The Herd Purity Technology Of Laboratory Mini-pig And The Establishment And Application Of Its Various Models

1. The study on the safety-control technology of SPF mini-pigThe study on the safety-control techniques of SPF mini-pig was carried out on a herd of the scale of 20 sows, with the amount of livestock on hand 350 a year. We basically aim to control the chronic infectious disease on which the vaccines have low effect, including: Mycoplasma Pneumonia of Swine, Atrophicrhintis, Swine dysentery, Pseudorabies, Transmissible gastroenteritis of pigs, etc. The main control method is through the control of the source (by Caesarean birth), environment, personnel, vehicles, articles, rodents, birds, flies and other insects, feeds and water, and the assay monitors. We realized the favourable environment of bio-security for the SPF mini-pig through setting up an artificial barrier. The experiment continued on three years with 1016 pigs, among which the total survival amount was 965 (the total survival rate is 95%). Moreover, we have eliminated two decades of acute or chronic infectious disease besides the 7 aimed specific pathogenies. The experiment indicates that as long as we strictly adhere to the safety epidemic preventive measures, the protection of SPF mini-pig is definitely reachable.2. Grade of pathogenic microorganism and parasite for experimental mini-pigs and its monitoring StandardsThe two national standards《The microorganism grading and monitoring for experimental animals》(GB14922.2 2001 ),and《The parasite grading and monitoring for experimental animals》(GB14922.1 - 2001) authorized by the National Technical Supervise Bureau on Aug, 29th, 2001 are mainly used with the aspect of rodents. However, the standards could not serve to the microorganism classification or monitoring for the mini-pig. Given that at present more and more domestic mini-pig producers or institutions require the experimental animal license to supply the standard experimental pigs but have no national standard to refer to, it is high time that we should constitute the specific standards or criterion for the mini-pig with the aspect of microorganism and parasite grading and monitoring. After looking up to the relevant national standards and making sound investigation of the research and development status of the mini-pig, we submit National Technical Supervise Bureau the overture and make the standard draft. In this standard, we prescribe the definition, grading of the experimental mini-pig and the monitoring regulation of the pathogenic microorganism and parasite for them.3. The entirely artificial nursing experiment of SPF mini-pigThe caesarean operation is the route to obtain the sterile piglets. The effect of the operation is largely relevant to the anaesthesia methods.To verify the feeding effect of the artificial milk, we adopted an entirely artificial nursing technology for the mini-pig in isolated environment. We compared its growth performance, livability, feed intake and conversion efficiency, blood physiological and biochemical parameters with conventional natural delivery piglets. The result indicated that we could produce qualified experimental SPF mini-pig by using the artificial milk in the light of scientific managemen..4. Molecular Analysis of Intestinal Mieroflora of SPF PigletsObjective: To monitor and characterize the structural features of intestinal microbial communities in fecal samples of SPF( specific pathogen free) piglets and conventional piglets. Methods: The newborn piglets were transferred from the womb into a barrier system immediately after Caesarean operation.The genomic DNA of bacteria was extracted from each sample and ERIC-PCR amplification was then carried out. The PCR fingerprints were southern-blotted with a mixed dig-tagged probe from the labeled ERIC-PCR product of one sample. Results: The banding-patterns of samples of SPF piglets and conventional piglets elucidate poor similarities before, during and after weaning respectively. Simultaneously there really existed much difference in the aspect of the structural features of intestinal microflora between SPF piglets and conventional piglets on the DNA level.5. Growth, blood physiological and blochemical parameters of SPF mini-pigTo investigate the growth and blood physiological and biochemical parameters in different age of BAMA SPF mini-pig, 50 pigs aging from 10～150 days were used in this experiment. The blood samples were collected via the superior vena cava, while the blood parameters, hormone contents and biochemical parameters in plasma were analyzed.Growth: BAMA mini-pig grew faster during the first 10～50 day. The RGR at day 20 was the highest, and it declined to the slowest on day 40. Hormone contents: The GH level was the lowest on day 10, and it reached its highest on day 120. The T₃ level during day 10～90 was higher than those on the other days, it was significantly the highest on day 20. The T₄ level was lowest at day 60. The TSH level on day 80 and day 150 were evidently higher than those on the other days; The Ins level climbed to its highest on day 30 and day 100, and dropped to the lowest on day 40; The Glu level was higher during day 10～60, it increased on day 70, then decreased gradually, and finally reached the