Prevalence of Angiostrongylus cantonensis infection in snails in Fujian Province from 2017 to 2021 / 中国血吸虫病防治杂志

Objective To investigate the prevalence of Angiostrongylus cantonensis infection in market-sold and field-captured snails in Fujian Province, so as to provide the scientific basis for the formulation of the angiostrongyliasis control measures. Methods In each month from May to October during the period from 2017 through 2021, Pomacea snails were collected from two field fixed surveillance sites and Bellamya aeruginosa collected from one agricultural product market in Fuzhou City, while Pomacea and B. aeruginosa snails were collected from two agricultural product markets and four restaurants in Xiamen City. At least 50 Pomacea snails and 500 g B. aeruginosa were sampled each time. A. cantonensis infection was detected in Pomacea snails using lung microscopy, and in B. aeruginosa using a tissue homogenate method. Results A total of 9 531 Pomacea snails were detected for A. cantonensis infection in two cities of Fuzhou and Xiamen, and the overall prevalence of A. cantonensis infection was 4.40%, with the highest prevalence in 2017 (6.82%, 116/1 701) and the lowest prevalence in 2019 (3.46%, 83/2 400). The prevalence of A. cantonensis infection was significantly higher in Pomacea snails sampled from Fuzhou City (11.23%, 326/2 903) than from Xiamen City (1.40%, 93/6 628) (χ2 = 461.48, P < 0.01). A. cantonensis larvae were detected in larval Pomacea snails in two cities of Fuzhou and Xiamen each month. The prevalence of A. cantonensis infection in Pomacea snails appeared an overall tendency towards a rise with month in Fuzhou City, with the highest prevalence in October (15.24%), and there was a significant difference among month (χ2 = 14.56, P < 0.05), while the prevalence of A. cantonensis infection in Pomacea snails was low in Pomacea snails sampled from Xiamen City each month, with the highest prevalence in June (2.64%), and there was a significant difference among month (χ2 = 23.17, P < 0.05). A total of 18 966 B. aeruginosa snails were detected for A. cantonensis infection in two cities of Fuzhou and Xiamen, and the overall prevalence of A. cantonensis infection was 0.01%. Conclusions A. cantonensis infection was identified in Pomacea and B. aeruginosa snails in Fujian Province from 2017 to 2021, and there is a potential risk of human A. cantonensis infection.

A comparative study of lung-microscopy and tissue homogenate in detecting Angiostrongylus cantonensis in the Pomacea canaliculata / 中国热带医学

@#Abstract: Objective　To compare the efficiency of lung-microscopy and tissue homogenate in the detection of Angiostrongylus cantonensis larvae from Pomacea canaliculate, with the amin of finding a simple and rapid method suitable for different scenarios. Methods　Pomacea canaliculata was caught and collected from ponds, ditches, rivers and other environments in the confirmed epidemic areas reported by predecessors. After each snail was weighed one by one, and dissected into two parts: lung sac and muscle. Firstly, each snail lung sac was check for nodules with lung-microscopy, and the nodules were picked out with anatomical needle and press them separately. Then, the worm was found and identified for worm species under microscope, and the lung sac and snail meat then was rechecked with tissue homogenate. Results　A total of 330 snails were detected, with 19.1% (63/330) snails with Angiostrongylus cantonensis stage Ⅲ larvae were detected by tissue homogenate and 15.8% (52/330) snails with nodules were detected by lung-microscopy. Among them, 36 snails with nodules and larvae were detected by the lung-microscopy, and all of them were positive by the tissue homogenate, with a coincidence rate of 100% (36/36); 16 snails with nodules but no larvae, among which 6 snails were positive and 10 snails were negative by the tissue homogenate, The false detection rate was 19.2% (10/52). 278 snails with no nodules, but 21 of them were detected by the tissue homogenate, the missing rate was 7.6% (21/278). There was no significant difference between tissue homogenate and lung-microscopy (taking the positive determination of nodules as the standard) (χ2=1.27, P=0.26, P>0.05). There was significant difference between tissue homogenate and lung-microscopy (taking the detection of larvae as the standard)(χ2=8.66, P = 0.003, P<0.01). There was no significant difference between the two methods and tissue homogenate in the detection rate of large snails ( ≥25 g, χ2=0.08，P=0.777; χ2=2.58, P=0.108), but there was significant difference between the two methods and tissue homogenate in the detection rate of small snail (≤10 g, χ2=5.63, P=0.02). Conclusions　Compared with the tissue homogenate, lung-microscopy is simple in the detection of large snails, requires less instruments and equipment, and its detection speed is faster. It is suitable for the field investigation of the natural focus of Angiostrongylus cantonensis. The tissue homogenate has high sensitivity, can directly display the insect state and activity, has strong insect vitality and high detection rate, it's more suitable for food safety risk monitoring.

