| As a special local animals living mainly by grass,Tibetan pigs have become the main food and economic source of the Tibetan Plateau herdsmen because of their excellent adaptability to extreme weather such as cold and hypoxia in high altitude areas,which make them very indispensable.Tibetan pigs have been suffering various diseases in recent years,especially parasitic diseases due to the free grazing and poor management model,unreasonable herd structure,the degradation of Tibetan pigs' breeds and the weak awareness of disease prevention and control for residents.The most common parasites are Ascaris and Cysticercus tenicollis that not only can impact on the growth and production of Tibetan pigs seriously,but can bring huge economic losses and improve the risk of parasitic infection.In this study,the prevalence of parasitic diseases in Tibetan pigs,the isolation and identification of Ascaris and Cysticercus tenicollis,and the mitochondrial genomics were investigated.The results were as follows: 1.Epidemiology of parasitic parasites in Tibetan Pigs in Nyingchi,TibetA total 112 of Tibetan pigs from slaughter house and 73 faecal samples were examined from Tibet by complete parasitological necropsy method and parasitological stool examination.The results showed that eleven species of parasites were detected which belong to 5 phylums,6 classes,9 orders,10 families and 10 genus,including 5 nematodes,2 proscolex,2 protozoa,1 trematode and 1 acanthocephalan.The predominant helminths were Cysticercus tenuicollis(42.9%),Metasrongylus apri(38.4%),Hydatid cyst(33.0%),Ascaris suum(30.4%),Fasciola hepatica(26.8%),Oesophagostomum sp.(18.8%),Trichocephalus suis(15.2%),Eimeria suis(15.1%),Balantidium coli(6.8%),M.hirudinaceus(5.6%)and Physocephalus sexalatus(2.7%),respectively.The present survey indicated that intestinal parasite infections were highly prevalent in Tibetan pigs of Tibet.2.Amplification and sequence analysis of nad1,cox1 and cox2 genes of Ascaris in Tibetan PigsThe current study was designed to determine the nematode by the genetic characterization of three mitochondrial(mt)genes namely NADH dehydrogenase subunit 1(nad1),cytochrome oxidase subunit 1(cox1)and cytochrome oxidase subunit 2(cox2).The nad1,cox1 and cox2 genes sequences collected from adult Ascaris individuals were amplified by polymerase chain reaction.The cloned-amplicons and the positive products were sequenced and phylogenetic analysis was performed.The results indicated that the sequences similarities of three genes were all 99% and the Ascaris infecting the Tibetan pigs were Ascaris suum(A.suum).This is the first report on the isolation,identification and genetic characterization of three mitochondrial genomes(nad1,cox1,and cox2)of A.suum originated from Tibetan pigs at high altitudes in Tibet.3 Epidemiological investigation and sequence analysis of cox2 gene of Cysticercus tenicollisThe aim of this study was to investigate the prevalence and phylogenetic analysis of Cysticercus tenicollis in Tibetan pigs.For this purpose,PCR amplification and sequence analysis was performed on cox2 gene of Cysticercus tenicollis,for the first time.The overall prevalence of Cysticercus tenicollis was 43.93% in Tibetan pigs,with 42.86% in 2014 and 45.35% in 2015.In Tibetan male and female pigs,the prevalence of Cysticercus tenicollis was 43.39% and 44.56%,respectively.The prevalence of Cysticercus tenicollis in different growing stage(juveniles,sub adults and adults)varied from 30.20% to 63.79%.The phylogenetic analysis of the Cysticercus tenicollis isolates showed very close resemblance to 16 reference isolates from Gansu,Qinghai and Sichuan province of China.To the best of our knowledge,this is the first report on the prevalence and genetic characterization of Cysticercus tenicollis derived from Tibetan pigs.The data of present study provide baseline information for controlling Cysticerci infections in pigs in Tibetan Plateau,China.4.Analysis of Mitochondrial Genome Sequences of Ascaris and Cysticercus tenicollisThe present study therefore was designed to determine and analyze the mt genomes of ascaris suum and Cysticercus tenuicollis,and to reveal the phylogenetic relationships of these commonly parasites using mt DNA sequences.Results showed that the complete mt genomes of ascaris suum was 14128 bp,containing 12 protein-coding genes(cox1-3,nad1-6,nad4 L,atp6 and cytb),35 transfer RNA genes,two ribosomal RNA genes(rrnL and rrnS).The complete mt genomes of Cysticercus tenuicollis was 13607 bp,containing 12 protein-coding genes(cox1-3,nad1-6,nad4 L,atp6 and cytb),22 transfer RNA genes,two ribosomal RNA genes(rrnL and rrnS).The overall A+T content are significant higher than C+G,with 71.74% and 70.90 in ascaris suum and Cysticercus tenuicollis,respectively.A total of 4387 and 4194 codons are found by the ascaris suum and Cysticercus tenuicollis,respectively.The most common condon is TTT,with 15.51% and 10.44% in ascaris suum and Cysticercus tenuicollis,respectively.The least use of codons in ascaris suum are CAA(0.05%)and CGC(0.05%),while CGC(0.05%)is the least use of codons in Cysticercus tenuicollis.The three highest encoded amino acids are Phe(17.13%),Leu(16.03%),Val(10.86%)in ascaris suum,while three most in Cysticercus tenuicollis are Leu(16.48%),Phe(11.76%),Val(11.06%).The least are Gln(0.73%),His(0.62%)in ascaris suum and Gln(1.29%),Trp(1.67%),Ala(1.81%)in Cysticercus tenuicollis.The phylogenetic analysis indicated that the identity of ascaris suum isolated from Tibetan pigs were 99% with Ascaris lumbricoides(NC-016198.1 and X54253.1);98% with ascaris suum isolated in Zhanjiang,China(HQ704901.1).The current results reveal that the high homology between Ascaris suum and Ascaris lumbricoides is in line with previous results,which demonstrate that the two parasites is the same species.The identity of Cysticercus tenuicollis isolated from Tibetan pigs are 99% with previous reported isolates(GQ228819.1,Isolated from Gansu,China).These data may help for the control of ascaris suum and Cysticercus tenuicolli,especially on the Qinghai Tibetan plateau. |
PDF Full Text Request