| Cystic echinococcosis(CE)and alveolar echinococcosis(AE)are the two common forms of the diseases caused by the metacestode larval stage of Echinococcus species.The disease has a significant impact on livestock and public health and is globally distributed,except in Antarctica.Various carnivores are definitive hosts for the adult stage,while domestic and wild mammals act as intermediate hosts in the general life cycle.In regions with diverse host assemblages and transmission cycles,the differences in host specificity across genotypes and species of Echinococcus are unquestionably significant.Mitochondrial DNA(mt DNA)is the most effective genetic marker for analyzing genetic diversity,genetic differentiation,and the evolution of species.In this study,three mt DNA markers were employed to investigate the genetic diversity and population structure of Echinococcus species in sheep slaughtered in three different regions of western China,as well as the species diversity and genetic variation of Echinococcus species from canines in two different counties in Gansu Province,China.Echinococcus cysts were collected from sheep from slaughterhouses in Gansu province(n = 354),Tibet Autonomous Region(TAR,n = 154),and Xinjiang Uygur Autonomous Region(XUAR,n = 160)in north-and south-west China during routine animal CE surveys from 2019 to 2021.Additionally,a total of 115 faecal samples,comprising 90 dogs and 25 foxes from two counties of Gansu Province,were examined.Total genomic DNA was extracted using the TIANamp Genomic DNA Kit following the manufacturer’s protocols.Infection with E.granulosus sensu lato(s.l.)in intermediate hosts was detected and discriminated using mt genes(cox1,nad1,and nad5).Dna SP v6 was used to compute the population diversity and neutrality indices.Median-joining networks using population analysis with reticulate trees(Pop ART)were constructed to analyze intraspecific phylogenies based on nucleotide sequences of mt genes and their concatenated sequences according to genotypes and locations.The phylogenetic relationship among species,genotypes and haplotypes was constructed using the maximum likelihood(ML)method in MEGA 7 software and/or Bayesian inference in the Mr Bayes program with Markove Chain Monte Carlo(MCMC)sampling to assess the posterior distribution of parameters.Of the total isolates,317,322,and 326 were successfully amplified and sequenced for the cox1,nad1,and nad5 genes,respectively.BLAST analysis revealed that the majority of the isolates were E.granulosus sensu stricto(s.s.),and using the cox1,nad1,and nad5 genes,respectively,17,14,and 11 isolates corresponded to E.canadensis(genotype G6/G7).In the three study areas,G1 genotypes were the most prevalent.There were 233 mutation sites along with 129 parsimony-informative sites.A transition/transversion ratio of 7.5,8.0,and 3.25,respectively,for cox1,nad1,and nad5 genes was obtained.Every mitochondrial gene had intraspecific variations,which were represented in a star-like network with a major haplotype and observable mutations from other distant and minor haplotypes.The Tajima’s D and Fu’s Fs values were significantly negative in all populations,indicating a substantial divergence from neutrality and supporting the demographic expansion of E.granulosus s.s.in the study areas.The phylogeny inferred by the ML method using the nucleotide sequences of cox1–nad1–nad5 further confirmed their identities.The nodes assigned to the G1,G3,and G6 clades,as well as the reference sequences utilized,had maximal posterior probability values of 1.00.Interestingly,during the routine investigation of cystic echinococcosis(CE)in Gansu Province,China,an unusual finding involving E.multilocularis was observed in sheep.Using the nad1(584 bp)gene,the isolates displayed ≥ 98.87% similarity to reference sequences deposited in Gen Bank.In furthering this course,twelve protein-coding region primer pairs were utilized for the analysis.Utilizing these genes,at least three genes and as many as nine genes were successfully amplified and sequenced for a single sample.In the meantime,the current isolates were correctly identified as E.multilocularis by the concatenated sequences of nad4–nad2(2,089 bp)and nad5–cox3–cob–nad4L–nad4–nad2–nad1–nad3–nad6(7,266 bp),which revealed 99.30%similarity with the reference sequence.Three nuclear genes,pepck(1,545 bp),elp-exons VII and VIII(566 bp),and elp-exon IX(256 bp),were successfully amplified,and the resulting sequence in one of the samples confirmed infection with E.multilocularis with a≥ 98.42% similarity with reference gene sequences.The Bayesian phylogeny based on sequences of nad1,nad2 and cob confirmed that all of the present isolates of E.multilocularis were in the same clade as other E.multilocularis from different hosts and countries retrieved from Gen Bank.On the other hand,DNA sequences of E.granulosus(n = 25),E.multilocularis(n = 3),and E.shiquicus(n = 18)were detected in canine fecal samples by copro-PCR analysis and were ≥ 97% identical to the reference sequences.Echinococcus granulosus and E.shiquicus were the main Echinococcus species detected in dog and fox feces,respectively.In conclusion,the present study confirms the existence of a significant major haplotype of E.granulosus s.s.and,to some extent,E.canadensis(G6),where G1 is the predominant genotype.The result of the detection of E.multilocularis in an unusual intermediate host clearly demonstrated the necessity for expanded surveillance in the area.It is also recommended to maintain continuous and intensive epidemiological monitoring to clarify the true infection rate of canine echinococcosis in various regions of China. |
