| Cryptosporidiosis,an important and one of the most common zoonotic intestinal protozoan diseases with a worldwide distribution,is caused by the genus Cryptosporidium.About 300 species animals including human can be attacked.Cryptosporidium oocysts commonly employ a variety of transmission routes,mainly including direct contact with infected patients or animals and intake of contaminated food or water,and the diarrhea is the common clinical symptom.Wildlife infected Cryptosporidium has showed the potential zoonotic concern,and nearly half of Cryptosporidium species and genotypes have been confirmed in wildlife,suggesting the necessity and importance of molecular epidemiologic studies of Cryptosporidium to wildlife.These works will provide essential data for assessing the transmission dynamics of Cryptosporidium infecting wildlife.However,there is scarcely report of Cryptosporidium infection in wildlife in Sichuan province located in the basin.To date,there is still no effective treatment drug for cryptosporidiosis,increasing the difficulty of prevention and treatment of the pathogeny.The environmental solution is developing the vaccine to decrease the infection of Cryptosporidium.The food-grade Lactococcus lactis(L.lactis)possesses some advantages,including very strong adhesive force,direct oral,high safety,mature antigen-presenting system,and stimulating the intestinal immune respone.These adventages make the excellent live carrier of oral vaccine of Cryptosporidium a reality.This study consists of four parts as follows:Exp.1 Detection of wildlife fecal samples in Sichuan provinceIn the present study,2678 fecal samples were collected from different native wildlife in various regions of Sichuan province from 2012 to 2015.The fecal oocysts were examined for Cryptosporidium through the Sheather's sucrose flotation technique and PCR amplification to Cryptosporidium 18S rRAN gene of feces DNA.Results showed thirteen fecal samples were detected positive,involving five wildlife species:three bamboo rat feces/isolates(YA01,YA02,and YA03),three camel feces/isolates(derived from two camels)(SCBC02 and CDBC01),one squirrel monkey fece/isolate(SCSM01),three wild boar feces/isolates and three miniature pig feces/isolates.Exp.2 Identification of Cryptosporidium genotype and subtypeThe Cryptosporidium spp.obtained from Exp.1 were genotyped by nested PCR and nucleotide sequencing of the 18S rRNA,70-kDa heat shock protein(HSP70),oocyst wall protein(COWP),and actin genes.Further subtyping was performed by PCR amplification and DNA sequence analysis of the 60-kDa glycoprotein(gp60)and MLST(MS1,MS2,MS3,and MS4)genes.By aligning with each other and reference sequences,adjusting the obtained sequences,and phylogenetic analysis using neighbor-joining(NJ)or maximum likelihood(ML)method,the Cryptosporidium genotype and subtype for each positive isolate were determined.The results showed that:1.For the YA01,YA02,and YA03 isolates,in the 18S rRNA gene,there were 1-5 nucleotide differences between the bamboo rat isolates and other known C.parvum isolates.At the HSP70 and actin loci,14,17,11 and 4,4,9 nucleotide substitutions occurred in this study.The COWP genes of YA01 and YA02 isolates were in accordance with those of published C.parvum;differently,two nucleotide mutations noted in the YA03 sequence.The similarly of each isolate at the each locus was>99%.Similar topologies produced in the NJ trees of each of the four loci,and the three isolates and known referenced C.parvum formed a cluster.Thus,based on our genotyping data,all three isolates were classitified as C.parvum.The YA03 isolate shared a nucleotide identity of 98.6%with a Swedish C.parvum subtype ?o from diarrheal patients,and had three less TCA repeats.Therefore,the isolate YA03 belonged to subtype ?o and named subtype ?oA13Gl.Meanwhile,our research confirmed the subtype IIo family as zoonotic subtype firstly.Isolates YA01 and YA02 yielded identical gp60 gene sequences and had 9 TCA repeats.In addition,YA01 and YA02 shared a maximum nucleotide identity of 93.9%to a previously characterized C.parvum subtype IInA8,and had an extensive sequence polymorphism in the non-repeat regions.Furthermore,phylogenetic analysis showed YA01 and YA02 grouped together separately.Therefore,YA01 and YA02 were