Epidemiological Survey Of The Common Protozoans Infection In Yaks And Tibetan Pigs,and Analysis Of The Mitochondrial Genome Of Lungworm Derived From Tibetan Pig

As one of the artiodactyl animals with limited geographical distribution,yak is a unique cattle species in the high plateau region.The yaks as a pillar industry in the plateau region,is of great importance in economic to the local herdsmen.Tibetan pig is a rare local breeding pig in the high remote plateau region and a unique precious miniature pig species in China.However,parasitic disease not only threaten the health of yaks and seriously limitate the development of the industry of yaks and Tibetan pigs.The zoonotic parasites infection in yaks and Tibetan pigs may also threaten the health of human beings.In recent years,diarrhea is frequent occurring in yaks.Diarrhea in animals caused by parasites accounts for 40% of all diarrhea diseases.To master the prevalence of the common protozoans infection in yaks and Tibetan pigs,investigate the parasitic pathogens of diarrhea yaks,uncover the molecular characterization of parasites infection in yaks and Tibetan pigs,reveal the mitochondrial molecular markers of parasites and discover potential drug targets,we preformed researches of investigating of the common protozoans infection in yaks and Tibetan pigs,high-throughput sequencing of fecal samples from diarrhea yaks,mitochondrial genome sequencing of lungworm.The results were as follows: 1.Epidemiological investigation of the common protozoans infection in yak and Tibetan pig on the Qinghai Tibetan plateauSerum samples from yaks?n=3582?and Tibetan pigs?n=454?were obtained from 2011-2017 and were assayed for antibodies against Toxoplasma gondii,Neospora caninum,Theileria,Babesia,Echinococcus granulosus,Besnoitia,and Sarcocystis,respectively by piloting commercial IHA or ELISA kits.Results revealed that the seroprevalence of T.gondii,N.caninum,Theileria,Babesia,E.granulosus,Besnoitia,and Sarcocystis was 25.0%,6.3%,15.1%,21.5%,6.5%,1.1%,and 0.9%,respectively.2.Mitochondrial gene amplification and sequence analysis of five parasites infection in yaks and Tibetan pigsThis research was carried out to uncover the molecular characterization of lungworms,intestinal nematodes,ascarid,hydatid cyst collected from a Tibetan pigs slaughter house in Tibetan and ascarid isolated from yaks in Hongyuan,Sichuan based on methods of gene clone and sequencing.By employing multiple sequence alignment and phylogenetic analysis via DNAMAN and Mega,we found that lungworm infection in Tibetan pigs was demonstrated to be Metastrongylus pudendotectus.M.pudendotectus isolated from Tibetan pigs in Milin,Nyingchi and Gongbo'gyamda revealed high homologous?97.5%-97.7%?to M.pudendotectus?NC₀13813.1?derived from Australian wild boar.Three Oesophagostomum spp?O.quadrispinulatum,O.dentatum and O.stephanostomum?were uncovered in Tibetan pigs and O.stephanostomum was first found in pigs.The prevalenc of Oesophagostomum spp infected in stomach,cecum and colon was 81.13%,66.04%,and 77.36% based on gene markers of nad1,cox3,and ITS1,respectively.Ascaris suum isolated from Tibetan pigs showed high homology?98.4%-99.9%?with A.suum isolated from Japan,99.8% with A.suum isolated from Zhanjiang,99.4% with A.lumbricoides isolated from Linshu,99.5-99.7% with A.lumbricoides isolated from Brazil.Echinococcus granulosus infection in Tibetan pigs were revealed to be genotype of G4 and G6 by utilzing gene markers of cox1 and atp6,respectively.Ascarid infected in yaks were demonstrated to be Toxocara vitulorum,with high homologyof 99.7% with T.vitulorum?AJ937266.1?.3.Revealing the parasitic pathogens of diarrhea yaksIn the current study herein,we obtained fecal samples from healthy adult yaks?HA?,diarrheic adult yaks?DA?and diarrheic yak calves?DC?in Gannan Tibetan Autonomous Prefecture.We preformed gDNA extraction,amplification of 18 S SSU rDNA,and high-throughput sequencing to reveal the parasitic diversity in the 3 groups of yaks.The results showed that,at Phylum level,Nematoda and Apicomplexa were detected in all three yak groups;however,Apicomplexa was found to be conspicuously higher in the DC yaks as compared to DA yaks?p<0.05?.At Class level,Litostomatea,Chromadorea and Gregarinasina were found in all the three yak groups,while Spirotrichea and Colpodea were only found in the DC yaks.At Order level,Vestibuliferida,Tritrichomonadida,Rhabditida and Eugregarinorida were observed in all the three yak groups;and Neogregarinorida was observed in HA and DC yaks while Trichomonadida and Hypotrichomonadida were only found in DA yaks.Tritrichomonadida was found to be relatively higher in the DA yaks when compared with HA yaks?p<0.05?and DC yaks?p<0.05?.At Family level,Simplicimonadidae and Haemonchidae were found in all the three yak groups.Trichostrongylidae was observed in both HA and DA yak groups.Syncystidae