Development Of Loop-Mediated Isothermal Amplification For Detection Canine Distemper Virus And Study Neutalization For Chicken Egg Yolk Antibody Of Tandem Epitope

Canine distemper (CD) is acute and highly contagious infectious disease caused by canine distemper virus (CDV). In recent years, CD natural host range increased from canine and fur-bearing animals to many animals such as lion, tiger and even to primate. CDV has often been detected by electron microscopy, indirect immunefluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and reverse transcription polymerase chain reaction (RT-PCR). However, these methods are time-consuming and complicated operation, special equipment and only experienced operators are needed to perform a clinical diagnosis. In addition, in some countries and regions reported that dogs vaccinated can still be infected with CDV and vaccine failure is a common problem. Egg yolk antibody as an effective biological control agents in prevention and treatment for animal diseases which shows significant result to provide a new idea for CD prevention.Three canine distemper virus field strains were isolated from clinically distemper suspected dogs that named J7、A8and S9, respectively. In order to study isolates and vaccine strains, the CDV H gene was amplified and analysed of nucleotide、amino acid homology and evolutionary tree. The result showed that nucleotide homology of isolates was99.1～99.3%which homology with A75/17was highest and with vaccine strain Onderstepoor、CDV3was lowest. Compared with the clinical distemper vaccine, homology was quite different. Evolutionary tree showed that relationship between isolates and5804P、5804were closest, farthest to Onderstepoort、CDV3vaccine strain. Analysis of nucleotide homology among isolates and CDV seven different genotypes, isolates has the highest homology that was99.2%～99.3%with domestic Asia-1HLJ3-08. Nucleotide homology comparison result showed that isolates among America-2、European wildlife、Europe、Arctic-like、 Asia-2、Vaccine decreased gradually and isolates belongs to Asia-1.CDV H gene of isolates encoded607amino acids which have nine potential N-glycosylation sites. Convac、Onderstepoort have seven and four N-glycosylation sites, respectively. There were six-seven potential N-glycosylation sites for clinical distemper vaccine. Different number of H protein asparagine glycosylation maybe caused the antigen change that lead CD outbreak.In this study, a loop-mediated isothermal amplification (LAMP) method for the rapid detection of CDV was developed. A set of two pairs LAMP primers, internal primers (FIP, BIP) and external primers (F3, B3) were designed according to the conserved sequences of CDV N gene. The test of optimum condition, specificity, sensitivity and clinical samples assay of the LAMP were investigated. The results showed that the optimal reaction conditions for LAMP amplifying CDV N gene was achieved at63℃for1h in water bath. The detection virus limit of the LAMP assay was approximately100.5TCID50, ten times more sensitive than RT-PCR, a thousand times more sensitive than rapid CDV Ag test kit, and no cross-reaction with rabies virus (RV), canine parvovirus (CPV) and canine adenovirus Ⅱ(CAV Ⅱ). In addition, the results gathered by detecting the clinical samples using this way were totally in agreement with the RT-PCR assay. As it is an easy and simple method, the LAMP is potentially applicable for canine distemper diagnosis.Using tandem epitope and prokaryotic expression technology to obtain the CDV F gene B cell linear epitopes of the recombinant protein. Tandem epitope antigen、isolates-J7and the vaccine strain CDV-11were used as immunogen to immunize the seven months old Highland laying hens for three times. Each time interval was two weeks. The chicken of tandem epitope antigen group were immunized0.40mg、0.60mg、0.80mg per chicken in the first immunization second immunization and third immunization. The chicken of isolates-J7group was immunized3.0ml per chicken in three times (1035TCID50/ml). The chicken of vaccine strain CDV-11group was immunized3.0ml per chicken in three times (1045TCID50/ml).The eggs were collected and the yolk antibodies were extracted by chloroform, then the titers of the yolk antibodies were detected by indirect ELISA. The highest titer of three egg yolk antibodies were1:2048、1:512、1:256, respectively. Experiment in vitro results showed that the ability for neutralized CDV of three kinds of egg yolk antibody with highest titers was poor, with the increase concentration (5and10times), egg yolk antibody neutralized ability enhance to protect the cell. Protective effect was effective within24h, after48h the cytopathic effect began. MDCK cells without inoculation with CDV grew normally, Non-immunized yolk antibody as the same as cytopathic effect inoculation with CDV and immunized egg yolk antibody almost no damage to the cell. Experiment in vivo results showed that5times concentrated egg yolk antibody protect Kunming mice that were challenged intracranially with CDV.Results of this study indicated that the LAMP assay for detection of CDV was a rapid, sensitive and specific. It could make the technique far more suitable for practice in the field. Three kinds of egg yolk antibody was prepared against canine distemper virus, it provided material basis for prevention and speicific experimental to next clinical application.