| Canine parvovirus(CPV)is one of the important pathogens causing canine diarrhea,resulting in significant losses for the dog industry.At present,CPV is co circulating with many antigenic strains in China,and epidemiological surveys and studies on the pathogenic characteristics of circulating strains can help prevent and control the disease,whereas therapeutic antibodies are an effective means of treating CPV.The aim of this study is to investigate the prevalence and pathogenic biological characteristics of CPV in Chengdu City during 2019-2020,and to prepare an exquisite high-level yolk antibody against the circulating strains.The results obtained were as follows:1.Distribution of CPV and its anti-prototypeTo understand the current prevalence of CPV and its anti-prototype in Chengdu,Sichuan Province,China,121 canine diarrheal fecal samples collected from Chengdu in2019-2020 were tested by PCR method to specifically detect CPV,the PCR products of positive samples were sequenced and the phylogenetic tree was constructed.The prevalence of new cpv-2a,cpv-2a,and cpv-2c infection was 52.1% for CPV,and 31%,34%,35% for new cpv-2a and cpv-2c among the 63 positive samples,including 8% of samples with a mixture of cpv-2a and cpv-2c.Appeal results showed that CPV is still one of the important pathogens causing canine diarrhea in Chengdu City at present,there are three kinds of anti-prototype CPV epidemic,and there is mixed infection,which illustrated that the anti-prototype of CPV epidemic strains in Chengdu City is complex and diverse.2.A cd-dy6-2019 isolate was successfully isolated and identifiedTo further characterize the biology of CPV,a mixed infection positive sample of type1 cpv-2a and cpv-2c was inoculated and subcultured into f81 cells,which were tested by PCR,transmission electron microscopy and indirect immunofluorescence were performed after the appearance of stable lesions.The results showed that after continuous passage to the sixth generation,CPE stably appeared at 96 h after toxification,and the initial CPV infected f81 cells appeared round and shrunk,and the cells appeared disintegrated and sloughed with time,typical of dragline lesions.Sequencing typing results of the PCR products of the cultures showed cpv-2a pattern.The results of indirect immunofluorescence test showed that the isolated strain showed bright specific fluorescence.Virions that conformed to the characteristics of CPV could be observed by TEM,which confirmed the successful isolation of a novel cpv-2a type strain from the mixed infection positive samples and provided a material basis for subsequent exploration of its biological characteristics and pathogenicity.3.Genomic characteristics of cd-dy6-2019 isolateTo further characterize the molecular features of the cd-dy6-2019 isolate,eight pairs of primers were designed to amplify the complete gene sequence of the cd-dy6-2019 isolate segmentally using the software premier 5.0,and five pairs of primers were designed to double check,which revealed that the cd-dy6-2019 isolate contained two complete reading frames of 4269 BP in length.The nucleotide similarity of the isolates to the reference strains ranged from 98.8% to 99.9%.The deduced amino acid sequence revealed mutations at 4 amino acid(23,247,248,443)sites on the NS1 protein and 8 amino acid sites(80,91,93,103,232,305,564,568)on the.The impact of these mutations on CPV awaits further study.Notably,genetic evolution analysis revealed that this isolate is most recently related to a 2019 feline panleucopenia virus(FPV)isolate from China(accession number: mn908257,mt614366)and more distantly related to CPV in Gen Bank,suggesting that this isolate evolved from FPV.This study enriched the epidemiological data of CPV in Chengdu City,and provided a reference for the study of genetic variation of CPV.4.Development of cpv-2c type purified high immune yolk antibodyThe goal of this study was to produce exquisite high titer yolk antibodies to cpv-2c that are broadly neutralizing and protective against new cpv-2a,new cpv-2b,and all circulating cpv-2c strains.The 201 adjuvant inactivated vaccine was prepared from sc-14 /2017(cpv-2c,TCID50 104.5 / 0.1ml).The chickens were immunized with 1 ml of 201 adjuvant inactivated vaccine every 14 days.The results showed that the neutralizing titers of the original vitelline antibody obtained on the 15 th,28d,and 57 d after the three immunizations were 1:46286,1:23141,1:10953,respectively,and those of the refined vitelline antibody purified by the octanoic acid-peg6000 method were 1:8111,1:5758,1:4490,respectively.Refined yolk antibodies from this study against new cpv-2a,new cpv-2b,and cpv-2c The neutralization titers of serotype IIV were 1:1024,1:1780,1:8111,indicating that this refined high titer ovalbumin antibody has good broad-spectrum neutralization,and in addition to high protective titers against homotypic strains,it also has better cross protective titers against two other different antigenotypic strains,and is superior to commercially available m Abs and high titers,providing broad-spectrum neutralization for further production The foundation for the action of anti CPV refined yolk antibodies. |
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