Isolation And Identification Of Canine Parvovirus (CPV) And Preparation Of Highly Immunized Egg Yolk Antibodies

Canine parvovirus(CPV), one of the smallest and the simplest single stranded DNA virus, is a kind of lethal virus which can cause acute infectious canine diseases with clinical symptoms such as bloody enteritis and cardiac muscle inflammation. According to the statistical data of veterinary office in Shihezi region, CPV is the primar cause of morbidity and mortality of various infectious diseases in dogs. At present, there is not effect CPV treatment method .Development of CPV antibodies is considered as an important role for the diseases prevention and treatment.CPV samples were collected from infected dogs according to the clinical symptoms. The embryonic feline kidney cell line F81 was used to multiplicate the viruses. The virus was than purified by Sepharose-4B column chromatography and Fractogel EMD TMAE(M) Anion exchange chromatography. HA/HI, immune-electron microscope (IEM) and PCR methods were used to identify the CPV. Laying hens were immunised by purified CPV samples and the egg yolk antibodies (IgY) were extracted by modified PEG6000 method and water dilution method. The titres of IgY was tested with agar spread method and indirect ELISA. The anti- CPV IgY antibodies were than used to treat 2~3 months old dogs which were challenged with CPV perversely. Furthermore, field investigation using 34 CPV infected dogs was done in order to get the enlarged statistical data.The present research show that the cell supernatant fluid can be detected by HA, the diluter of HA range is 2~9. The purified virus can reacted with serum CPV antibody in immune-electron microscope observation (IEM) and the virus diameter is 20-24nm. The VP2 gene of the virus was amplified by PCR reaction, The sequences data of the gene were than submitted to GenBank(EU170352). The gene sequences were classified as a CPV-2a type with a few diversities. It is named as CPV-SHZ in the present paper. Different IgY antibody extraction methods were compaired. Higher protein concentration is observed with the use of modified PEG6000. However, there is higher IgY consentration by the use of water dilute method. The titer of IgY was tested as 1:32 by agar spread method and which is equaled to 1:24000 by indirect ELISA. The clinical results showed that four dogs in the IgY group were cured and one in the control group died, and the protection rate reached to 100%. The enlarged animal test with 34 dogs shows protection rate of 79.4%.The above showed results indicate that the CPV-SHZ strains have been successfully isolated. Furthermore, highly titred and neutralized IgY antibodies can be purified,and show good virus protection effects in both in vitro and in vivo. The present research suggests that anti-CPV IgY have the potential for further development and use of CPV prevention and treatment.