| Feline panleukopenia is an acute, highly contagious infectious disease caused by FPV, with high pathogenicity and wide-ranged infection. The incidence of the diease is especially high in kettens with a mortality of 85% or more. Both of large felids such as tigers, leopards, lions and small felids such as smaller lynx, leopard cats, wild cats, and minks or ferrets are susceptible animals to FPV, the virus that is most widely distributed and with the strongest pathogenicity amongst carnivore parvoviruses. To date, there is still no specific remedy for this disease. It is of great significance to develop highly effective anti-FPV antibodies.A strong strain of feline parvovirus was obtained from the isolations of a cat which was suspected to be died of FPV infetion in March, 2008, by cell sensitivity test, physical and chemical tests, morphological examination, HA and HT tests, animal infections, and sequencing of PCR produtcs for characteristic sequence.Suspensions were prepared from the tissues of kidney, intestine, and liver respectivesly and tested by FPV Golden sign indicator paper (made in Korea), which gave rise to a positive result. The F81 cells were used for propagation of FPV, and typical pathological changes appeared at the fourth generation. Electronic microscopy showed that thediameter of the virion was about 20-24nm. Physical and chemical features was coincide with characteristics of DNA viruses, such as free of envelope, high resistance to heat, chloroform ,ether and trypsin. The virus made blood clot easily to porcine blood cells under 4℃. The HI test showed a hemagglutinin titer of 256 folds, and HA test with FPV positive serum showed a inhibitory titer ofmore than 32 folds. As the primary structural protein, VP2 is the main protection antigen inducing the organism to produces the neutralizing antibody and determining the specificity of host. The result of PCR with universal primer was positive, indicating the virus was a kind of parvovirus. Two pairs of specific primers were used for further PCR testing, results showed consistency with the expected size of the design. The complete sequence of VP2 amplified by PCR was about 1755bp. Compared with the sequence of the 11 FPV strainws published previously , their homology was up to 99%. Only 5 to 10 base pairs were mutated but there is no change in the key bases and amino acids determining its host range,pathogenicity and hemagglutinin.The virus was highly susceptible to experimental cats,because infection rate was 100% of the kittens tested by PCR for feces samples 5 days after artificial infection, and mortility was 66.7%. Conclusively, the strain we isolated is a strong strain of parvovirus. Basic and high degree immunizations against the live FPV were conducted in two horses with the 4-time-concentrated FPV culture medium as antigen, white oil - Twain and astragalus polysaccharide as adjuvants. Antibody titers were detected by ElISA on d 1,7,14,40, 60 and 90 and the results showed that the highest titer appeared on d 60 and decreased gradually thereafter and finally trended to stable.There was no singificant difference in antibody titers between two adjuvants. During the early stage of immunization, greater immune response was found in white oil–Twain treated horse while no response in astragalus polysaccharide treated horse. Experiments showed that horses have good immunogenicity for FPV, effectively resisting the strain of feline parvovirus withoutany clinical symptoms and uncomfortable signs.It suggests that immunization against FPV is safty and reliable.Plasma was seperated by natural sedimentation at 4℃and cold ethanol method were compared with the traditional IgG preparation methods like the ammonium sulphate precipitation and bitter - ammonium carbonate precipitation. The optimal restriction tempreture and enzyme dosage was exploreed as well.The result showed that it was best to use pepsin at 37℃for 1h,or 30℃for 2h. We optimized a new method of purification of IgG, by digesting the serum at37℃with pepsase, removing albumin in caprylic acid, and the twice ammonium sulfate precipitation. According to the character that Protein A and Fc of immunoglobulin can connect with IgG of most mammals, the F (ab)'2 was collected and purified by Protein A column filter.The spectrophotometry showed that 12.59mg IgG was obtained from 1mL serum by the new method which increased by 30% than the traditional methods of caprylic acid and ammonium sulfate precipitation. SDS-PAGE gel electrophoresis and thin-layer scanning demonstrated that IgG prepared by optimized bitter - ammonium sulfate salting-out precipitation method showed clear electrophoretic bands and high purity.According to the relavant items of "People's Republic of China States Pharmacopoeia" (2005 version), The physical properties, heat antigenicity, safety, aseptic, and shelf life of this biological product of horse anti- FPV IgG were examined.The results showed that: the product is a milk white solution that is non-layered, free of visible particles, bacteria, fungi, mycoplasma and other microbial contaminations. There is no changes in physical and chemical properties and no reducction of hemagglutination inhibition titer after store for 90 days at room temperature. Rabbits, mice, and guinea pigs injected with the product was observed for 10 successvie days and showed no significant increase in body temperature, no reduction in body weight, and good status of appetite, excrement, and spirit, suggesting the safty and stabilityThermogenic test was conducted by injected with the product in 3 rabbits. The results showed that the average increase in temperature of rabbits were lower than 0.6℃, the total increase was d 1.2℃, less than the standard limitation of 1.4℃. The potency was evaluated by HA and HI test, agarose double diffusion, cells neutralization test and animal treatment test. The results showed that the product is highly specific with the titer of 128-fold. Six artificial FPV- infected cats onsetted the disease on scheduled time. Five of the six cats were treated by 3 injections of 2ml of the product on every other day. The clinical symptoms of all the 5 cats disappeared 7 days later with the recovery rate of 100%, indicating the high effectiveness of the product in treatment of feline panleukopenia.Conclusively, a strong FPV strain was isolated and identified and a safty, highly specific product of horse anti-FPV antibody was produced by improved techniques. |
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