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The infection rate of C. sinensis in Hunan Province between 2016 and 2020 is shown in Table 1. The infection rate in the province, except in Yongzhou and Huaihua, was less than 1.5%. The infection rate in Yongzhou and Huaihua year-by-year showed a downward trend because of prevention, intervention, health education, and other reasons. Tongdao county, Lengshuitan District, and Qiyang County were monitored ashigh infection areas in Yongzhou and Huaihua; the systematic investigation of population infections in these three regions was of great significance in controlling the spread of C. sinensis (Table 2).
Table 1. Infection status of C. sinensis in Hunan Province from 2016 to 2020
Item 2016 2017 2018 2019 2020 5 years in total Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Changsha 2,017 0 0 0 2,156 0 3,227 0 2,017 0.05 9,417 0.01 Zhuzhou 3,205 0 1,000 0 1,000 0 3,033 0 2,413 0 10,651 0 Xiangtan 3,014 0 0 0 1,041 0 987 0 0 0 5,042 0 Hengyang 2,554 0.04 0 0 1,000 0 5,000 0 0 0 8,554 0.01 Shaoyang 4,103 0 3,014 0 2,000 0 4,002 0 0 0 13,119 0 Yueyang 9,768 0 2,013 0.15 2,000 0 4,000 0 1,000 0 18,781 0.02 Changde 4,099 0 0 0 1,000 0 3,004 0 3,000 0 11,103 0 Zhangjiajie 0 0 1,000 0 1,109 0 1,006 0 1,000 0 3,115 0 Yiyang 3,731 0 0 0 2,619 0.04 3,500 0 1,000 0 10,850 0.01 Chenzhou 7,031 0 1,029 0.29 2,052 0.05 5,023 0 4,000 0 19,135 0.02 Yongzhou 5,979 7.26 1,006 5.47 4,166 5.59 5,035 5.54 10,102 2.15 26,288 4.63 Huaihua 7,233 7.47 1,000 23.80 5,072 3.21 5,229 4.25 8,294 2.50 26,828 5.11 Loudi 4,635 0.02 0 0 3,580 0 4,500 0 1,000 0 13,715 0.01 Xiangxi 2,276 0 2,006 0.15 2,135 0 3,006 0 2,018 0 11,441 0.03 Hunan 59,645 1.64 12,068 2.50 30,930 1.29 50,552 0.99 35,844 1.19 188,039 1.38 Table 2. Infection status of C. sinensis among populations in Tongdao, Qiyang, and Lengshuitan
Region Number of examinees Number of infected people Infection rate
(%)Tongdao County 5,385 1,449 26.90 Qiyang County 3,761 713 18.96 Lengshuitan District 3,200 699 21.80 Total 12,346 2861 23.17 The results showed that 1,449 people were infected in Tongdao County, with an infection rate of 26.90%; 713 people in Qiyang County, with an infection rate of 18.96%; and 699 people in Lengshuitan District, with an infection rate of 21.80%. Significant differences (χ2 = 82.91, P < 0.01) were observed in the infection rates in these three regions.
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The infection of fish metacercariae by C. sinensis in Qiyang County, Lengshuitan District, and Tongdao County was investigated (Table 3). Common freshwater fish cultured in fish ponds were purchased from Tongdao County, Qiyang County, and Lengshuitan District. Differences in infectiosity invarious freshwater fish metacercariae were investigated; specifically, both infection rate and average infection rate were the highest in crucian carp (90.41% and 73.54 piece/g, respectively), followed by Parabramis pekinensis (78.26% and 46.16 piece/g, respectively), cyprinuscarpio (59.26% and 11.33 piece/g, respectively), and grass carp (25.00% and 1.83 piece/g, respectively). The infection rates of these four fishwere significantly different (χ2 = 103.56, P < 0.01); the infection rate and infectiosity were higherin Qiyang County than in the other regions.