lowest level on day 100, it was only 25ï¼…of day 10(p＜0.05). The Cor level fiuctated during the whole experiment.Biochemical parameters: The Blood sugar level on day 10～80 was the highest. On day 70 the blood sugar was higher than those on day 10 and day 80～150. it reached its highest on day 30; The GPT level were significantly higher on day 70 and day 80 than those on the other days.Blood parameters: RBC, The Hb and HCT levels were the highest on day 90. The WBC level was higher on day 40 than those on the other days. The largest resistance of EOF on day 10～80 were stable, and they were observably greater on day 140 and day 150 than those on the other days. The smallest resistance of EOF on day 10, day 20, day 140 and day 150 were remarkably the higher. ESR on day 10～90 were lower than those on the rest days. The results indicated that hormone contents, physiological and biochemical parameters in blood are changed with the growth of the piglets.6. Profiling of In-vivo Waning and Waxing of Porcine Transmissible Gastroenteritis Virus Inoculated Via Different PassagesThis experimentation studied the waning and waxing of TGEV inoculated by ocular passage, tracheal passage and oral passage respectively.TGEV rejuvenated on the ST cell layer before inoculating via different passages in order to pinpoint viral replicative sites and in-vivo waning and waxing. 25 days old specific pathogen-free (SPF) piglets were inoculated via ocular, tracheal or oral passages respectively. One SPF piglet was killed every 24 hours after inoculation, and samples of tissues and blood were collected and detected by immunofluoescent antibody assay (IFA) and reverse-transcription PCR (RT-PCR). The tissues collected included stomach, duodenum, jejunum, ileum, lung, lymph node, heart, trachea, liver, spleen and tonsil. TGEV antibody titers of all piglets were measured by ELISA. The results are as follows: Ocular passage: None of the fifteen SPF piglets showed any clinical signs and lesions. The IFA results of all samples were negative, but the RT-PCR results of lung and intestine were positive 30 days after inoculation. OD value of antibody didn't increase on post inoculation days (PID) 1-20, of which the lowest OD value was 0.21 and the highest was 0.30, but suddenly boosted on PID 22. The result showed that TGEV couldn't replicate in vivo after inoculating ocularly. The virus exuded with tears, which caused the spread of TGEV in the environment after uptaking it. This study confirmed that TGEV could not be infected by ocular passage firstly.Tracheal passage: All 25-day old piglets developed respiratory symptoms 24 hours after inoculation, but eventually recovered. There were different lesions of the lungs. TGEV Antibody titers of all pigs were measured by ELISA. Therefore, there was age-dependent resistance to TGEV infection. IFA and RT-PCR results of samples of trachea (PID 2-10), lung (PID 2-30), and spleen (PID 2-5) were positive, while all samples of intestinal tract were negative. The result confirmed viral waning and waxing in the respiratory system.Oral passage: Four of thirty 25-day old piglets developed diarrhea on PID 3, and three died on PID 6, 9 and 17 respectively. There were significant lesions of intestines, three of which had the different lesions of lungs. IFA results of samples collected from intestinal tract and lymphatic node were positive 48 hours after inoculation, and the number of positive cells continuously increased. The IFA results of tonsil, spleen and systemic lymphatic nodes were also positive. The results of RT-PCR showed that spleen of 3-5 days was positive. The OD value of antibody boosted from 0.07 to 1.54. There is age-dependent resistance to TGEV infection. The result confirmed viral waning and waxing in the digestive and respiratory system, and oral passage was the main way resulting this diseases.TGEV in spleen could be detected on PID 2-5 by both tracheal passage and oral passage, which showed that TGEV could not replicate in spleen.7. Study on establishment of Human flora associated mini-piglet model and molecular analysis of SPF piglet's intestinal microbiotaTo investigate the availability of HFA piglet model, we selected five litters of Bama Xiang mini-pigs that were delivered by caesarean section. This study included two parts. On one hand, man-rearing procedures of caesarean piglets in barrier facility were optimized and colonizing status of donor' intestinal microbiota in the piglets' gut was evaluated by molecular methods as ERIC-PCR and microbial community molecular hybridization. On the other hand, the structural features of SPF piglets' intestinal microbiota were analyzed by the above techniques.Five litters of Bama mini-piglets were delivered by caesarean section and then man-reared in barrier facility. During the first three days, donor' fecal suspension was inoculated orally into piglets daily and then geared to once every other day until on 11^th day. On the 5^th, 9^th, 12^th day piglet's fecal samples were collected and frozed at -70℃. The results showed that human microbiota could be partly colonized and stabilized relatively in the piglets' gut by ERIC-PCR and microbial community molecular hybridization analysis while the difference between the piglets were negligible.