| Human sparganosis is a neglected food-borne parasitic disease caused by the larval forms?procercoid/plerocercoid?of the species Spirometra.Frogs,as the second intermediate host in the life cycle of Spirometra,play an important role in the spread of the disease.Humans can be infected through the consumption of raw or undercooked frog meat or by using raw frog flesh in traditional poultices.In recent years,human sparganosis has increased in China.Although with medical important,our knowledge about the prevalence of sparganum infection in frogs remains fragmentary,the taxonomic identification of the parasite and the systematics of spirometrid cestodes has long been controversial.In this study,we firstly surveyed the prevalence of sparganum infection in frogs in 145geographical locations from 28 of the 34 provinces/autonomous regions/municipalities in China;Then,the collected sparganum isolates from the different locations were subjected to molecular identification by a multiplex PCR assay and clustering analysis;Next,one sparganum sample was used to infect a cat to obtain an adult worm,and the taxonomic identification was confirmed based on the morphological characteristics.In the second,all publicly available cox1 sequences available in the Gen Bank and 127 new sequencing genes from China were used as the dataset.The haplotype identify,network,genetic differentiation and phylogenetic analysis were conducted successively,to determine the current knowledge on the evolution and genetic structure of Spirometra tapeworms.Materials and Methods1.Sample collection and examination of frogs:From July 2013 to September 2018,we surveyed frogs for sparganum infection in 28 of the 34 administrative regions in China.The frogs were caught in paddy fields or other wild environments,and then the collected frogs were euthanized using ethyl-ether anaesthesia,weighed and skinned.The presence of spargana in the skeletal muscles was carefully observed visually.The number of identified spargana,parasitizing sites,infection intensity and length of each sparganum were counted and measured.The measurements are expressed as ranges,with means followed by SDs.2.Multiplex PCR and sequencing analysis:In total,72 spargana were used for the molecular identification.The multiplex PCR assay was performed using two sets of species-specific primers,Se/Sd-1800F+Se-2018R plus Se/Sd-7955F+Se-8356R,and Se/Sd-1800F+Sd-2317R plus Se/Sd-7955F+Sd-8567R.The amplified PCR products were sequenced to confirm the results of electrophoretic analysis.The clustering analysis was performed with two methods:maximum likelihood?ML?and Bayesian inference?BI?.3.Morphological analysis:A 6-month-old female domestic cat was used as the definitive host.A single plerocercoid from a positive frog?Pelophylax nigromaculatus?was orally fed to the cat.The amount of discharged eggs was counted using the Kato-Katz technique.After 40 days of infection,the cat was preanaesthetized and then euthanized.And the adult worm was removed alive,and washed in saline solution.The collected adult worm was pressed under a glass plate and fixed with AFA overnight for alum-carmine staining.The vaginal opening,uterus,uterine pore,genital pore and testes of mature and gravid proglottids were observed.The morphologic data were compared to previously published data of other Spirometra species.4.Genetic diversity analysis:To perform a worldwide genetic variable comparative analysis of Spirometra tapeworms,all publicly available cox1 sequences available in the Gen Bank and 127 new sequencing genes from China were used as the dataset.The variable sites,nucleotide compositions and pairwise distances were also estimated in MEGA v.6.06.The number of haplotypes,haplotype diversity?Hd?,and nucleotide diversity?Pi?were analysed with Dna SP v.6.Network v.5.0 was employed to draw a median-joining network to explore the relationships among the detected haplotypes.To explore the levels of genetic differentiation among the geographical populations,the pairwise FST values between populations were calculated by using Arlequin v.3.5.5.Phylogenetic analysis:The phylogenetic pattern of all cox1 haplotypes was estimated through maximum likelihood?ML?and Bayesian inference?BI?.The ML analysis was performed in MEGA v.6.06.BI was performed in Mr Bayes v.3.2.The neutrality tests using Tajima's D and Fu's FS were also applied through Arlequin v.3.5 as an assessment of possible population expansion.In addition,to estimate changes in population size over time,a Bayesian Skyline Plot analysis?BSP?implemented in BEAST v1.8.2 was performed.Results1.Prevalence of