Table 3. Infection of fish metacercaria in Tongdao, Qiyang, and Lengshuitan (
$\overline x$ ± s)Region Category Number of examinees (mantissa) Number of infected (mantissa) Infection rate (%) Average infectiosity (piece/g) Tongdao County Crucian 25 22 88.00 72.32 ± 38.05 Parabramispekinensis 12 10 83.33 46.08 ± 22.89 cyprinuscarpio 7 4 57.14 7.14 ± 5.82 Grass carp 4 1 25.00 0.75 ± 1.00 Qiyang County Crucian 18 17 94.44 77.78 ± 26.95 Parabramispekinensis 10 9 90.00 49.10 ± 18.56 cyprinuscarpio 8 5 62.50 10.25 ± 6.20 Grass carp 5 1 20.00 1.40 ± 2.09 Lengshuitan District Crucian 30 27 90.00 70.52 ± 33.82 Parabramispekinensis 24 17 70.83 43.28 ± 26.00 cyprinuscarpio 12 7 58.33 16.58 ± 10.17 Grass carp 7 2 28.57 2.00 ± 2.40 Total Crucian 73 66 90.41 73.54 ± 33.43 Parabramispekinensis 46 36 78.26 46.16 ± 23.21 cyprinuscarpio 27 16 59.26 11.33 ± 8.33 Grass carp 16 4 25.00 1.38 ± 1.95 -
C. sinensis adults were collected from cats and dogs (age, more than 5 years) and observed under a microscope (Figure 2). The body of an adult C. sinensis long and narrow, with a flat dorsal abdomen, narrow front end, blunt and round rear end (like a sunflower seed), and no spines on the surface. The oral suckers are slightly larger than the abdominal suckers, which are located at the front 1/5 of the body. The alimentary canal is simple, with the mouth at the center of the oral sucker, the pharynx is spherical, and the esophagus is short, followed by the intestinal branches. The intestinal branches are divided into two, which reach the posterior end along both sides of the worm without confluence and with a blind end. Judging from morphology, it is the adults of C. sinensis.
Figure 2. Adult Clonorchis sinensisobtained from hosts. (A) Naked eye of C. sinensis. (B) Clonorchis sinensisfrom Tongdaounder a microscope (magnification, 10×). (C) Clonorchis sinensisfrom Qiyang under a microscope (magnification, 10×). (D) Clonorchis sinensisfrom Lengshuitan under a microscope (magnification, 10×).
Morphological differences were detected among the C. sinensis adults from the three regions. The some adults in Qiyang County are similar to those in Lengshuitan District. The some adults of Tongdao county was more slender and brighter in color.
The mitochondrial genes Cox1 and Nad1 of C. sinensis were used as genetic markers for amplification. The results showed that the length of Cox1 and Nad1 are approximately 1,000 bp and 800 bp, respectively, which isconsistent with the predicted values; the electrophoresis results showed clear bands with no tailing phenomenon or unspecific bands (Figure 3).
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To verify the amplification accuracy of the two genes, the PCR products were sequenced and compared with homologous sequences from GenBank, and the amplified products were the Cox1 and Nad1 sequences of C. sinensis.
A phylogenetic tree was constructed using Cox1 as the genetic marker and Gyrodactylusas the outgroup for C. sinensis and other trematodes in Tongdao County, Qiyang County, and Lengshuitan District as well as Guangdong Province, Gansu Province, China, the United States, and Russia (Figure 4A). C. sinensisspecimens from Qiyang and Lengshuitan were clustered together without any regional differences; both exhibited high homology with those from Guangdong Province and Gansu Province, China, with a high bootstrap value. The regional clustering in Tongdao County was high and demonstrated low homology with that in Qiyang County and Lengshuitan District but high homology with that in the United States and Russia. The relationships of the other liver flukes, schistosomes, and Gyrodactylus with C. sinensis gradually decreased, which is consistent with the taxonomic records. No significant specificity was observed in the genetic relationships of the parasitic C. sinensis in different definitive hosts (dog and cat).