determined to belong to a novel subtype family of C.parvum,named IIpA9.2.The homolgoys of the obtained 18S rRNA and COWP genes between the camel isolates and those of C.andersoni were both 100%.Phylogenetic analysis showed that the isolates from this study clustered in the clade of C.andersoni,and demonstrated that the two camel-derived isolates did indeed represented C.andersoni.For the MLST subtype,SCBC02 and CDBC01 shared 100%homology with A4,A4,A4,A1 subtype from cattle and A6,A5,A2,A1 subtype from camel in the GenBank database,respectively.In addition,phylogenetic analysis of each subtype locus supported these identifications.3.The COWP sequence of the Cryptosporidium SCSM01 isolate had an identical sequence to that of known C.cuniculus,C.hominis,and the monkey genotype.However,for the 18S rRNA,HSP70 and actin loci,the most nucleotide differences were 2,10,and 6 by BLAST.The results of RFLP-PCR of 18S rRNA gene indicated that the SCSM01 was very close to the classification of C.hominis.Inconsistent topologies were produced by the neighbor-joining method.The SCSM01 and C.hominis referenced,but not the monkey genotype,formed a cluster in the 18S rRNA and COWP genes.However,for the HSP70 locus,the isolate from this study formed its own cluster.Remarkably,with respect to the actin gene,the SCSM01 was grouped together with the monkey genotype,which then formed a large cluster with known C.hominis.Therefore,this SCSM01 was identified by maximum likelihood analysis at each locus again based on differential evolution,and similar phylograms occurred compared to the neighbor-joining trees,respectively.In view of the genetic differentiation,the squirrel monkey isolate was a previously unknown C.hominis genotype,distinct from the monkey genotype,monkey genotype ?.In the gp60 gene,the SCSM01 shared only 88.3%maximum homology with C.hominis subtype IdA10G4,which was far lower than that between the known C.hominis subtypes,In the topology trees producted by the ML and NJ trees,the classification of the SCSM01 was consistent,and meanwhile,the current SCSM01 evolved alone at the phylogenetic level.Therefore,a novel C.hominis subtype family was identified,named IkA7G4.4.By BALST research in the NCBI,the Cryptosporidium wild boar and miniature pig isolates yielded the 100%homologies to known C.scrofarum in the 18S rRNA gene.However,for the HSP70 gene,the wild boar isolates shared 100%simility with C.ubiquitum,and in the phylogenetic tree,the two species grouped together.Differently,miniature pig isolates and those C.suis referenced from GenBank formed a cluster.The data from our study indiacated that both wild boar and miniature pig harbored mixed infection in Cryptosporidium.Exp.3 The construction of food-grade lactococcus lactisstrain for CryptosporidiumIn this study,two pairs of primers including different restriction enzyme sits were devised based on the P23 gene sequence,and the vectors pNZ8149 and pET-3 2a characteristics.The C.parvum RNA was extracted,and the P23 gene was amplified by RT-PCR using the two pairs of primers,respectively.Subsequently,the obtained P23 genes were subcloned into the vector PMD19-T,respectively,and were sequenced by the primers of T vector.The P23 genes included in recombinant T vectors purified using the kit.The duplicated P23 fragments,with different restriction enzyme sites,were inserted the pNZ8149 and pET-32a vectors,and then correspondingly transformed into L.lactis NZ3900 and E.coli BL21 by electroporation and thermal stimulation,respectively,to construct the recombinant L.lactis P23-pNZ8149/NZ3900 strain and E.coli P23-pET32a/BL21 strain.The recombinant L.lactis P23-pNZ8149/NZ3900 strain was respectively induced 3h,6h,9h,12h by lOng/mL nisin,and the tarbet recombinant protein was analysed by SDS-PAGE after induction.At the best induction time,the density of the induced recombinant L.lactis was determined by testing the OD600.By IPTG induction,the expressed protein of the recombinant P23-pET32a/BL21 strain was injected into rabbit.The serum from rabbit was isolated to analyze the immunogenicity of expressed P23 protien in L.lactis P23-pNZ8149/NZ3900 recombinant strain by Western-blot.In addition,through the IIF experiments,the location of expressed P23 protien in L.lactis P23-pNZ8149/NZ3900 