.94) was greater than isolates from Guangdong (Hd = 0.78) or Jiangsu (Hd = 0.67) (Table 2). This could be attributed to the difference in the number of farms examined in different regions. In contrast, IIdA20G1 isolates had relatively low genetic diversity (Hd = 0.48). Among them, isolates from Hebei and Heilongjiang showed high genetic homogeneity (Hd = 0.00) within each population. In contrast, The IIdA15G1 isolates from Xinjiang was highly heterogeneous (Hd = 1.00) (Table 2).
Table 2
Genetic diversity within Cryptosporidium parvum populations based on analysis of the concatenated sequences from eight genetic loci.
Population | N | H | Hd | LD (|D’|) | Rms |
Location | | | | | |
| Guangdong | 10 | 6 | 0.78 | Y = 1.0000 + 0.0000X | 0 |
| Shanghai | 9 | 7 | 0.94 | Y = 0.9295 + 0.0350X | 1 |
| Jiangsu | 4 | 2 | 0.67 | / | 0 |
| Hebei | 10 | 1 | 0.00 | / | / |
| Heilongjiang | 5 | 1 | 0.00 | / | / |
| Xinjiang | 3 | 3 | 1.00 | Y = 1.0000 + 0.0000X | 0 |
gp60 subtypes | | | | | |
| IIdA19G1 | 23 | 12 | 0.85 | Y = 0.8811 + 0.0177X | 1 |
| IIdA20G1 | 15 | 2 | 0.48 | / | 0 |
| IIdA15G1 | 3 | 3 | 1.00 | Y = 1.0000 + 0.0000X | 0 |
Total | 41 | 17 | 0.89 | Y = 0.9994–0.0041X | 1 |
Population structure of IId subtypes of C. parvum
In the analysis of the genetic structure of IId subtypes with VD and L measurements, an epidemic genetic structure was obtained in the overall population (ISA = − 0.0421, PMC = 0.889, and VD: 1.1307 < L: 2.3307) (Table 3). In further analyses, most of the subpopulations by region or gp60 subtype also had the epidemic genetic structure, except for the subpopulations of Heilongjiang, Hebei, and Xinjiang which could not be determined due to the small sample size (Table 3).
Table 3
Results of linkage disequilibrium analysis of allelic profile data from Cryptosporidium parvum at eight genetic loci.
Populations | N | ISA | PMC | VD | L | VD > L | LD or LE |
Jiangsu | 4 | 0.0286 | 1 | 0.2667 | 0.2667 | N | LE |
Guangdong | 10 | − 0.0137 | 0.537 | 0.7091 | 1.3455 | N | LE |
Shanghai | 10 | 0.0816 | 0.067 | 1.9727 | 2.0182 | N | LE |
Hebei | 12 | − 0.0110 | 1 | 0.2564 | 0.5641 | N | LE |
Heilongjiang | 5 | / | 1 | / | / | / | / |
Xinjiang | 5 | − 0.0128 | 0.472 | 1.2111 | 3.2111 | N | LE |
IIdA19G1 | 24 | 0.0272 | 0.175 | 1.2794 | 1.483 | N | LE |
IIdA20G1 | 17 | 0.0867 | < 0.001 | 0.9638 | 0.8008 | Y | LD |
IIdA15G1 | 5 | − 0.0128 | 0.472 | 1.2111 | 3.2111 | N | LE |
Total | 46 | 0.0672 | < 0.001 | 2.202 | 1.8848 | Y | LD |
Jiangsu* | 2 | / | < 0.001 | / | / | / | / |
Guangdong* | 6 | − 0.051 | 0.898 | 0.6 | 1.7429 | N | LE |
Shanghai* | 7 | − 0.0158 | 0.589 | 0.9476 | 2.2476 | N | LE |
Hebei* | 2 | / | / | / | / | / | / |
Heilongjiang* | 1 | / | / | / | / | / | / |
Xinjiang* | 3 | 0.2321 | 0.029 | 2.3333 | 2.3333 | N | LE |
IIdA19G1* | 12 | − 0.0695 | 0.986 | 0.5979 | 1.8287 | N | LE |
IIdA20G1* | 2 | / | < 0.001 | / | / | / | / |
IIdA15G1* | 3 | 0.2321 | 0.029 | 2.3333 | 2.3333 | N | LE |
Total* | 17 | − 0.0421 | 0.889 | 1.1307 | 2.3307 | N | LE |
Abbreviations: *, Considering isolates with the same MLG as one individual. N: number of isolates; ISA, standardized index of association calculated using the program LIAN 3.5; PMC, significance for obtaining this value in 1,000 simulations using the Monte Carlo method; VD, variance of pairwise difference; L, 95% critical value for VD; VD > L, presence of linkage disequilibrium. |
Subpopulations of IId subtypes ofC. parvum
Maximum likelihood analysis of the sequences grouped the 41 isolates into several evolutionary clusters (Fig. 1). Among them, the IIdA20G1 isolates from Heilongjiang and Hebei formed two clusters separated from other isolates. Another cluster was formed by IIdA15G1 isolates from Xinjiang. In contrast, there was no significant geographic clustering among IIdA19G1 isolates from Jiangsu, Shanghai, and Guangdong (Fig. 1).
