3.1 Rate of infection and Sequence variation
Isolates from sheep represented the majority of samples (172 out of 183 isolates), meanwhile 11 were from goats. The infection proportion of C. tenuicollis in slaughtered small ruminants was 46% in the omentum, 36.6% in mesentery, 16.8% in liver and 0.6% in lungs (Table 1). In the current study high proportion of cysts was collected from omentem followed by mesentry which is in agreement with some previous reports (Alvi et al., 2020; Radfar et al., 2005). Cysts were round with a white thin wall containing a scolex (bladder worms) which appeared as a white dot. The sizes were from 1–11 cm in the omentum and 1–2 cm in the liver and lung which is similar to previous cyst sizes reported by Al-Hamzawi and Al-Mayali (2020).
Table 1
Distribution pattern of Taenia hydatigena cysticerci in various organs in sheep and goats.
Host
|
Organs
|
Omentum
|
Mesentery
|
Liver
|
Lung
|
Total
|
Sheep
|
79 (45.9%)
|
63 (36.6%)
|
29 (16.9%)
|
1 (0.6%)
|
172
|
Goats
|
11 (100%)
|
0
|
0
|
0
|
11
|
Sheep and goats
|
90 (49.2)
|
63 (34.4%)
|
29 (15.8)
|
1 (0.5%)
|
183
|
PCR amplification of the 114 isolates from China, demonstrate 100% (114 from 114 isolates) and 85.1% (97 out of 114 isolates) successful amplification of the complete nad4 (1,254 bp) and nad5 (1,569 bp) mitochondrial genes respectively. Examination of the entire nad4, nad5 and concatenated nad4-nad5 (2,823 bp) genes sequences showed 182, 223 and 385 segregating sites or polymorphic sites, respectively.
PCR amplification of the sixty-nine isolates from Mongolia, demonstrated 100% (69 from 69) and 85.5% (59 out of 69) successful amplification of nad4 (1,254 bp) and nad5 (1,569 bp) genes, respectively. Examination of the complete nad4 (1,254 bp), nad5 (1,569 bp) and concatenated nad4-nad5 (2,823 bp) gene sequences indicated 161, 168 and 318 polymorphic sites, respectively for each gene (Table 2).
Table 2
Diversity and neutrality indices for Cysticercus tenuicollis isolates from Mongolia and China using of nad 4, nad5 and concatenated nad4-nad5 mitochondrial gene sequences (Indicative haplotypes sequences from current investigation have been deposited in the GenBank database under the accession numbers ON379092 - ON379244 and ON379245 - ON379369, respectively) .
Index
|
China
|
Mongolia
|
nad4 (1,254 bp)
|
nad5 (1,569 bp)
|
nad4nad5 (2,823 bp)
|
nad4 (1,254 bp)
|
nad5 (1,569 bp)
|
nad4nad5 (2,823 bp)
|
Sheep
|
Goats
|
Total
|
Sheep
|
Goats
|
Total
|
Sheep
|
Goats
|
Total
|
Sheep
|
Sheep
|
Sheep
|
No. of isolates
|
103
|
11
|
114
|
87
|
10
|
97
|
87
|
10
|
97
|
69
|
59
|
59
|
No. of mutations
|
177
|
69
|
191
|
223
|
31
|
233
|
375
|
94
|
400
|
165
|
173
|
326
|
Parsimony informative sites
|
97
|
44
|
106
|
116
|
7
|
118
|
206
|
48
|
217
|
81
|
58
|
128
|
No. of haplotypes, H
|
87
|
11
|
98
|
69
|
10
|
76
|
80
|
10
|
90
|
62
|
53
|
56
|
Haplotype diversity (Hd)
|
0.995
|
1.000
|
0.996
|
0.987
|
1.000
|
0.989
|
0.998
|
1.000
|
0.998
|
0.994
|
0.991
|
0.996
|
Variance of haplotype diversity, Var Hd
|
0.000
|
0.001
|
0.000
|
0.000
|
0.002
|
0.000
|
0.000
|
0.002
|
0.000
|
0.000
|
0.000
|
0.000
|
Nucleotide diversity (π)
|
0.013
|
0.017
|
0.013
|
0.009
|
0.004
|
0.009
|
0.011
|
0.009
|
0.010
|
0.011
|
0.007
|
0.008
|
Tajima’s D
|
-1.709
|
-0.226
|
-1.733
|
-2.199
|
-1.483
|
-2.257
|
-1.997
|
-0.898
|
-2.050
|
-1.956
|
-2.464
|
-2.337
|
Fu’s Fs
|
-34.252
|
-2.014
|
-32.908
|
-45.412
|
-4.350
|
-55.309
|
-45.272
|
-1.234
|
-32.626
|
-34.098
|
-33.846
|
-32.626
|
Fst
|
0.000
|
0.013
|
0.000
|
0.000
|
0.015
|
0.000
|
0.000
|
0.013
|
0.000
|
0.000
|
0.007
|
0.005
|
The nBLAST search for the resulting sequences accurately identified all isolates as T. hydatigena with 98.7 to 100% similarity to GenBank deposited sequences.