Population genetics of invasive Pomacea spp. in Hangzhou City, Zhejiang Province / 中国血吸虫病防治杂志

Objective To investigate the distribution and identify the genetic genetics of invasive Pomacea species in Xihu District, Hangzhou City, so as to understand the spread tendency of Pomacea species. Methods The specimens of Pomacea species were collected from five sites in water systems (lakes, rivers and wetlands) and its costal lands in Xihu District, Hangzhou City in 2017 for morphological identification. Total DNA was isolated from the foot tissues of adult snails for amplification of the COI gene, and haplotype diversity and nucleic acid diversity analyses were performed. In addition, a phylogenetic tree was created based on the haplotype captured from GenBank and those from this study to investigate the phylogenetic relationships. Results Pomacea specimens, which were preliminarily characterized as Pomacea, were found in ponds, rivers and wetlands in Xihu District of Hangzhou City. A total of 16 sequences were captured from the DNA samples of Pomacea specimens, which belonged to 3 haplotypes, including Hap1, Hap2 and Hap3. A high frequency was seen in Hap1 and Hap3, and a low frequency was found in Hap2. The Pomacea specimens collected from the 5 sites in Xihu Districts included P. canaliculata and P. maculate. The Pomacea specimens with a Hap1 had a close genetic relationship with the P. canaliculata from Argentina, Guangdong Province and Hong Kong Special Administrative Region of China, and the Pomacea specimens with a Hap2 had a close genetic relationship with the P. canaliculata from Argentina, Japan and Guangzhou City of Guangdong Province, China, while the Pomacea specimens with a Hap2 had a close genetic relationship with the P. maculate from Argentina and Brazil. Conclusions P. canaliculata and P. maculata are present in Xihu District of Hangzhou City. P. maculata may spread to Xihu District through multiple introductions or water flow.

Progress of research on biologically invasive medical molluscs in China / 中国血吸虫病防治杂志

Invasion of alien species has increasingly become a major environmental problem that damages human health, economic development and ecological security in China, which has been paid much attention. Until now, there have been 753 invasive species reported in China, and invasive medical molluscs include Pomacea canaliculata, Achatina fulica, and Biomphalaria straminea. This review summarizes the status of biologically invasive species in China, describes the species and distribution of invasive medical molluscs, analyzes the damages of the invasive medical molluscs-transmitted diseases, and proposes the preventive and control measures.

Quantitative analysis of gene expression in Pomacea canaliculata infected with Angiostrongylus cantonensis and α - tubulin gene expression in various tissues / 中国血吸虫病防治杂志

Objective To investigate the expression of some genes in Pomacea canaliculata infected with Angiostrongylus cantonensis, so as to provide insight into the preliminary understanding of the interactions between Angiostrongylus cantonensis and its intermediate host Pomacea canaliculata. Methods P. canaliculata was fed with rat faces containing the first-stage larvae of A. cantonensis. Three to five P. canaliculata was sampled 1, 10 days and 20 days after feeding, and the hemolymph, hepatopancreas, kidney, intestinal tract, head-foot and gill tissues were collected, while uninfected P. canaliculata served as controls. Total RNA was extracted from various tissues of P. canaliculata at different time points post-infection, and transcribed reversely into cDNA. Based on previous transcriptome sequencing results, 10 genes associated with immune defense, signal transduction, cell growth and metabolism, stress response were selected, and the gene expression was determined in the hemolymph tissues of P. canaliculata 1, 10 days and 20 days post-infection with A. cantonensis using real-time fluorescent quantitative PCR assay, and the α-tubulin gene expression was quantified in the hepatopancreas, kidney, head-foot, intestinal tract and gill tissues of P. canaliculata infected with A. cantonensis. Results Higher CELA1 gene expression was detected in the infection group than in the control group 1 (t = 12.32, P < 0.05), 10 days (t = 23.51, P < 0.05) and 20 days post-infection (t = 34.92, P < 0.05), and the CELA1 expression increased with the time of infection. The GST gene expression was (7.26 ± 1.80) times higher in the infection group than in the control group 1 day post-infection, and was significantly lower in the infection group than in the control group 10 days (t = 23.89, P < 0.05) and 20 days post-infection (t = 19.83, P < 0.05). Higher ferritin gene expression was found in the infection group than in the control group 10 days post-infection (t = 32.76, P < 0.05), and higher CRT gene expression was seen in the infection group than in the control group 1 (t = 7.23, P < 0.05), 10 days (t = 5.78, P < 0.05) and 20 days post-infection (t = 6.32, P < 0.05). In addition, the greatest α-tubulin gene expression was observed in the the hepatopancreatic tissues of P. canaliculata (F = 17.58, P < 0.05), and the α-tubulin gene expression altered in various tissues of P. canaliculata post-infection with A. cantonensis, with the most remarkable reduction of α - tubulin gene expression seen in the hepatopancreatic tissues (P < 0.05). Conclusions Following A. cantonensis infection in P. canaliculata, the expression of multiple genes is altered, and the expression of α-tubulin gene is inhibited in multiple tissues. The findings provide a basis for the further elucidation of the interactions between P. canaliculata and A. cantonensis.