Figure 4. Phylogenetic relationships of C. sinensis with Cox1 and Nad1 as the genetic marker. (A) Phylogenetic relationships of C. sinensis with Cox1 as the genetic marker. (B) Phylogenetic relationships of C. sinensis with Nad1 as the genetic marker.
With Nad1 as the genetic marker (Figure 4B), C. sinensis from Qiyang County and Lengshuitan District showed few regional differences and high homology with those from Guangdong Province and Gansu Province, China. the regional clustering in Tongdao County was high and demonstrated high homology with that in the United States and Russia. The relationships of the other liver flukes, schistosomes, and Gyrodactylus with C. sinensisgradually decreased, which is consistent with the taxonomic records. The parasitic C. sinensis in different definitive hosts (dog and cat) showed no significant specificity in the genetic relationship. This is consistent with the phylogenetic relationship results with Cox1 as the genetic marker.
doi: 10.3967/bes2021.121
Interspecies Phylogenetic Analysis of Clonorchis sinensis in High-incidence Areas of Hunan Province, China
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Abstract:
Objective This study aims to investigate the infection of Clonorchis sinensis (C. sinensis) in high-incidence areas of Hunan Province, China. The phylogenetic analysis of the C. sinensis species in the highly infected areas was carried out. Method Infection of the definitive human host and intermediate fish host by C. sinensis was investigated, and the mitochondrial genes cox1 and Nad1 were used as genetic markers for phylogenetic analysis. Results In 2016–2020, the average population infection rate of Hunan was 1.38%, while in Tongdao County the rate was up to 26.90%, and the highest fish infection rate was detected in Qiyang County (99.44% in the dorsal fin of crucian carp). High genetic sequence similarity was observed in the samples from Qiyang and Lengshuitan which exhibited high homology with those from Guangdong and Gansu, whereas the parasitic species from Tongdao was highly homologous with those located in high-latitude areas. Moreover, no significant difference was found in the gene sequence of the parasitic species in definitive hosts dogs and cats. Conclusion The systematically study of C. sinensis infection in the high-incidence areas will contribute greatly to the prevention and effectively controlling the spread of Clonorchis sinensis in Hunan Province The endemic of C. sinensis infection in Hunan Province is the result of co-action of local and foreign parasite species. -
Key words:
- Clonorchis sinensis /
- Mitochondrial genes /
- Cox1 /
- Nad1 /
- Phylogeny
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Figure 1. Sampling map for Tongdao, Qiyang, and Lengshuitan. Samples were obtained from the following towns in Tongdao: Shuangji Village of Pingtan Township (cats, sample numbers TD1 and TD2), Qiaozhai Village of Yatunpu Town (cat, sample number TD3), Shuinan Village of Jingwuzhou Town (cats, sample numbers TD4 and TD5), Xinghua Village of Wanfoshan Town (dogs, sample numbers TD6 and TD7), and Pipa Village of Shuangjiang Town (cats, sample numbers TD8 and TD9). Samples were collected from the following towns in Lengshuitan: Wujiayuan Neighborhood Committee of Yangjiaqiao Sub-district (dogs, sample numbers lST1, lST2, and lST3), Guanzitou of Yangcundian Township (cats, sample numbers lST4, lST5, and lST6), Yangshanguan Village of Shanglingqiao Town (cats, sample numbers lST7, lST8, and lST9), Hongxing Village of Renwan Street (cats, sample numbers lST10, lST11, and lST12), and Tianbatang Village of Huangyangsi Town (dog, sample number lST13). Samples were obtained from the following towns in Qiyang: Fulian Village of Maozhu Town (dogs, sample numbers QY1, QY2, and QY3), Xinlian Village of Lijiaping Town (dogs, sample numbers QY4, QY5, and QY6), Qingzhu Village of Longshan Street (dogs, sample numbers QY7, QY8, and QY9), Yanghua Village of Meixi Town (dogs, sample numbers QY10, QY11, and QY12), and Shili Village of Dazhongqiao Town (cats, sample numbers QY13, QY14, and QY15).
