Moleular Identincation Of Roundworms From Eight Wild Animals, Expression Of Antigen Genes From Baylisascaris Schroderi And Ascaris Lumbricoides And Immunization With Recombinant Antigens Induces Protection Against Roundworms After Vaccination In Mice

Wild animals are valuable natural resources,the protection of wild animals is the common responsibility of all mankind.Giant pandas,chimpanzees and gibbons are the world endangered species,Ailurus fulgens,Ursus maritimus,Ursus arctos pruinosus,Ursus thibetanus mupinensis and Ursus arctos lasiotus are listed in AppendicesⅡof china's national list for specially protected wild animals.Ascarid,the most prevalent of the intestinal helminth infections that colonize the small intestine,has been reported in many wild animals(Ailuropoda melanoleuca,Ailurusfulgens,Ursus maritimus,Ursus arctos pruinosus,etc.).This ubiquitous nematode is a particular problem for wild and captive populations.The ascarids may enter some of the pipelines linked to the intestines,including the pancreatic tube and bile duct.This may cause intestinal obstruction,inflammation,and even death.To effectively control parasitic diseases,it is necessary to identify types of parasites.Identification of the traditional types of parasites was mainly based primarily on morphological characteristics.But the traditional methods of morphological classification was difficult to accurately reflect the real situation in Ascaris species.The ascarids remain a major health problem worldwide and the feasible strategies for treatment are limited. In this paper,the second internal transcribed spacer sequences of the ribosomal DNA (ITS-2) and the mitochondrial gene NADH dehydrogenase subunit 1(ND1) were used to determine the taxonomic status of ascarids from Ailuropoda melanoleuca, Ailurus fulgens and four kinds of bears(Ursus maritimus,Ursus arctos pruinosus, Ursus thibetanus mupinensis and Ursus arctos lasiotus).So far methods for the control of the ascarids infection are mainly based on chemotherapy,but these drugs cause side effect,drug-resistence and drug-remaining.It is necessary to find a new way to treating Ascaridasis.Here we attempted to clone antigenic genes of roundworms from Ailuropoda melanoleuca and Pan troglodytes by reverse transcription-polymerase chain reaction(RT-PCR).The Escherichia coli-expressed recombinant protein showed highly immunoreactive by Western-blot analysis and can be considered as potential vaccine candidates.The results of research are summarized as following:1.Molecular phylogenetic studies on roundworms from Ailuropoda melanoleuca and seven rare wild animals based on ITS-2 and ND1 geneThe taxonomic status of roundworms from some rare wild animals has been an open question.The second internal transcribed spacer(ITS-2) of the rRNA gene and the mitochondrial partial nahdl gene for NADH dehydrogenase subunit 1 genes of roundworms from Ailuropoda melanoleuca,Ailurus fulgens,Ursus maritimus,Ursus arctos pruinosus,Ursus thibetanus mupinensis,Ursus arctos lasiotus,Pan troglodytes and Hylobates hoolock were sequenced.Homology analyses indicated that the identity levels of ITS-2 genes sequences ranged from 80.4%to 98.6%and ND1 genes sequences ranged from 90.4%to 99.7%among roundworms from Ailuropoda melanoleuca,Ailurus fulgens,Ursus maritimus,Ursus arctos pruinosus,Ursus thibetanus mupinensis and Ursus arctos lasiotus,the 100%identity levels of nucleotide of ITS-2 and 99.2%identity levels of nucleotide of ND1 genes among Pan troglodytes roundworms and Hylobates hooloc roundworms.The alternative trees indicated that roundworms from Ailuropoda melanoleuca,Ailurus fulgens and four species of bears belong to Baylisascaris roundworm,so Toxascaris selenarctis or Toxascaris transfuga in previous studies may be Baylisascaris transfuga;the Ailurus fulgens roundworm should be designated to Baylisascaris ailuri.The Hylobates hoolock roundworm and Pan troglodytes roundworm should be Ascaris roundworm. The present study also showed that a coevolutionary relationship may link roundworm and their corresponding hosts together.2.Analysis of sequence of Bs-Ag1 gene from Bayliscaris schroederi and immunization with recombinant Bs-Ag1 antigen induces protection against Bayliscaris schroederi larval migration after vaccination in miceThe gene of Bs-Ag1 from Baylisascaris schroederi(Ailuropoda melanoleuca) was amplified,cloned and sequenced.A search of the public databases revealed that Bs-Ag1 shared 92%similarity with the nucleotide sequence of L2R59(GenBank accession no:AB057441) from Ascaris suum,and the deduced amino acid sequence of Bs-Ag1 shared the highest amino acid sequence similarity with an AS14 (BAB67769)(91%) from Ascaris suum.A coding region of Bs-Agl eDNA except for the signal