| ObjectivesThe density and infection status of Angiostronglyus cantonensis in Pomacea canaliculata,Achatina fulica and rats from Nan'ao Island of Guangdong Province were surveyed and the spatial distribution characteristics and rules of hosts were analyzed on small scale.The applicability of spatial analysis technology and spatial modeling technique in the spatial studies of the hosts of Angiost onglyus cantonensis were explored.The genetic diversity of Angiostrongylus cantonensis from Nan'ao Island and the phylogenetic relationship of Angiostrongylus cantonensis from other places in china were analyzed with the aim of exploring the type of geographic strain and its source.So it could provide methodology reference and theoretical basis for the prevention and control of the hosts of Angiostronglyus cantonensis.Methods1.Three villages were selected as the study areas from Nan'ao Island based on the stratified random sampling method.The density and natural infection status of Angiostrongylus cantonensis in Pomacea canaliculata and Achatina fulica as intermediate hosts and rats as definitive hosts were surveyed in every quarterly from December of 2015 to September of 2016.Pomacea canaliculata collected from Nan'ao Island were examined for the third stage larvae by lung-microscopy,tissue homogenate and enzyme digestion method,and Achatina fulica were examined by enzyme digestion method.Rats,which were captured by cage-trap method,were drowned and dissected,and then its hearts and lungs were checked for the infection status of adult worms after species identification.In addition,the geographical coordinates(longitude and latitude)of each sampling site was also recorded by GPS map 60CS(Garmin Corp.,Kansas,USA).Some geographic environmental data,such as season,environmental types,distance between residential areas and water areas,were also collected.2.Using ArcGIS10.2(ESRI Corp.,USA)and its spatial analysis technology to analyze the spatial correlation of infection rates of Pomacea canaliculata and rats on small scale by establishing semivariogram model.In addition,SaTScan9.4(NCI,USA)was used to detect the spatial aggregation of the infection rate,which was based on Possion model.Monte Carlo simulated method was used to construct the statistics of Likelihood Ratio(LLR)and to judge if they were statistically significant.Ordinary Least Squares regression model(OLS)and Geographically Weighted Regression model(GWR)were constructed by geostatistical analyst tools in ArcGIS10.2(ESRI Corp.,USA).The spatial distribution characteristics and the influence factors of infection rates of Angiostrongylus cantonensis in Pomacea canaliculata and rats were analyzed,and the fitting degree of the models was compared.3.Rats,which were captured from Nan'ao Island,were dissected and its hearts and lungs were checked for adult worms.Meanwhile,Pomacea canaliculata and Achatina fulica which were collected from fields were examined for the third stage larvae,and then adult worms were collected from SD rats which were infected by the third stage larvae after 6 weeks.To extract genomic DNA and amplify internal transcribed spacer 2(ITS2)and cytochrome c oxidase subunit I(COI)gene sequence by PCR.Sequences were aligned by Clustal X1.83 and the features of the sequences and genetic diversity was analysed by DnaSP5.10.The sequences of Angiostrongylus cantonensis from other places in china were obtained from GenBank and published reports.To count the genetic distances of Angiostrongylus cantonensis from different places by Mega6.06.In addition,phylogenetic trees were constructed by Neighbor Joining method and Maximum Likelihood method to analyze the phylogenetic relationship of Angiostrongylus cantonensis from different places in china.Results1.Totally 2192 Pomacea canaliculata were gathered from fields and 1190 were randomly chosen to be examined for the third stage larvae of Angiostrongylus cantonensis,and 72 positive individual were found,with a larva infection rate of 6.05%(72/1190).Totally 24 Achatina fulica was collected and 20 positive individual were found,with a larva infection rate of 83.33%(20/24).Totally 110 rats including 85 Rattus norvegicus,10 Rattus flavipectus,1 Rattus losea and 14 Suncus murinus were captured and 32 positive individual were found,with an average infection rate of 29.09%(32/110).Adult worms were found in Rattus norvegicus and Rattus favipectus with a prevalence of 36.47%(31/85)and 10%(1/10),but adult worms were not found in Rattus losea and Suncus murinus.There was a significant difference between different species of rats(Fisher's ExactTest,P=0.0051).There was a significant difference between different reasons in density(P=0.0089)and infection rate(P<0.0001)of Pomacea