sparganum infection in wild frogs:A total of 4665 frogs belonging to13 species were collected.Sparganum infections were found in 8 species?Pelophylax nigromaculatus,Pelophylax plancyi,Fejervarya limnocharis,Sylvirana latouchii,Boulengerana guentheri,Quasipaa spinosa,Odorrana margaretae,Hoplobatrachus chinensis?.In general,Spirometra spargana were found in 9.58%?447/4665?of all the examined frogs.Among the 8 positive species,the highest infection rate was found in Pelophylax nigromaculatus?14.07%?.The highest mean infection intensity of sparganum in infected frogs was also found in P.nigromaculatus?4.27±3.02?.The prevalence of sparganum infection in frogs ranged from 0 to 66.67%,with an infection intensity of 1-49 spargana per frog in different geographical locations.Most of the spargana were present in the thigh muscles of the frogs.The levels of sparganum infections in frogs in South and Southwest China were higher than those in Central and East China.However,no infected frog has been found in Northeast China.2.Molecular identification:All the sparganum isolates collected from the 72 different geographical locations in China revealed two specific bands?540 bp and 644 bp?.The clustering analysis of the sequenced PCR products showed two phylogenetic patterns:most of the isolates from South and Southwest China formed one clade with high support value,and isolates from Central and East China formed the other clade.The Chinese isolates had a close relationship with S.erinaceieuropaei.3.Morphological identification:The eggs were light brown or grey in colour and measured 50-65?m by 30-45?m in transverse diameter.After the first detection,the amount of discharged eggs continued to increase until reaching a stationary phase on the 19th day after infection,with an average of 70,425 eggs per gram of stool per day.The scolex of the living worm was capable of substantial extension and contraction.The neck between the scolex and the first segment was slender and had no proglottids.In the mature proglottid,the excretory canals?ECs?,genital pouch?GP?,and uterus were conspicuous.The uterus of the gravid proglottid was coiled 4-6 times with a ball-shaped terminus.There were three genital pores:the anterior-most cirrus pore?CP?,the next vaginal pore?VP?and the last uterine pore?UP?.The two anterior pores were united and formed the genital pouch,which was situated on the midline in the anterior 1/5 of the gravid proglottid.Through a morphological comparison of features among S.erinaceieuropaei,S.decipiens,S.ranarum and the Chinese isolate,the Chinese isolate was more similar to the S.erinaceieuropaei in morphology.4.Haplotypic variability:A total of 45 haplotypes?Haps?were identified from these488 sequences.All samples had high haplotype diversity?0.652±0.023?accompanied by low nucleotide diversity?0.01619±0.00156?.The median-joining network analysis showed that Hap12 was the most prominent haplotype represent 55.3%of all sequences and originated from China,Thailand,Japan,Australia,Korea and Iran.Most of the Fst values between American countries?Brazil,Argentina,Venezuela and USA?and Asian countries+Australia,American and African countries,African countries and Asian countries+Australia were very high?>0.5?.5.Phylogeny:The genetic analysis revealed that there are four clades of spirometrid cestodes:Clade 1?Brazil+USA?and Clade 2?Argentina+Venezuela?included isolates from America,Clade 3 contained African isolates and one Korean sample,and the remainders from Asia and Australia belonged to Clade 4;unclassified Spirometra from America and Africa should be considered the separate species within the genus;and the taxonomy of two Korea isolates?S.erinaceieuropaei KJ599680 and S.decipiens KJ599679?was still ambiguous and needs to be further identified.In addition,the demographical analyses supported population expansion for the total spirometrid population.Conclusion1.The sparganum infection rates in wild frogs in several regions of China were still high,especially in South and Southwest China.Eating wild frogs especially with improper cooking methods,is associated with considerable health risks in China.2.The sparganum isolates from China were more likely the species of S.erinaceieuropaei.3.The molecular identification suggested that the taxonomic positions of S.decipiens?KJ599679?and S.erinaceieuropaei?KJ599680?are controversial.4.The Spirometra tapeworms had high haplotype diversity.5.There are four clades of spirometrid cestodes:Brazil+USA clade,Argentina+Venezuela clade,Africa clade and Asia+Australia clade. |
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