recombinant strain was determined.The results showed that:1.Successfully cloned the P23 gene and constructed T vector of P23 gene with different restriction enzyme sits.The gene fragment of obtained 333bp revealed one nucleotide change,but the translated amino acids were consistent with published P23 protein.2.Successfully constructed the food-grade L.lactis P23-pNZ8149/NZ3900 recombinant strain.3.Successfully constructed the E.coli P23-pET32a/BL21 recombinant strain.4.After induction by nisin,a 23kDa protein expressed was detected by SDS-PAGE from the recombinant L.lactis P23-pNZ8149/NZ3900 strain,indicating the P23 protein expressed was glycosylated.The clearest protein band was presented at 9h,illustrating that 9h was the best induction time,and the OD600=2.7.Whereas,no P23 protein in the non-induced L.lactis P23-pNZ8149/NZ3900 and pNZ8149/NZ3900 strains.5.For the P23-pET32a/BL21 strain,the recombinant protein was also normally expressed by IPTG induction.The weight was 38kDa.No P23 protein was in non-induced E.coli P23-pET32a/BL21 recombinant strain.6.Western-blot and IIF experiments revealed the tarbet protein maintained the preferable antigenicity of Cryptosporidium.Meanwhile,the expressed P23 protien was located on the surface of recombinant L.lactis P23-pNZ8149/NZ3900 strain.Exp.4 Preliminary immune index of food-grade L.lactis P23-pNZ8149/NZ3900 recombinant strain to BALB/C miceThe induced L.lacti P23-pNZ8149/NZ3900 recombinant strain from Exp.3 immunized ten-week old BALB/c mice by oral.A dose of 1010CFU L.lacti recombinant cell suspension was used to one mouse per day for five consecutive days(0d was the first vaccination),and the mice were sacrificed on day 5d,10d,15d,and 20d.Blood,intestinal mucus,spleen,and fresh feces were collected on these days after immunization.The IgG,IgA,IgGl and IL-4,IL-12,TNF-?,IFN-? in blood were analyzed by similar ELISA(enayme-linked immunosorbent)assay.Likewise,sIgA and IgM in feces and intestinal mucus were tested by the use of the same mathod.IL-4,IL-12,TNF-a,IFN-y,CD4+,and CD8+ in spleen were analyzed for evaluating of their changes.Simultaneously,pNZ8149/NZ300 strain and PBS were set as negative and control.Our results showed that:1.The sIgA and IgM in intestinal mucus and feces rised significantly from the fifth day(p<0.01),and then gradually increased compared with the pNZ8149/NZ3900 strain and PBS.These results revealed that the P23 protein was effectively presented to intestinal mucosa by our food-grade L.lactis P23-pNZ8149/NZ3900 living-vector strain,and the intestinal immune respone was strongly stimulated.Simultaneously,the preferable antigenicity for P23 protein exposed again.2.All of the IgG,IgA,IgGl and IL-4.IL-12,TNF-a,IFN-y in blood of BALB/c mice obviously increased until to the end of the test by the oral vaccine strain-L.lactis P23-pNZ8149/NZ3900.However,these indicators in pNZ8149/NZ3900 strain and PBS groups had no difference(p>0.05).Thus,the humoral immune response in BALB/c mice was stimulated significantly.3.The changes of cytokines(IL-4?IL-12?TNF-?,and IFN-y)and T cell(CD4+ and CD8+)in spleen were consistent with the antibodies in blood in BALB/c mice(p<0.01).The CD4+/CD8+ rised slowly and had no significant difference to the negative and control groups(p>0.05).Therefore,the cellular immune response in BALB/c mice was also obviously stimulated,and the Thl and Th2 immune response occured together.Besides,CD4+ increase was gaven priority to the cellular immune response in BALB/c mice.In summary,this study was the first wide-range surgey about the Cryptosporidium infection in native wildlife in Sichuan province,and the public health concern did not present.However,based on the current molecular characteristic,the Cryptosporidium isolated reveealed the genetic diversity.Our research successfully constructed the food-grade L.lactis P23-pNZ8149/NZ3900 recombinant strain for cryptosporidiosis.The secreted P23 was a fully biologically active protein,as demonstrated by its capacity to stimulate intestine mucosal immune,humoral immune and cellular immune in BALB/c mice.Thus,the constructed vaccine strain provided a good basic for the broad application of an environmental food-grade vaccine to prevent cryptosporidiosis. |
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