3.2 Population and diversity indices
The highest number of mutations, haplotypes and nucleotide diversity were observed in sheep of China as compared to Mongolia (Table 2).
The overall number of mutations in Chinese sheep and goats was 191 (177 and 69 for sheep and goats, respectively), and in Mongolian sheep was 165 for nad4. It was 233 in Chinese sheep and goats (223 and 31 for sheep and goats, respectively), and 173 in Mongolian sheep for nad5. Previous report in sheep (Ohiolei et al., 2021a) from China showed lower number of mutations (93). On the other hand, a study from Nigeria indicated lower number of mutations from sheep (1) and higer from goats (33) (Ohiolei et al., 2019). This variation maybe due to the variation of sample size. For concatenation of nad4-nad5, mutations observed were 400 in Chinese sheep and goats (375 and 94 for sheep and goats, respectively), and 326 in Mongolian sheep.
The overall parsimony informative sites in Chinese sheep and goats were 106 (97 and 44 for sheep and goats, respectively), and in Mongolian sheep were 81 for nad4. They were 118 in Chinese sheep and goats (116 and 7 for sheep and goats, respectively), and 58 in Mongolian sheep for nad5. An investigation from China showed lower parsimony informative sites with 52 (Ohiolei et al., 2021a). A study in Nigeria indicated that the parsimony informative sites from sheep were lower and from goats were higher, which were 1 and 12 respectively (Ohiolei et al., 2019). For concatenated nad4-nad5 there were 217 parsimony informative sites in Chinese sheep (206) and goats (48), and 128 in Mongolian sheep.
The all-over number of haplotypes in Chinese sheep and goats was 87 and 11, respectively, and in Mongolian sheep was 62 for nad4. For nad5, it was 69 and 10 in Chinese sheep and goats, respectively, and 53 in Mongolian sheep. Previous report in sheep by Ohiolei et al (2021a) from China showed lower number of haplotypes (17), followed by Nigeria from sheep (2) and goats (8) (Ohiolei et al., 2019). For concatenation of nad4-nad5 were 80 and 10 in Chinese sheep and goats, respectively, and 56 in Mongolian sheep.
The all-over haplotype diversity (Hd) in Chinese sheep and goats was 0.995 and 1.000 for sheep and goats, respectively, and in Mongolian sheep was 0.994 for nad4. It was 0.987 and 1.000 in Chinese sheep and goats, respectively, and 0.991 in Mongolian sheep for nad5. Previous report from China showed lower haplotype diversity (0.979) in sheep (Ohiolei et al., 2021a), followed from Nigeria in sheep (0.536) and in goats (0.766) (Ohiolei et al., 2019). For concatenated nad4-nad5 was 0.998 and 1.000 in Chinese sheep and goats, respectively, and 0.996 in Mongolian sheep.
The overall Variance of haplotype diversity (Var Hd) in Chinese sheep and goats was 0.000 (0.000 and 0.001 for sheep and goats, respectively), and in Mongolian sheep was 0.000 for nad4. Similarly, it was 0.000 in Chinese sheep and goats (0.000 and 0.002 for sheep and goats of China respectively), and 0.000 in Mongolian sheep for nad5. For concatenation of nad4-nad5 was 0.000 in Chinese sheep and goats (0.000 and 0.002 for sheep and goats, respectively), and 0.000 in Mongolian sheep.