A similar result was obtained in STRUCTURE analysis of allelic data. At all K values used in the analysis, the IIdA20G1 isolates from Heilongjiang were clearly separated from isolates of other regions, including those from Hebei that had the same gp60 subtype. The best separation of subpopulations by gp60 subtype was seen at K value of 3; all three C. parvum subtypes formed their own clusters (Fig. 2). In addition, regardless the K values (3–5) used in the analyses, IIdA19G1 isolates from Guangdong, Shanghai, and Jiangsu clustered together (Fig. 2). This was supported by results of the PCoA and median-joining network analyses, in which isolates from Heilongjiang, Hebei, and Xinjiang formed their own clusters while those from Shanghai, Jiangsu, and Guangdong clustered together (Figs. 3 & 4).
The results of Fst analysis supported the occurrence of geographically-associated subpopulations of C. parvum IId subtypes (Table 4). By gp60 subtype, isolates of IIdA15G1, IIdA19G1, and IIdA20G1 were genetically segregated from each other with high statistical significance. Within the IIdA20G1 subtype, there was a significant differentiation between isolates from Hebei and Heilongjiang. In contrast, the differentiation among IIdA19G1 isolates from Guangdong, Shanghai, and Jiangsu was low. There was also reduced differentiation of between IIdA20G1 isolates from Hebei and IIdA19G1 isolates from other areas (Table 4).
Table 4
Pairwise genetic distance (Fst, lower diagonal) and P values (Significance Level = 0.05, upper diagonal) between subpopulations of Cryptosporidium parvum by gp60 subtype and geographic origin.
Location | Guangdong | Jiangsu | Shanghai | Hebei | Heilongjiang | Xinjiang |
Guangdong | | 0.00901 ± 0.0091 | 0.02703 ± 0.0194 | 0.23423 ± 0.0411 | 0.00000 ± 0.0000 | 0.00000 ± 0.0000 |
Jiangsu | 0.14401 | | 0.04505 ± 0.0244 | 0.01802 ± 0.0121 | 0.00000 ± 0.0000 | 0.00000 ± 0.0000 |
Shanghai | 0.05817 | 0.09422 | | 0.01802 ± 0.0121 | 0.00000 ± 0.0000 | 0.00000 ± 0.0000 |
Hebei | 0.02455 | 0.32678 | 0.08043 | | 0.00000 ± 0.0000 | 0.00000 ± 0.0000 |
Heilongjiang | 0.67935 | 0.85765 | 0.5247 | 0.87905 | | 0.00000 ± 0.0000 |
Xinjiang | 0.36149 | 0.29526 | 0.28499 | 0.36733 | 0.47694 | |
Subtype | IIdA19G1 | IIdA20G1 | IIdA15G1 | | | |
IIdA19G1 | | 0.00000 ± 0.0000 | 0.00000 ± 0.0000 | | | |
IIdA20G1 | 0.14262 | | 0.00000 ± 0.0000 | | | |
IIdA15G1 | 0.34028 | 0.32851 | |