Genetic Diversity Of Cysticercus Tenuicollis Isolated From Sheep And Goats In China And Mongolia Based On Mitochondrial Nad4 And Nad5 Genes

One of the major difficulties facing livestock production around the world is a parasitic infestation.Taenia hydatigena is the cause of the non-zoonotic disease of cysticercosis tenuicollis.Through their feces,infected canines release eggs,and the eggs then infect animals through food and water.Then,infected animals,particularly sheep and goats,evolve into the metacestode or larval stage(Cysticercus tenuicollis)in the peritoneum,mesentery,liver,and other locations.Both domesticated animals and wild ruminants are frequently infected with C.tenuicollis.There are,however,limited details on the genetic differentiation of this cestode.The main aim of this study was to identify the mitochondrial DNA differences or genetic diversity of isolates from sheep and goats in China and Mongolia based on nucleotide sequences of the mitochondrial genes of NADH dehydrogenase subunits 4(nad4)and 5(nad5)and the recently described PCR-RFLP method for species and haplotype identification and to provide theoretical basis and data support for the epidemiological survey,control and so on of the disease.A total of 114 cysticerci samples(isolates)were collected from sheep and goats from China.Moreover,from the Modern Slaughterhouses in Erenhot city,Inner Mongolia,69 cysticerci were also procured from Mongolian sheep as gifts during routine veterinary meat inspections.parasites were collected from the omentum,liver,and lung of the intermediate hosts.Genomic DNA was extracted from each sample,and the nad4 and nad5 genes were amplified with PCR.At last,nad4 and nad5 amplicons respectively from 183 and 156 isolates were successfully sequenced.Generated nucleotide sequences were edited and aligned using the Bioedit v7.2.6 and/or Unipro UGENE v1.29.0 software.Using the NCBI BLAST algorithm,each isolate’s identity was verified using its nucleotide sequence in the Gen Bank database.One hundred eighty-three and one hundred-fifth six samples for nad4 and nad5 mitochondrial genes,respectively.The results of Chinese isolates showed that haplotype diversity(Hd)and nucleotide diversity(π)were as follows: nad4(Hd = 0.996,π = 0.013),nad5(Hd = 0.989,π = 0.009)and nad4+nad5(Hd = 0.998,π = 0.010).Populations of T.hydatigena from sheep and goats had negative Tajima’s D results for all sequences.Fu’s Fs was negative for the genes of all isolates from sheep and goats indicating the presence of rare haplotypes coming from a recent population expansion.The concatenated gene sequences(nad4+nad5: 2,823 bp)also revealed that Tajima’s D(-2.050)was negative which is statistically significant(P < 0.05).which further proves population expansion.The remarkably negative Fu’s Fs(-32.626,P < 0.02)calculated for the nad4+nad5 sequences reveals the presence of haplotypes for isolates of sheep and goats in China.Similarly,the results of Mongolian isolates showed that haplotype diversity(Hd)and nucleotide diversity(π)values were as follows: nad4(Hd = 0.994,π = 0.011),nad5(Hd = 0.991,π = 0.007)and nad4+nad5(Hd = 0.996,π = 0.008).The population of T.hydatigena from sheep showed negative Tajima’s D results for all sequences.Fu’s Fs was negative for all genes isolates from sheep indicating the presence of rare haplotypes expected from a recent population expansion.The concatenated gene sequences(nad4+nad5: 2,823 bp)were analyzed,and it was found that Tajima’s D(-2.337)was negative for the nad4+nad5 sequences(P < 0.05),further proving the population expansion.The remarkably negative Fu’s Fs(-32.626)calculated for the nad4+nad5 sequences reveals the presence of haplotypes for isolates of sheep in Mongolia(P < 0.01).The PCR-RFLP approach of the nad5 showed nine mitochondrial sub-clusters A1,A2,A3,A5,A8,A9,A10,A11 and B of T.hydatigena isolates from sheep and goats from Mongolia and China.Meanwhile,the haplogroup A1 RFLP profile was more widespread than other variants.It was inferred in this study that the genetic variants of T.hydatigena are being undoubtedly spreading mostly in different parts of China such as Tibet Autonomous Region,Jiangxi and Gansu provinces.In conclusion,analysis of the mitochondrial loci demonstrates the similar genetic composition of C.tenuicollis in sheep and goats in both countries compare to other geographical locations suggesting that animal movement and migration between the border countries contribute to the existing genetic population diversity.Therefore,it is important to dispose of contaminated viscera,restrict dog movement and manage the slaughterhouses properly for the prevention and control of cysticercosis.These data supplement existing information on the molecular epidemiology of T.hydatigena.This study also offers theoretical basis and data support for epidemiological investigations and prevention of C.tenuicollis infections in animals through analysis of genetic variation and diversity of nucleotide sequences of mitochondrial genes of C.tenuicollis isolates from sheep and goats in China and Mongolia,and the PCR-RFLP method for identification of their haplotypes or haplogroups.