Progress of research on biologically invasive medical molluscs in China / 中国血吸虫病防治杂志

Invasion of alien species has increasingly become a major environmental problem that damages human health, economic development and ecological security in China, which has been paid much attention. Until now, there have been 753 invasive species reported in China, and invasive medical molluscs include Pomacea canaliculata, Achatina fulica, and Biomphalaria straminea. This review summarizes the status of biologically invasive species in China, describes the species and distribution of invasive medical molluscs, analyzes the damages of the invasive medical molluscs-transmitted diseases, and proposes the preventive and control measures.

Quantitative analysis of gene expression in Pomacea canaliculata infected with Angiostrongylus cantonensis and α - tubulin gene expression in various tissues / 中国血吸虫病防治杂志

Objective To investigate the expression of some genes in Pomacea canaliculata infected with Angiostrongylus cantonensis, so as to provide insight into the preliminary understanding of the interactions between Angiostrongylus cantonensis and its intermediate host Pomacea canaliculata. Methods P. canaliculata was fed with rat faces containing the first-stage larvae of A. cantonensis. Three to five P. canaliculata was sampled 1, 10 days and 20 days after feeding, and the hemolymph, hepatopancreas, kidney, intestinal tract, head-foot and gill tissues were collected, while uninfected P. canaliculata served as controls. Total RNA was extracted from various tissues of P. canaliculata at different time points post-infection, and transcribed reversely into cDNA. Based on previous transcriptome sequencing results, 10 genes associated with immune defense, signal transduction, cell growth and metabolism, stress response were selected, and the gene expression was determined in the hemolymph tissues of P. canaliculata 1, 10 days and 20 days post-infection with A. cantonensis using real-time fluorescent quantitative PCR assay, and the α-tubulin gene expression was quantified in the hepatopancreas, kidney, head-foot, intestinal tract and gill tissues of P. canaliculata infected with A. cantonensis. Results Higher CELA1 gene expression was detected in the infection group than in the control group 1 (t = 12.32, P < 0.05), 10 days (t = 23.51, P < 0.05) and 20 days post-infection (t = 34.92, P < 0.05), and the CELA1 expression increased with the time of infection. The GST gene expression was (7.26 ± 1.80) times higher in the infection group than in the control group 1 day post-infection, and was significantly lower in the infection group than in the control group 10 days (t = 23.89, P < 0.05) and 20 days post-infection (t = 19.83, P < 0.05). Higher ferritin gene expression was found in the infection group than in the control group 10 days post-infection (t = 32.76, P < 0.05), and higher CRT gene expression was seen in the infection group than in the control group 1 (t = 7.23, P < 0.05), 10 days (t = 5.78, P < 0.05) and 20 days post-infection (t = 6.32, P < 0.05). In addition, the greatest α-tubulin gene expression was observed in the the hepatopancreatic tissues of P. canaliculata (F = 17.58, P < 0.05), and the α-tubulin gene expression altered in various tissues of P. canaliculata post-infection with A. cantonensis, with the most remarkable reduction of α - tubulin gene expression seen in the hepatopancreatic tissues (P < 0.05). Conclusions Following A. cantonensis infection in P. canaliculata, the expression of multiple genes is altered, and the expression of α-tubulin gene is inhibited in multiple tissues. The findings provide a basis for the further elucidation of the interactions between P. canaliculata and A. cantonensis.