canaliculata,and there was also a significant difference between different reasons in density of rats(P=0.0002).Pomacea canaliculata which was living in irrigation canals and ditches and rats which were captured in reclamation depot had the highest infection rate.There was a significant difference of infection rate of rats in different breeding environments.The infection rate of Pomacea canaliculata and rats were closely related to the distance from breeding grounds to residential areas,and it revealed that the shorter the distance was,the higher the infection rate grew.2.In the semivariance model for infection rate of Pomacea canaliculated,the value of Nugget,Partial Sill and Sill were 0.008833,0.014418 and 0.023251.The ratio of Nugget to Sill was 37.99%and the ratio of Partial Sill to Sill was 62.01%.In the semivariance model for infection rate of rats,the value of Nugget,Partial Sill and Sill were 0.032829,0.093567 and 0.093567,and the ratio of Nugget to Sill was 25.97%and the ratio of Partial Sill to Sill was 74.03%.Both of them showed that the spatial heterogeneity was mainly caused by spatial autocorrelation.9 spatial clusters areas and 2 spatial cluster areas were detected in the distribution of positive Pomacea canaliculata and positive rats,and the maximum radius of accumulation areas in the infection rates of Pomacea canaliculated and rats were 0.049 km and 0.069 km.The positive Pomacea canaliculata cluster areas was mostly in artificial channels near villages and positive rats are more cluster in the places where environment was messy,such as rubbish heap and recycling center.To make a compare between OLS mode and GWR model,the compare results showed AICC?R2?R2 adjusted and ?2 in GWR model were superior to it in OLS model.3.The amplified part of ITS2 and COI gene of Angiostrongylus cantonensis were 693 bp and 1174 bp in length.The ration between variable sites and conservative sites were 4.7%and 1.0%.The values of haplotype diversity index,nucleotide diversity and average number of nucleotide difference were 0.927,0.007 and 4.549 in ITS2.Haplotype diversity index,nucleotide diversity and average number of nucleotide difference were 0.440,0.004 and 3.743 in COI.4.The genetic distance based on ITS2 sequence of Angiostrongylus cantonensis from some regions of china was 0.000?0.797 and the genetic distance based on ITS2 sequence was 0.003?0.051.Phylogenetic trees based on ITS2 showed the isolates of Angiostrongylus cantonensis from Shenzheng,Qingyuan,Jiangmen,Puer,Naning,Qiongzhong and Wenzhouwere clustered into one branch,while isolates from Fuqing and Taiwan were clustered into other two branchs.Phylogenetic trees based on COI showed the isolates of Angiostrongylus cantonensis from Nan'ao Island were clustered into one branch with isolates from Yulin and Nanning,while isolates from Wenzhou and Taiwan were clustered into other two branchs.5.There was of difference between the sequences of Angiostrongylus cantonensis from the rats which were captured from Nan'ao with the sequences of Angiostrongylus cantonensis from SD rats which were inoculated by positive Pomacea canaliculata and Achatina fulica,which were colleated from Nan'ao Island.Conclusions1.There was a risk of Angiostrongyliasis in Nan'ao Island of Guangdong Province as the intermediate hosts and definitive hosts of Pomacea canaliculata were widely distributed in there and positive infection has been found in the hosts,so more monitoring work about the hosts of Pomacea canaliculata should be taken in there.2.There existed spatial correlation and spatial clusters in the spatial distribution of positive Pomacea canaliculated and rats.The maximum radius of spatial clustersareas of positive rats was basicallyconsistent with the rats' sphere of activity.GWR model had advantage over OLS model in the spatial analysis of hosts of Angiostrongylus cantonensis.3.There existed different geographical strains or sub-strain of Angiostrongylus cantonensis in china and it maight have three strains or sub-strain in china:Fuqing strains,Wenzhou strains and Taiwan strains.4.There existed some genetic differentiation and genetic diversity within the population of Angiostrongylus cantonensis in Nan'ao Island.It maight have various types and sources in Angiostrongylus cantonensis from Nan'ao Island,but the population of Angiostrongylus cantonensis in Nan'ao Island still follow neutral theoryover the course of evolution.5.There were of difference between Angiostrongylus cantonensisfrom rats which were captured from Nan'ao with Angiostrongylus cantonensis from SD rats inoculated by positive snails which werer collectded from Nan'ao,and it maight have choose for different hosts. |
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