The overall nucleotide diversity (π) in Chinese sheep and goats was 0.013 (0.013 and 0.017 for sheep and goats, respectively), and in Mongolian sheep was 0.011 for nad4. Similarly, it was 0.009 in Chinese sheep and goats (0.009 and 0.004 for sheep and goats, respectively) and 0.007 in Mongolian sheep for nad5. This finding is almost similar with previous report (nucleotide diversity = 0.010) in sheep from China (Ohiolei et al., 2021a), however, higher from Nigerian sheep (π = 0.000 and goats (π = 0.003) (Ohiolei et al., 2019). For concatenation of nad4-nad5 was 0.010 in Chinese sheep and goats (0.011 and 0.009 for sheep and goats, respectively), and 0.008 in Mongolian sheep.
The overall Tajima’s D was negative for all sequences and in Chinese sheep and goats was − 1.733 (-1.709 and − 0.226 for sheep and goats, respectively), and in Mongolian sheep was − 1.956 for nad4. It was − 2.257 in Chinese sheep and goats (-2.199 and − 1.483 for sheep and goats respectively) and − 2.464 in Mongolian sheep for nad5, Meanwhile previous report showed (higher Tajima’s D -1.583) in sheep from China (Ohiolei et al., 2021a), in another study from Nigeria showed higher Tajima’s D in sheep (1.166) and goats (-1.646) (Ohiolei et al., 2019). For concatenation of nad4-nad5 was − 2.050 in Chinese sheep and goats (-1.997 and − 0.898 for sheep and goats, respectively), and − 2.337 in Mongolian sheep, which is signifying an excess presence of low-frequency polymorphisms indicative of an expanding population (Table 2).
The overall Fu’s Fs was negative for all isolates and in Chinese sheep and goats was − 32.908 (-34.252 and − 2.014 for sheep and goats, respectively), and in Mongolian sheep was − 34.098 for nad4. It was − 55.309 in Chinese sheep and goats (-45.412 and − 4.350 for sheep and goats, respectively) and in Mongolian sheep were − 33.846 for nad5. Previous report showed higher Fu’s Fs (-2.997) in sheep from China (Ohiolei et al., 2021a), followed by Nigeria from sheep (0.866) and goats (0.934) (Ohiolei et al., 2019). For concatenation of nad4-nad5 was − 32.626 in Chinese sheep and goats (-45.272 and − 1.234 for sheep and goats respectively) and − 32.626 in Mongolian sheep, which indicates the presence of rare haplotypes as expected from a recent population expansion (Table 2).
Average number of differences (K) was 17.14, 14.84 and 30.77 for nad4 (1,254 bp), nad5 (1,569 bp) and concatenated nad4-nad5 (2,823 bp) mitochondrial genes respectively in China, while it was 14.96, 11.24 and 24.14 for nad4, nad5and nad4-nad5 respectively in Mongolia.
Pairwise fixation index or Fst between China and Mongolia was also estimated for the determination of genetic differentiation which was 0.000 and 0.003 for nad4 respectively. For nad5 it was 0.000 and 0.007 for China and Mongolia respectively. Regarding the concatenated nad4-nad5 it was 0.000 and 0.005 respectively, between China and Mongolia (Table 2).
3.3 Haplotype networks
The haplotype network of nuclear nad4 and nad5 genes and their concatenation of C. tenuicollis showed haplotypes arranged in a star-like configuration of with three extending groups (A1, A2 and B) (Fig. 1a, b and c). For nad4, the network formed a star-like configuration of 153 haplotypes of C. tenucollis of Mongolian and Chinese origin, of which seven haplotypes H6, H69, H5, H8, H46, H61 and H57 were present in both countries. For nad5, of the 125 haplotypes, four haplotypes H8, H15, H51 and H47 appeared in both countries. For the spliced nad4-nad5 gene network, only one haplotype H76 of the 144 was common to both countries, and interstingly, similar findings were reported previously (Sgroi et al., 2020). The majority of sequences from 93 out of 114 Chinese isolates were as haplogroup A variants, while 15 and 6 were as haplogroup B and A2 for the nad4 gene. However, out of 69 isolates from Mongolia 60, 5 and 4 belonged to haplogroups A, A2 and B respectively. The most isolates, 85 out of 97 were as haplogroup A variants, 8 as haplogroup B variants, and 4 as haplogroup A2 for the nad5 gene from China, meanwhile, whereas from Mongolia 55, 1 and 3 from 59 isolates were as haplogroups A, A2 and B, respectively. On the other hand, for concatenated nad4-nad5, 85, 8 and 4 out of 97 Chinese isolates were grouped in to haplogroups A, B and A2 variants whereas for Mongolia, 55, 1 and 3 out of 59 isolates were as haplogroups A, A2 and B, respectively (Fig. 1a, b and c).
