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@#Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.
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Objective To evaluate the effectiveness of urban construction projects on Oncomelania snails control in Wuhan City, so as to provide insights into the development of an integrated control strategy for urban schistosomiasis. Methods The data pertaining to the endemic situation of schistosomiasis, schistosomiasis control data, the construction of the Yangtze River Bridge, marshland management and park constructions were collected from Wuhan City from 1990 to 2020. The changes of areas of snail habitats and high-risk settings were compared before and after implementation of these urban construction projects to evaluate the effectiveness of urban construction projects on Oncomelania snails control. Results The number of schistosomiasis cases decreased by 97.35%, and the number of cattle with schistosomiasis decreased by 100% in Wuhan City from 1990 to 2020, with a 41.99% reduction in areas of snail habitats, including 94.97% and 34.40% reductions in snail habitats inside and outside the embankment decreased. During the period from 1990 through 2020, a total of 10 Yangtze River bridges were built in Wuhan City, and areas of snail habitats around the bridges reduced from 11 699.05 hm2 before the bridge building to 8 726.14 hm2 after the building (a 25.41% reduction), while the areas of high-risk settings reduced from 411.69 hm2 before the bridge building to 276.78 hm2 after the building (a 32.77% reduction). Following treatment of three marshlands in Hankou, Hanyang and Wuchang, the areas of snail habitats reduced from 225.80 hm2 before the management to 199.47 hm2 after the management (a 11.66% reduction), and the areas of high-risk settings reduced from 23.14 hm2 before the treatment to 17.73 hm2 after the building (a 23.38% reduction). Following the building of 5 parks in snail-infested settings, the areas of snail habitats reduced from 319.61 hm2 before the construction to 280.80 hm2 after the construction (a 12.14% reduction), and the areas of high-risk settings reduced from 35.00 hm2 before the construction to 27.73 hm2 after the construction (a 20.77% reduction). Conclusion Simultaneous implementation of urban construction projects and schistosomiasis control measures is effective to shrink snail-infested setting and high-risk settings and control the endemic situation of schistosomiasis in endemic foci in Wuhan City.
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Objective To study the effects of environmental temperature and humidity on the survival and reproduction of Oncomelania snails, and to provide a basis for the development of scientific strategies and measures for snails control. Methods Based on the field experimental base of Oncomelania snails in Hubei Province, through long-term and systematic ecological observation of snails, the living conditions of snails at different temperatures were recorded, and the average densities of live snails and young snails as well as the amount of spawning were calculated. Results In a certain temperature range (15°C-30°C), the average densities of live snails and young snails were positively correlated with the changes of temperature and humidity, and the effect of temperature on the spawning of snails was also significant. The optimal breeding temperature of Oncomelania snails was 25°C. Conclusion In a certain temperature range (15°C-30°C), the environmental temperature and humidity have a significant impact on the survival and reproduction of snails in the field.
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Objecitve To observe the diffusion and distribution of imported Oncomdania snails in non-endemic areas of schistosomiasis.so as to provide scientific basis for developing the scheme of snail surveillance.Methods Oncomelania snails from the msrshland of Dongting Lake were imported in a waste land in March,2008,Qingxin County,which belonged to non-endemic areas of schistosomiasis.and their diffusion and distribution condition were observed from May to December in the same year.Resuits A total of 1 600 Oncomelania snails were imported.The occurrence rate of frame with snails in sunny days and cloudy rainy days were22.88%and 29.24%,respectively,and there was a statistically significant difference between them(χ~2=4.113,P=0.043).Those in waste Land and the sides/bottoms of the ditches were 9.38%and 35.99%,respectively,and the difference between them also showed a statistical significance(χ~2=67.246,P<0.001).The average density of living snails in sunny days and cloudy rainy days were 0.79 and 1.11 No./0.1 m~2,respectively,and those in waste land and the sides/bottoms of the ditches were0.11 and 1.34 No./0.1 m~2.The furthest diffusion distance of snailswas 4.5 m.Conclusions The detective opportunity of imported snails is relatively high in cloudy rainy days,which mainly distribute at the sides/bottoms of ditches and diffuse along the ditches.
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Objective To study the compliance of forbidden depasturing livestock on the marshland with Oncomelania snails in schistosomiasis endemic areas. Methods According to 3 levels of human infection rates as > 10% ,5%-10% and <5% , 2 204 residents selected randomly from the schistosomiasis endemic villages were sampled with the stratified cluster sampling method in Hunan, Hubei, Jiangxi, Anhui, Jiangsu, Sichuan and Yunnan provinces, and investigated by questionnaire. The contents of the questionnaire included the recognition and implementation of forbidden depasturing livestock on marshland with Oncomelania snails and breeding livestock in bam. Results A total of 78.4% residents agreed forbidden depasturing livestock on marshland with snails, but 3. 7% residents disagreed it. A total of 83. 9% residents considered the relationship between breeding livestock in bam and schistosomiasis control, but 3. 1% residents thought that it was no relationship. The main reasons of depasturing livestock on marshland with Oncomelania snails were the high cost of breeding livestock in bam (36. 2% ) , unaccustomed (26.4% ) and no room for breeding livestock in bam (25.4% ). Conclusion Forbidden depasturing livestock on the marshland with Oncomelania snails should be strengthened according to the local economic, nature environment, agriculture, residents'culture degree and agriculture habit.
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A total of 13 allozymes of 7 populations of Oncomelania snails trom 7 provinces in the mainland China were examined by horizontal starch gel electrophoresis. Results demonstrated that out of 17 loci examined, 10 were monomorphic loci: Aldh,Ao,G6pd ,Gpdh,Hbdh,Ldh, Sdh,Est-2,Est-3,and Est-6,and 7 were polymorphic loci:Est-4,Est-5,Xdh,Mdh,Idh,Got and Ap. Banding pattern showed that six enzymes were polymorphic accounting for 46.15 % (6/13) of total enzyme systems examined. Results indicated that a certain of relative relation-ship was existed along with the significant variation presented based on their environment and location among the 7 populations of snails from the mainland of China. It was revealed that the 7 polymorphic loci were the important loci affecting the gentic variation in the differ-ent populations of Oncomelania spp.
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Objective To evaluate the effect of elimination of schistosomiasis infectivity in water body by applying molluscicide on transmission marshland outside an embankment in Dongting Lake.Methods Two patches of transmission marshland with similar Oncomelania snails and infectious snails density in the northern part of eastern Dongting Lake regions were selected as molluscicide trail and control groups,respectively,and the snails and water body infectious indicators were investigated and compared between them.Results Adjusted death rate of snails in the trail group 15 days after applying molluscicide was 94.13%,without infected snails.The infection rate of sentinel mice in the trial group 50 days after applying molluscicide was 100.00%,with average 56.6 worms per mice.The infection rate of sentinel mice in the control group without molluscicide was also 100.00%,with average 147.8 worms per mice.Conclusions In transmission marshland areas outside the embankment of rivers or lakes,it is impossible for focus mollusciciding to eliminate the infectivity of schistosomiasis in water body,yet possible to reduce the degree of infectivity to a certain extent.