Genotyping and polymorphism analysis of cytochrome c oxidase subunit Ⅰgene of Pomacea canaliculata from Lincang City in Yunnan Province / 中国血吸虫病防治杂志

Objective To analyze the genetic diversity of Pomacea canaliculata based on the mitochondria DNA cytochrome c oxidase subunitⅠ(mtDNA COⅠ)gene as a molecular marker in Lincang City of Yunnan Province,so as to provide the scien-tific data for monitoring Angiostrongylus cantonensis in local areas. Methods The genotypes and polymorphisms of 38 speci-mens of P.canaliculata collected from Mengding Town of Lincang City were analyzed by sequencing COⅠgene.The phylogenet-ic tree and genetic distances were produced based on the haplotypes from GenBank and the present study by using the neighbour-joining method with the software MEGA version 6.06. Results Totally 31 sequences were acquired in the present study and they produced 3 unique haplotypes.Haplotype 1 showed a higher frequency compared to the others and it accounted for 83.9 % (26/31).The data showed that the least genetic distances ranged from 0 to 0.052 between P.canaliculata and 3 haplotypes,as well as the largest genetic distances ranged from 0.021 to 0.239 between Pila conica and 3 haplotypes.Otherwise,the analysis of the phylogenetic trees based on COⅠgene sequences of P.canaliculata indicated that all of 3 haplotypes clustered into one big clade with that from Japan(GenBank accession number: AB433769),China(GenBank accession number: KT313034)and USA(GenBank accession number:EU523129),which owned the closet relationship amongst them.Their genetic relationships were distantly related to the GenBank's reference sequences of P.insularum(GenBank accession number:EF514942),P.cam-ena(GenBank accession number: EF515059)and so on. Conclusion There is a P. canaliculata species in Lincang City of Yunnan Province as well as a high genetic diversity amongst the acquired 3 haplotypes in this study.

Observation of animal model of life cycle of Angiostrongylus cantonensis / 中国血吸虫病防治杂志

Objective To observe the effect of artificially construction of the life cycle of Angiostrongylus cantonensis in the laboratory condition so as to provide the basis for the research of angiostrongyliasis. Methods SD rats were infected orally with the third?stage larvae of A. cantonensis collected from infected Pomacea canaliculata. Six weeks after the infection the first?stage larvae were isolated and counted from fresh feces of the rats and then were used to infect P. canaliculata. Three weeks lat?er the snails were dissected for counting the third?staged larvae of A. cantonensis. Results The first?stage larvae were detect?ed in the feces of the rats 6 weeks after the infection and the third?staged larvae were successfully isolated after the infection of P. canaliculata. Conclusion The animal model of the entire life cycle of A. cantonensis is successfully established in the labo?ratory with the infection of 50 larvae per rat.

Comparison on the methods of DNA extraction from lungs of Pomacea canaliculata used in PCR assay / 中国人兽共患病学报

To compare the efficiency of methods of DNA extraction from lungs of Pomacea canaliculataused in PCR assay, 80 P.canaliculata collected in field were divided into 8 groups and the lungs of each snail were separated from the soft body. Six methods of DNA extraction from lungs of P. canaliculata were used to extract DNA from lungs, i.e. With Qiagen, Tiangen,and Omega commercial DNA extraction kits, guanidine thiocyanate method, Chelex 100 resin method and Chelex-silica particle method. The 16S rDNA of C.canaliculata was amplified by PCR and the concentration of PCR-products relative to marker was determined in order to evaluate the efficiency of each method. It was demonstrated that each method was valid to extract DNA from lungs used in PCR assay, but the concentrations of PCR-products were different. The concentrations of PCR-products obtained by Qiangen kit, Omega kit, Chelex 100 resin method and Chelex-silica particle method were significantly higher than those of other 4 methods of DNA extraction, in which Qiangen and Omega kits were suitable for small sample size. In term of efficiency and cost, Chelex 100 method and Chelex-silica particle method were feasible for large sample scale, while the guanidine thiocyanate method was preferred due to its fast extraction and low cost, but on account of its toxicity, it is used in urgent status or in large scale of sample extraction.