sequence was ligated into the pET32a(+) protein expression vector,the resultant plasmid was transferred into E.coli BL21(DE3) and induced with IPTG.The recombinant Bs-Agl proteins can be successfully expressed in BL21(DE3).The rBs-Agl mixed with Freund's Complete Adjuvant(FCA),were tested in a vaccine trial in mice,can elicited immune responses and provided protection against challenge infections as manifested by a 69.26%reduction(P＜0.01) in recovery of Baylisascaris schroederi L3 at day 7 post-challenge.Specific anti-Bs-Agl antibodies from immune protected mice had a significantly increased level of immunoglobulin G (IgG)(P＜0.01).Our data supported the use of Bs-Agl as a potential candidate for vaccination against Baylisascaris schroederi infection.3.Analysis of sequence of Bs-Ag2 gene from Bayliscaris schroederi and immunization with recombinant Bs-Ag2 antigen induces protection against Bayliscaris schroederi larval migration after vaccination in miceThe gene of Bs-Ag2 from Baylisascaris schroederi was amplified,cloned and sequenced.Sequence analysis indicated that the nucleotide sequences of the Bs-Ag2 from adult and L2 larvae of B.schroederi were completely identical.A homology search performed by BLAST revealed that Bs-Ag2 shared 96%similarity with the nucleotide sequence of L2R37(AB089179) from Ascaris suum,Bs-Ag2 shared the highest amino acid sequence identity with Asl6 protein from Ascaris suum (94%)(BAC66614).The recombinant Bs-Ag2 proteins can be successfully expressed in Escherichia coli BL21(DE3).The rBs-Ag2 was used to evaluate their ability to induce immune protective responses in mice against L3-challenge infection in a mouse-B,schroederi model.There was a 65.54%reduction(P＜0.01) of recovery of larvae compared with that in the control group.Specific anti-Bs-Ag2 antibodies from immune protected mice had significantly higher levels of immunoglobulin G(IgG) (P＜0.01).Our data supported the use of Bs-Ag2 as a potential candidate for vaccination against B.schroederi infection. 4.Analysis of sequence of Al-Ag1 gene from Ascaris lumbricoides and immunization with recombinant Al-Ag1 antigen induces protection against Ascaris lumbricoides larval migration after vaccination in miceThe gene of Al-Ag1 from Ascaris lumbricoides was amplified,cloned and sequenced.A search of the public databases revealed that Al-Ag1 shared 99% similarity with the nucleotide sequence of L2R59 from Ascaris suum(GenBank accession no:AB057441),and the deduced amino acid sequence of A1-Agl shared the highest amino acid sequence similarity with an AS14(BAB67769)(97%) from Ascaris suum.A coding region of Al-Agl eDNA except for the signal sequence was ligated into the pET32a(+) protein expression vector,the resultant plasmid was transferred into E.coliBL21(DE3) and induced with IPTG.The recombinant Al-Ag1 proteins can be successfully expressed in BL21(DE3).The rAl-Ag1 mixed with Freund's Complete Adjuvant(FCA),were tested in a vaccine trial in mice,can elicited immune responses and provided protection against challenge infections as manifested, by a 69.34%reduction(P＜0.01) in recovery of Ascaris lumbricoides L3 at day 7 post-challenge.Specific anti-Al-Ag1 antibodies from immune protected mice had a significantly increased level of immunoglobulin G(IgG)(P＜0.01).Our data supported the use of Al-Ag1 as a potential candidate for vaccination against Ascaris lumbricoides infection.5.Analysis of sequence of Al-Ag2 gene from Ascaris lumbricoides and immunization with recombinant Al-Ag2 antigen induces protection against Ascaris lumbricoides larval migration after vaccination in miceThe gene of Al-Ag2 from Ascaris lumbricoides was amplified,cloned and sequenced.A search of the public databases revealed that Al-Ag2 shared 96% similarity with the nucleotide sequence of L2R37(AB089179) from Ascaris suum, and the deduced amino acid sequence of Al-Ag2 shared the highest amino acid sequence similarity with an AS16(BAC66614)(94%) from Ascaris suum.A coding region of Al-Ag2 eDNA except for the signal sequence was ligated into the pET32a(+) protein expression vector,the resultant plasmid was transferred into E.coliBL21(DE3) and induced with IPTG.The recombinant Al-Ag2 proteins can be successfully expressed in BL21(DE3).The rAl-Ag2 mixed with Freund's Complete Adjuvant (FCA),were tested in a vaccine trial in mice,elicited immune responses and provided protection against challenge infections as manifested by a 66.84%reduction(P＜0.01) in recovery ofAscaris lumbricoides L3 at day 7 post-challenge.Specific anti-Al-Ag2 antibodies from immune protected mice had a significantly increased level of immunoglobulin G(IgG)(P＜0.01).Our data supported the use of Al-Ag2 as a potential candidate for vaccination against Ascaris lumbricoides infection.