Epidemiological Investigation Of Canine Distemper In Giant Pandas And Experimental Immunication Research

Canine Distemper(CD)is an acute and highly contagious disease caused by the Canine Distemper Virus(CDV)in the order Carnivora.CDV has recently expanded its host range to the entire carnivorous and nonhuman primates from traditional Canidae,Procyonidae and Mustelidae.More recently,with an increase in the population or habitat of giant pandas,the risk of infection has greatly increased.Canine Distemper has become the first serious disease that threat to population and life safety of giant pandas.Over the period of December,2014 through April,2015,the Shaanxi Rare Wild Animal Rescue and Research Center,China,experienced a CDV outbreak in six captive endangered giant pandas.Five of six CDV-infected giant pandas died as a result of the infection.In this thesis,pathogen isolation and detection of CDV antibody were conducted in giant pandas from this outbreak,and first documentation that CDV can infect to giant pandas was recorded.Vaccination is the only effective measure to prevent CDV infection of domestic dogs and may have utility in captive giant panda populations.Currently,some attenuated vaccines are fatal to giant pandas and there are no vaccines in place that are safe,stable and effective for the prevention of CD in captive giant pandas in China.So,considering the rare and susceptibility of giant pandas and combining with the obtained experimental data,we developed an inactivated and virus-like particles subunit vaccines and evaluated its immunogenicity.In view of a canarypox-vectored recombinant distemper vaccine which recommended for the vaccination of ferrets has proved to be safe and effective in two giant pandas,so,we evaluated the immunogenicity of the commercial recombinant canine distemper vaccine which recommended for the vaccination of healthy dogs in giant pandas.Chapter one,isolation,identification and analysis of genetic evolution of the canine distemper virus from giant pandas.CDV RNA was detected by RT-PCR from samples collected from 22 Giant pandas in the Shaanxi Rare Wild Animal Rescue and Research Center from December,2014 through April,2015.Nucleic acids isolated from nasal swabs,urine,feces and blood collected from six affected pandas all tested positive for CDV by RT-PCR.Five of six CDV infected giant pandas presented with jaw trembling and violent convulsions of the limbs died.All affected giant pandas were housed in the same room or adjacent rooms,suggesting that CDV may have been transmitted between pandas via direct contact and/or respiratory droplets.Tissue samples were inoculated onto monolayers of Vero/DogSLAM cells,which have been previously used to culture and isolate CDV.The giant pandas CDV isolate was identified by electric microscope,CPE,IFA,genome sequence analysis and named as giant panda/SX/2014.Phylogenetic analysis based on the H gene sequence revealed that giant panda/SX/2014 belongs to the Asia-1 cluster.Sequencing of CDV from giant pandas revealed five unique amino acid changes(V26M,T213 A,K281R,S300 N,P340Q)encoded by the H gene that have not been observed previously in Asia-1 strains.Notably,giant panda/SX/2014 possessed a histidine(H)residue at position 549 within the SLAM binding region of the H gene,not tyrosine(Y)in wild canid strains.We therefore speculate that the fatal infection of giant pandas may be related to the emergence of highly pathogenic CDV and host range expansion associated with Y549 H substitution of giant panda/SX/2014 H protein.The effectiveness of CDV vaccination in giant pandas is supported by the survival of the single panda(Zhuzhu)during this outbreak was previously vaccinated against CDV and had high-titer VNAs(1:128).This animal did not display clinical signs despite recovery of CDV genomic material from blood and nasal swab samples,suggesting that the protective immune responses elicited by CDV vaccination may have survived giant pandas from the fatal infection.The study of a CDV outbreak among captive giant pandas suggests that vaccination should be considered to prevent the CDV infection in giant pandas.Chapter two,preparation and experimental immunological studies of CDV inactivated vaccine.In view of attenuated vaccines have caused morbidity and mortality in some susceptible wild animals,especially,vaccine-induced fatal canine distemper infection in giant pandas has been reported.Thus,in the present study,we developed inactivated vaccine of CDV.To ensure the dose of immunogen,the seed virus of CDV was cultured by Netherland Applikon bioreactor and GE Cytodexl spherical microcarriers,its titer as high as 107.8TCID50/mL.The immunity of vaccines which inactivated by formalin and beta-propiolactone solution were evaluated with three different doses in mouse.The results of immunological experiment showed that formalin was superior to beta-propiolactone.The immune effect was positively associated with dosage.The durable virus-neutralizing antibodies(VNAs)could also be induced by immunization of mouse at ten-fold dilution group.The immunogenicity and potential of inactived vaccine as novel vaccine was further evaluated by intramuscular vaccination in mink and fox models.Mink and fox studies demonstrated that the formalin-killed vaccine was not only safe but also could induce specific VNAs responses and possess the satisfied immunogenicity.The potential of inactivated vaccine developed as safe and efficacious canine distemper vaccine candidates for giant pandas may be further proved by efficacy test.Chapter three,construction and immunogenic evaluation of CDV viurs-like particles.Virus-like particles(VLPs)are one or several structural proteins self-assembly competent protein structures with identical or highly related overall structure to their corresponding native viruses.VLPs can stimulate both humoral and cellular immunity responses,so,it offers a unique advantage as vaccines.M protein is a main drive force of forming and budding of paramyxovirus VLPs,H protein is the main protective antigen of CDV.Thus,in the present study,We cloned M and H genes of giant panda/SX/2014 and rescued two recombinant baculoviruses that express M and H proteins by flashbac baculovirus expressed system,respectively.CDV VLPs were self-assemblyed and harvested by culture supernatants of Sf9 cells which co-infected by two recombinant baculoviruses that express M and H proteins,respectively.By electron microscopy,the CDV VLPs displays typical feature of paramyxovirus with diameter was approximately 100 nm and obvious surface spikes.By western blot,the construction of CDV VLPs were M and H proteins.The immunogenicity of CDV VLPs mixed with adjuvant was evaluated by intramuscular vaccination in mink and fox models.Mink and fox studies demonstrated that CDV VLPs immunized twice still could not induce specific VNA responses,boost and clarification of CDV VLPs may be needed.Chapter four,immunogenic evaluation of a canarypox-vectored recombinant canine distemper virus vaccine in giant pandas.The recombinant live canarypox virus is an non-replicating vector,it does not assemble progeny virus only can express the gene when immunized in mammal,so,it can be more safe and efficacious as for mammal.Currently,many kinds of canarypox-vectored vaccines express the F and H glycoproteins of CDV have been developed by Merial.In the present study,we choosed 23 giant pandas from one research center and 37 giant pandas from one research base of giant pandas for the immune study of the recombinant canarypox-vectored canine distemper virus vaccine(Recombitek? CDV).The immunity was evaluated by the VNAs of serum from giant pandas immunized.In antibody studies,results had shown that the Recombitek? CDV could induce specific VNAs responses in giant pandas.After booster vaccination,all giant pandas with or without the initial antibody before immunization of one center had detectable VNAs(geometric mean 65.1?33.1,range 8-256);9 of 37 giant pandas of one reaearch base had detectable VNAs(range 8-32).According to the seropositive rate and VNAs of the giant pandas immunized,the better immunization strategies of giant pandas was that one and two doses were recommended for primary vaccination of the giant pandas with and without preliminary antibodies,respectively.The next booster vaccination with one dose was recommended 3 weeks later.