3.4 Restriction fragment length polymorphism
Based on the genetic variation visualized in the median-joining networks, we digested a portion of the nad5 gene as recently described (Ohiolei et al., 2020, 2021). The in silico RFLP fragment generated for 156 sequences showed nine RFLP profiles namely A1, A2, A3, A5, A8, A9, A10, A11 and B. A comparison of the in silico RFLP fragments from Chinese and Mongolian isolates revealed seven common haplogroups (A1, A2, A3, A5, A8, A9 and B). Moreover, additional profiles (A10, A11) were recorded from China. Identified haplogroups including A1 from China, Nigeria, Pakistan and Sudan, A2 from China and Pakistan, A3 from China, Nigeria and Pakistan and B from China and Pakistan using nad5 were reported (Ohiolei et al., 2021b). Meanwhile, A4 from Nigeria and A6 and A7 from Pakistan were reported (Ohiolei et al., 2021b). In our investigation, A4, A6 and A7 were not present. Fragment sizes and profiles of the new haplogroups of T. hydatigena for nad5 gene withen Rsa1 and Acc1 restriction enzymes are showen in (Table 3). Haplogroup A1 was extensively spread than other haplogroupes which is in agreement with the previous study (Ohiolei et al., 2021b). The result as shown in (Fig. 2a, b). Lane one is haplogroup A1, lane two is A3, lane three is A5, lane four is A8, lane five is haplogroup A9, lane six is A10 and lane seven is haplogroup A11, lane eight till lane twelve haplogroup B and lane thirteen till lane seventeen haplogroup A2 demonstrate 9 RFLP profiles.
Table 3
Fragment sizes and profiles of T. hydatigena nad5 gene (1,444 bp) digested with Rsa1 and Acc1 restriction enzymes.
Haplogroup A
|
Rsa1 restriction site
|
Acc1restriction site
|
DNA fragments (bp)
|
RFLP-profile
|
Country
|
Host
|
|
226–1245
|
692
|
553,466,226,199
|
A5
|
China, Mongolia
|
Sheep
|
|
226–1090
|
692
|
466, 398, 354, 226
|
A8
|
China, Mongolia
|
Sheep
|
|
226–572
|
692
|
752, 346, 226, 120
|
A9
|
China, Mongolia
|
Sheep
|
|
226–1256
|
692
|
564, 466, 226, 188
|
A10
|
China
|
Sheep
|
|
226–725
|
-
|
719,499,226
|
A11
|
China
|
Sheep
|
3.5 Phylogenetic analyses
In total, 156 nucleotide sequences of the results of concatenated complete nucleotide sequences of (nad4-nad5) fragments and other homologous sequences from GenBank were used to constract phylogenetic tree. All obtained sequences diverged significantly in the concatenated genes from the isolates of other taeniid species, the type sequences of which were retrieved from GenBank and included in the analysis, T. solium (AB086256), T. asiatica (AF445798), T. saginata (AY684274), T. multiceps (GQ228818), T. pisiformis (GU569096) and T. ovis (NC_021138). The majority of nucleotide sequences of Cysticercus tenuicollis concatenatied nad4-nad5 genes withen phylogenetic tree, except for four isolates (haplotypes H30, H74, H108, H125, H136 and H141), clustered together and were divided in two subclades (Fig. 3). This study showed H119, H72, H109, H133, H2, H33, H100, H28, H56, H32, H117 and H50 as Haplogroup B, and H11, H70, H102, H29, H112, H124 and H77 from Mongolia and China as Haplogroup A2 of T. hydatigena, as represented in (Fig. 3), and others as Haplogroups A1, A3, A5, A8, A9, A10 and A11.