was established in both HA and DC yak groups except DA yaks.Trichomonadidae and Dictyocaulidae were only found in DA yaks.Plectidae,Strongylidae,Echinamoebidae,Lecudinidae,Pseudokeronopsidae and Panagrolaimidae were only discovered in DC yaks.Simplicimonadidae was found to be remarkably higher in DA yaks as compared to HA and DC yak groups?p<0.05?,and Haemonchidae was detected at higher levels in HA yaks as compared to DA and DC yaks?p<0.05?.At Genus level,Entamoeba,Buxtonella and Haemonchus were found in all three yak groups.Plectus and Echinamoeba were only found in DC yaks;while Trichostrongylus and Trepomonas were observed in HA and DA yak groups.The genus Gregarina was found in both diarrheic yak groups;while the genera of Tetratrichomonas and Dictyocaulus were observed in DA yaks only.The present study herein reported an insight of the change of parasitic diversity in diarrheic yaks at high altitude area,which shall make contribution towards the solid prevention of diarrhea in yaks.4.Discover the parasitic pathogens in acute diarrhea yak calvesIn the current study herein,we obtained fecal samples from healthy yak calves?H?and diarrhea yak calves?B?in Sichuan,China.We preformed gDNA extraction,amplification of 18 S SSU rDNA,and high-throughput sequencing to reveal the parasitic diversity in the two different yak groups.The results showed that,at Phylum level,Loukozoa,Bryozoa,and Apicomplexa were detected in the two yak groups;however,Apicomplexa was found to be conspicuously higher in group B yaks as compared to group H yaks?p<0.0001?,while Loukozoa was clearly higher in group H yaks?p<0.0001?.At Class level,Trichomonadea,Conoidasida,and Phylactolaemata were found in both yak groups,while Trichomonadea?p<0.001?and Conoidasida?p<0.0001?in group B was signifeicant higher the group H,respectively.Archamoebae,Aconoidasida,Blastocystidea were only discovered in group B,while Hypotrichomonadea was only found in group H.At Class level,both H and B group detected Tritrichomonadida,Eugregarinorida,and Plumatellida,while Tritrichomonadida and Eugregarinorid were much higher in group H?p<0.0001?.Pelobiontida,Piroplasmida,Blastocystida,Adeleorina were only uncovered in group B,while Hypotrichomonadida was only found in group H.At Family level,Simplicimonadidae,Gregarinidae and Plumatellidae were examed in the two groups,while Simplicimonadidae?p<0.0001?and Gregarinidae?p<0.01?in group H were obviously higher.Entamoebidae,Theileriidae,Trichomonadidae,Blastocystidae,Cryptosporidae were only detected in group B,while Hypotrichomonadidae was only found in group H.At Genus level,Simplicimonas and Gregarina were both found in group H and B,while both of the two genuses were much higher in group H?p<0.01?.Entamoeba,Theileria,Tetratrichomonas,Blastocystis,and Cryptosporidium were only discovered in group B,while Hypotrichomonas was only found in group H.5.Analysis the mitochondrial genome of lungworm derived from Tibetan pigsThe current study was carried out to preform mt genome sequencing of lungworm isolated from Tibetan pigs.The results found that the lungworm infected in Tibetan pigs was Metastrongylus salmi.The complete mt genome of M.salmi was 13722 bp,the overall A+T content was found to be 73.54%?Table 2?and the nucleotide composition was A?23.52 %?,C?6.14 %?,G?19.60 %?,T?50.02%?,and N?UCAG??0.73%?.The mt genome of M.salmi contains 12 protein-coding genes?cox1-3,nad1-6,nad4 L,atp6,and cytb?,22 transfer RNA genes,and two ribosomal RNA genes?rrnL and rrnS?,but lacks atp8.The nucleotide compositions of all mt DNA sequences for M.salmi are biased toward A and T,with T being the most common nucleotide and C the least common.The 3-end of genes nad4 L,atp6,nad2,cob,cox3,cox1,cox2,and nad5 are immediately adjacent to a downstream trn gene.A total of 4237 amino acids are encoded by Tibetan isolated M.salmi mt genome.The most common start codon is ATA at 41.7%?5/12 protein genes?,11 protein genes out of 12?91.7%?depicted a TAA or TAG translation termination codon.In protein-coding genes of M.salmi,the 63 possible codons were used;the UUU?Phe?codon is the most frequent whereas the CCC?Pro?codon is the least frequent;however,the CGC codon is not employed.In conclusion,the current research provided insight of the epidemic situation of seven parasites infection in yaks and Tibetan pigs,identified five species of parasites infection in yaks and Tibetan pigs based on molecular markers of mitochondrial,revealed the parasitic pathogens of diarrhea yaks,reported the mitochondrial genome informaton of lungworm infection in Tibetan pig and provided potential drug targets.The present results may make contribution to the prevention and control of parasitic diseases infection in yaks and Tibetan pigs on the high plateau,to promote the vigorous development of animal husbandry in China,and to drop the threat caused by parasitic zoonosis.