| Objective To establish a favorable condition for tree shrew's hand-breeding and then to explore the ways to optimize the tree shrew's model of HBV infection, by inoculating HBV positive serum to the perinatal and young tree shrews as well as by modulating the method for sensitively and reliably detect the maker of HBV infection.Method Part one: ①The male and female adult tree shrews were matched for breeding pairs. Files were made for each animal that recorded the reproductive situation. Calculated the numbers of all pups, the annually average childbirth and the average day between the first day of cohabitation and day of delivery. Recorded the pup's diet and body weight daily. ②The adult male and female tree shrews who had none of the pregnant record in the last year were randomly divided into two groups. Female animals in the group A were injected 10ug of PG Intramuscularly twice, with 10 days interval. The males in this group were with no treatment and cohabitated with females all the time. The female animals in the group B were treated in the same way as the females in group A. The males in this group were separated from the females at the beginning. Ten days later, when the females were getting the second dose of PG, each of the males were received 10U of Pregnant Mare Serum Gonadotropin (PMSG) intramuscularly and then cohabitated with females immediately. The reproductive ability between the two groups was compared. ③Compared the health condition of the pups that were only fed with the formulated milk and that were passively maternal milked combined with feeding formulated milk. Part two: ①The perinatal and young tree shrews were inoculated with human HBV-positive serum by the ways of injecting the serum to the newborns and cubs directly, or to the females in the terminal pregnant for vertical infecting the fetus. Using the technique of Enzyme-Lined Immunosorbent Assay (ELISA) to detect hepatitis B virus surface antigen(HBsAg), e antigen (HBeAg) and core antibody (Anti-HBc) in serum, as well as applying the nested Polymerase Chain Reaction (nPCR) to detect HBV DNA in the samples of liver biopsy and serum from the infected pups. The amplified products from PCR were sequenced and compared with the sequences of C and B fragments of human HBV respectively. The liver biopsies were observed histopathologically. Result Part one: ?35 female tree shrews gave birth to 400 pups in 147 litters during 16 months. The annual average of childbirth for each female was 3.2 litters and 8.7 pups. The average day between the first day of cohabitation and delivery was 68±32. A total of 400 newboras consisted of 95(23.8%) stillbirth, 83(20.8%) died of being eaten or bitten by females, 16(4.0%) abortion and 206(51.5%) survivals. ?During the 5 months of the non-reproductive season, none of the females in group A produced any pups after the drug treatment, while 8 of 13 females in group B gave birth to newborns. The effective rate of the ixeatment for group B was 61.5%, which was significantly higher than that for group A (/?<0.01) . (3)The average body weight of the newboras fed only by formulated milk in the ages of H^-day and 24*-day was 20.8±3.0g and 33.7±3.9g respectively, and the average body weight of the pups fed by passive maternal milk combined with formulated milk was 41.7±7.9g and 79.5±15.0g respectively. The latter was significantly higher than the former (all /?<0.01) . There was no significantly different between the quantities of daily diet among the pups. Until 25th-day old, the total survival rate of hand rearing tree shrew pups was 92.2% (190/206). Part two: 42 pups inoculated human HBV-positive sera were examined. ?The result of ELISA showed that HBsAg became transiently detectable in the serum samples from 16(38.1%) pups 4 weeks after inoculation, 2 of them(4.8%) were also detected HBeAg simultaneously. 15(35.7%) and 2(4.8%) pups were detected Anti-HBc and HBeAg respectively, 3(7.1%) were detected both Anti-HBc and HBeAg, otherwise there were 6(14.3%) pups that couldn't detect any makers of HBsAg, Anti-HBc or HBeAg. ?The result of nPCR on liver biopsy showed that 20 of 42 (47.6%) pups became HBV DNA positive during the period of 8 to 81 weeks after inoculation. 3(23.1%) from the 13 pups inoculated young were HBV DNA-positive, and 17(58.6%) from 29 pups inoculated perinatally were HBVDNA-positive, which included 2 pups transmitted vertically by the inoculated females. The positive rate of the two groups were significant difference (x2=4.546, p<0.05 ) .(3)The result of nPCR on serum showed 30 of 42 (71.4%) pups displayed HBV DNA-positive totally, while most of them (19/42, 45.2%) only displayed HBV DNA once within 4 to 6 week after inoculation. Part of them could detect twice (7/42, 16.7%) or thrice (4/42, 9.5%). 12 (28.6%) pups were negative for HBV DNA. There was one pup that could detect HBV DNA in both samples of serum and liver tissue simultaneously. ?The fragments of HBV C and S amplified from tree shrew serum and liver were similar to that of correspondent fragments of HBV genomes, with 98.0% and 99.0% homology respectively. (5) The leading pathological changes in liver tissue were acute hepatitis alike, since 58.7% of the samples showed cytoplasm swelling or loosening of liver cells, with inflammatory cells infiltrating, spotty necrosis and proliferation of liver cells, the hepatic architecture was still clear however. Lightly chronic hepatitis was seen in 24.0% of the samples, with the proliferation of fibrous cells and/or bile ducts, the expansion of portal tracts by inflammatory cells infiltration. Degeneration or death of hepatocyte was not conspicuous. 17.3% of the samples had no evident pathological changes. There was no extensive fibrosis in all of the liver samples.Concludes ?Hand breeding could improve the reproductive ability of the adult tree shrews, treated male and female tree shrews with hormone drugs simultaneously could promote females become pregnant during the non-breeding season. ?Hand-rearing tree shrew pups could enhance their survival rate, and the method of the passive maternal milking combined with formulated milk feeding is convenient and easy to operate. ?After inoculating the perinatal and young tree shrews with HBV-positive serum, HBV DNA can be detected in animals serum and/or liver tissue through the method of nPCR with proper primers, while HBsAg, HBeAg and Anti-HBc can be detected through the assay of ELISA, and the hepatitis-like changes could be observed histopathologically. ? HBV can be transmitted vertically to the newborns by inoculating HBV-positive serum into the females in terminal pregnant. The data revealed that the infection rate of newborns was superior to the youths. These results indicated that using hand-rearing...
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