Figure 2. Adult Clonorchis sinensisobtained from hosts. (A) Naked eye of C. sinensis. (B) Clonorchis sinensisfrom Tongdaounder a microscope (magnification, 10×). (C) Clonorchis sinensisfrom Qiyang under a microscope (magnification, 10×). (D) Clonorchis sinensisfrom Lengshuitan under a microscope (magnification, 10×).
Table 1. Infection status of C. sinensis in Hunan Province from 2016 to 2020
Item 2016 2017 2018 2019 2020 5 years in total Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Sample size Infection rate (%) Changsha 2,017 0 0 0 2,156 0 3,227 0 2,017 0.05 9,417 0.01 Zhuzhou 3,205 0 1,000 0 1,000 0 3,033 0 2,413 0 10,651 0 Xiangtan 3,014 0 0 0 1,041 0 987 0 0 0 5,042 0 Hengyang 2,554 0.04 0 0 1,000 0 5,000 0 0 0 8,554 0.01 Shaoyang 4,103 0 3,014 0 2,000 0 4,002 0 0 0 13,119 0 Yueyang 9,768 0 2,013 0.15 2,000 0 4,000 0 1,000 0 18,781 0.02 Changde 4,099 0 0 0 1,000 0 3,004 0 3,000 0 11,103 0 Zhangjiajie 0 0 1,000 0 1,109 0 1,006 0 1,000 0 3,115 0 Yiyang 3,731 0 0 0 2,619 0.04 3,500 0 1,000 0 10,850 0.01 Chenzhou 7,031 0 1,029 0.29 2,052 0.05 5,023 0 4,000 0 19,135 0.02 Yongzhou 5,979 7.26 1,006 5.47 4,166 5.59 5,035 5.54 10,102 2.15 26,288 4.63 Huaihua 7,233 7.47 1,000 23.80 5,072 3.21 5,229 4.25 8,294 2.50 26,828 5.11 Loudi 4,635 0.02 0 0 3,580 0 4,500 0 1,000 0 13,715 0.01 Xiangxi 2,276 0 2,006 0.15 2,135 0 3,006 0 2,018 0 11,441 0.03 Hunan 59,645 1.64 12,068 2.50 30,930 1.29 50,552 0.99 35,844 1.19 188,039 1.38 Table 2. Infection status of C. sinensis among populations in Tongdao, Qiyang, and Lengshuitan
Region Number of examinees Number of infected people Infection rate
(%)Tongdao County 5,385 1,449 26.90 Qiyang County 3,761 713 18.96 Lengshuitan District 3,200 699 21.80 Total 12,346 2861 23.17 Table 3. Infection of fish metacercaria in Tongdao, Qiyang, and Lengshuitan (
$\overline x$ ± s)Region Category Number of examinees (mantissa) Number of infected (mantissa) Infection rate (%) Average infectiosity (piece/g) Tongdao County Crucian 25 22 88.00 72.32 ± 38.05 Parabramispekinensis 12 10 83.33 46.08 ± 22.89 cyprinuscarpio 7 4 57.14 7.14 ± 5.82 Grass carp 4 1 25.00 0.75 ± 1.00 Qiyang County Crucian 18 17 94.44 77.78 ± 26.95 Parabramispekinensis 10 9 90.00 49.10 ± 18.56 cyprinuscarpio 8 5 62.50 10.25 ± 6.20 Grass carp 5 1 20.00 1.40 ± 2.09 Lengshuitan District Crucian 30 27 90.00 70.52 ± 33.82 Parabramispekinensis 24 17 70.83 43.28 ± 26.00 cyprinuscarpio 12 7 58.33 16.58 ± 10.17 Grass carp 7 2 28.57 2.00 ± 2.40 Total Crucian 73 66 90.41 73.54 ± 33.43 Parabramispekinensis 46 36 78.26 46.16 ± 23.21 cyprinuscarpio 27 16 59.26 11.33 ± 8.33 Grass carp 16 4 25.00 1.38 ± 1.95 -
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