Studies On Canine Distemper Virus, Canine Parvovirus And Canine Adenovirus Ⅰ Combined Live Vaccine

Canine distemper (CD), canine parvovirus(CPV) disease and canine adenovirus (CAV) disease, are highly contagious and fatal diseases of dogs and other terrestrial carnivores. CD can infect giant panda, panda, tiger, lion, the small leopard and other carnivora all 8 families, rtiodactyla pig family, primate called monkey, Pinnipedia Phocidae, with incidence rate of 100% and the mortality rate of 80%. It has been widely documented that CDV causes severe immunosuppression and persistent infection in the central nervous system in dogs. Moreover, the host spectrum of CDV is gradually increasing They have been causing tremendous economic losses to the canine farming, far cultivation and wildlife conservation industry throughout the world. CPV is responsible for canine hemorrhagic enteritis and myocarditis. Canine adenovirus occurs both in China and around the world among domesticated dogs, foxes and wild fox, bear, coyote and raccoons.With the development of society, as companion animals, economic animals, and special dog, dogs plays an important role in human life, therefore, it is imperative to strengthen the prevention and control of main diseases in dogs. Vaccination is considered to be the most direct and effective measures of prevention and control of canine disease.. At present, univalent and polyvalent attenuated vaccines is widely used around the world. Although widespread vaccination against CDV, CPV and CAV has been conducted for many decades, several outbreaks in vaccinated animals have been reported frequently. Besides the interference of maternal antibody, other viruses, vaccine potency and improper use of vaccines, Whether the epidemic virus variation strain caused by the urgent need to verify. At the same time, most of these vaccines currently used for copy foreign vaccine virus seed, the background is unclear and in cell passage number more, immunogenicity and safety are difficult to be guaranteed. Therefore, the development of suitable for domestic strains in the vaccine has been imminent.Studies on isolation, identification, attenuation and immune efficacy of CDV, CPV and CAV. A CDV, CPV and CAV strain was isolated with CEF cells, CRFK cells and MDCK cells respectively. The virus was confirmed by electron microscope and animal regression experiment and so on.A CDV strain was isolated with CEF cells culture from an affected dog with the syptoms of fever, diarrea and cough. The virus was confirmed by electron microscope. With the method of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals and experimental infection of animals, a wild CDV strain was confirmed. The virus was designated as CDV-YB. The virus titer was increased gradually to 1065 TCID50/mL. Meanwhile, the virulence of CDV-YB degraded gradually, designated as CDV-YBR. The result of immune efficacy indicated that CDV-YBR had good immunological activity and could provide dogs strong protection to resist the challenge of homologous virulent CDV-YB strain.A wild CPV strain was confirmed with experimental infection of animals. The virus was designated as CPV-YN. The CPV-YN virus titer was increased gradually to 106TCID50/mL after infection of CRFK cell for 110 passages. Meanwhile, the virulence of CPV-YN degraded gradually, designated as CPV-YNR. The result of immune efficacy indicated that CPV-YNR had good immunological activity and could provide dogs strong protection to resist the challenge of homologous virulent CPV-YN strain.A CAV strain was isolated with MDCK cells culture from an affected dog with the syptoms of fever and "Blue eyes". The virus was designated as CAV-H. The CAV-H virus titer was increased gradually to 107.5 TCIDso/mL after infection of MDCK cell for 110 passages. Meanwhile, the virulence of CAV-H degraded gradually, designated as CAV-HR. The result of immune efficacy indicated that CAV-HR had good immunological activity and could provide dogs strong protection to resist the challenge of homologous virulent CAV-H strain.Studies on CDV-CPV-CAV combined live attenuated vaccine:the efficacy of the CDV-CPV-C AV attenuated vaccine prepared by mix of three viruses infected cell culture supernatant and stabilizer at individual appropriate portion was evaluated by animal experiments. The combined vaccine demonstrated good safety and immunity. Firstly, the combined vaccine has similar immunological efficacy as the monovalent vaccine; secondly, it was more efficient compared with commercialized canine 5-mixed vaccine. The dose of virus antigen and safety of the combined vaccine are superior to the commercialized 5-mixed vaccine. Most of the commercialized vaccine is not suitable to the pregnant dogs, however, the combined vaccine in this study could be used to immunize the pregnant dogs safely. As some of the vaccines on the domestic market are manufactured with seed viruses derived from imported vaccines strains or isolated from foreign countries, the viral genes and antigenicity are significantly different from the field isolates. The combined vaccine seed viruses are isolated from clinical diseased animals and cloned from prevalent strains. The completely intellectual property fill the gap of the domestic canine vaccines. Now, the combined vaccine in this study is declaring a China Veterinary Drug Products License. This study provides the important material basis and technical reserves for the prevention and control of viral disease of the dogs in China, and which will produce good economic and social benefits.