| Cystic echinococcosis?CE?is a worldwide zoonosis that is highly prevalent in Asia,South America,Europe and Africa particularly in northern and eastern Africa where data is relatively more available.CE results in huge economic losses due to infected organ condemnation in the livestock industry and the cost of treatment in humans and it is caused mainly by Echinococcus granulosus sensu lato?s.l.?.A complex of species grouped into“genotypes”based on mitochondrial genome study.These genetic differences affect the species/genotype infectivity and preference to intermediate hosts.Therefore,knowledge of the species/genotypes in a range of hosts in a given location is invaluable in understanding and appreciating CE dynamics and could guide towards designing effective control and prevention measures.In this study,the species diversity and genetic variation of E.granulosus?s.l.?in four counties in the Tibet Autonomous Region?TAR?of China were investigated while for Nigeria,the study describes the genotypes/species responsible for CE for the first time.The study also investigated microsatellite markers that might be potentially useful in investigating intra-and interspecies genetic variation.Hydatid cyst samples were collected from different intermediate hosts in various abattoirs and slaughter slabs in the study locations?China and Nigeria?.Also,in some locations?Nigeria?,faecal samples from definitive hosts were examined.Infection with E.granulosus?s.l.?in intermediate hosts was detected and discriminated using mitochondrial?mt?genes?cox1,nad1,nad2 and nad5?and in some cases the complete mt genome sequences.Phylogenetic relationship among species/genotypes/haplotypes was constructed using Bayesian inference Mr Bayes program with a Markov Chain Monte Carlo?MCMC?sampling to assess the posterior distribution of parameters and/or maximum likelihood method in MEGA X software while median-joining networks were constructed in PopART using different mt genes as well as the complete mt genome datasets.Out of the 85 hydatid cyst samples collected from slaughtered sheep?n=54?and yaks?n=31?in TAR,83 were identified as E.granulosus?s.s.?G1?n=77?,G3?n=6?and2 were identified as E.canadensis G6.Analysis of the nad1/nad5 genes revealed 16/17 mutations with9/14 parsimony informative sites resulting in 15/14 haplotypes,respectively.Haplotype diversity?Hd?and nucleotide diversity???of E.granulosus?s.s.?population were 0.650 and 0.00127 for nad1 and 0.782 and 0.00306 for nad5,respectively,with an overall negative Tajima's D and Fu's Fs.A low FST indicated no genetic difference between isolates from sheep and yaks.In Nigeria,the prevalence of CE was investigated among 1,592 intermediate hosts and 58 definitive hosts.The genetic diversity and variation of Echinococcus spp.among the 32 isolates recovered were also investigated.The resulting nucleotide sequences via the NCBI BLAST algorithm and Bayesian phylogeny of the mt genes identified 31/32 of the isolates as E.canadensis?G6/G7?and were 99–100%identical to previously reported G6/G7haplotypes across Europe,Asia,North and East Africa.On analyzing the complete mt genome of representative G6/G7 samples,a total of 13,731 bp in a covalently closed circular molecule were realized.This total mt length was?10 bp than previously reported G6 and G7 mt genome sequences.Furthermore,the median-joining network and maximum likelihood phylogeny of the 12 protein-coding sequences correctly identified them as G6 genotype.On the other hand,The BLAST result of the nad1,nad5,cox1gene sequences confirmed the identity of 1/32 isolate as G1 genotype with a 99-100%similarity with other G1 sequences deposited in GenBank.The median-joining network based on the nad5 gene?680 bp?further distinguished the G1 genotype from the G3 genotype.In an attempt to investigate informative microsatellite markers with the potential to investigate genetic diversity and variation between and within species,the complete genome sequence of E.granulosus?v3?downloaded from the Sanger Institute database was screened for microsatellites using the Tandem Repeats Finder option of the Unipro UGENE v1.29.0 software.Primers were designed at each microsatellite flanking region.Finally,PCR was carried out with a fluorescent-labeled forward primer followed by capillary electrophoresis of PCR products.Sizes and height of the generated profiles were determined by GeneMapper 4.1.Out of the 6 screened microsatellite,two,EgSca6-II?GATA?and EgSca7-II?GCATG?showed proper amplification and gave a total of 6 and 12 alleles 157-198 bp and208-262 bp,respectively producing 5 and 19 genotypes/profile.However,the polymorphism information content?PIC?within the different species/genotypes of Echinococcus was very low?0.208 for the microsatellite EgSca6-II with an overall PIC of 0.679,and 0.295-0.660 for EgSca7-II,with an overall PIC of 0.811.Also,similar profiles were found in different genotype/species.The results demonstrated that the selected marker is not suitable for species/genotype identification.In conclusion,the study reports a demographically expanding population of the E.granulosus?s.s.?complex in TAR,China.While pockets of infection with E.canadensis?G6,G7,G8 and G10?have been previously reported in sheep,goats,yaks and humans in different parts of China.The G6 genotype has only been reported in sheep in TAR.The detection in a yak in this study represents the first in TAR to the best of our knowledge which suggests that future surveys and control efforts comprehensively investigate other potential intermediate hosts for the prevalence and genetic diversity of E.canadensis group?G6,G7,G8 and G10?across the country and their role in human infection in order to target them during CE control program.Furthermore,the G1 genotype believed to be responsible for the majority of CE burden globally was otherwise not responsible for the majority of CE in Nigeria which agrees with previous reports suggesting that E.canadensis G6 genotype could be the major causative agent of CE in the West Africa subregion.This study provides for the first time,insight into the genetic population structure of Echinococcus species as well as implications for CE control in Nigeria and identified the abundant genotype as the G6 genotype.Also,since longer mt genome datasets have shown some advantage over individual genes,the study provided the complete mt genome of representative G6 genotype from different Nigerian intermediate hosts which will be useful in future genetic population studies across the country and the West African sub-region.Additionally,the detection of a single G1 genotype in Nigeria strengthens the need for further investigation into the genetic diversity of Echinococcus spp.in Nigeria and across the West African sub-region where the G6 genotype is abundant,suggesting that the epidemiology of this highly zoonotic species?G1?could have a wider host range and distribution in the sub-region than previously thought. |
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