| Locoweed is one of the most serious toxic plants in the word, often caused a large number of livestock deaths, fetation miscarriage, premature birth, stillbirth and fetal dysplasia, high rate of empty fetation, Sire infertility etc, resulting in great loss of animal husbandry. So, many measures were taken to reduce the locoweed's harm, such as excavation, biological control, chemical elimination, adding antidotes, rotation grazing and so on. So far, however, this problem has never been completely resolved. Research has shown that swainsonine (SW) is the main Locoweed toxic ingredients. People carried out a lot of study on the physical and chemical properties of the SW, poisoning mechanism, pharmacological activity and toxicological. So how to prevent the locoweed poisoning of animals by the method of immunization has become a research hotspot. In this study, chloromethyl benzoic acid was choiced as spacerarm between the SW hapten and the bovine serum albumin (BSA) carrier. Synthetizing the artificial antigen SW-BSA, selecting the best combination of it and the immunological adjuvant was carried to do some immunological studies on rabbits and goats. The results are as follows:1. Acording to the principle of liquid-liquid extraction of various substances dispersed, the SW extracting from Locoweed toxic plants was optimized so that the extraction rate of SW was significantly improved to 50 mg/kg and 64 mg/kg or more from Astragalus Variabilis Bunge and Oxytropis kansuensis Bunge, which faciliate the further study of SW.2. The chloromethyl benzoate was first introduced as spacerarm between SW and BSA to synthesize SW quaternary ammonium salt compound , after every compounds were identified by poppen analysis, then to get SW-BSA by the methord of EDC. It was success by ultraviolet spectra.3. The four kinds of combination ratios between SW and BSA in artificial synthetic antigen SW-BSA were determined by UV method, and they were 14.7, 20.2, 25.3 and 32.6 respectively. Then the immunity test was carried out with SW-BSA in mice, the results of which showed that the four kinds of SW-BSA in mice produced quite high titers of antibodies by indirect EILSA, among of them the titer highest ratio was 20.2. So it indicated that the best coupling rate of SW-BSA was 20.2.4. Freund's, propolis and white oil adjuvant vaccine were made of coupling ratio of 20.2 in the SW-BSA, then immunized rabbits and the titers of rabbits serum were detected by indirect EILSA. The test results showed that both Freund's adjuvant vaccine and oil adjuvant vaccines had a higher titer. Because Freund's adjuvant vaccine, side effects, it was generally only used for the pilot study, This experiment use white oil adjuvant vaccine trial for further immunological studies, which provide the basis for the production practice.At the same time, indirect blocking ELISA test and agar double diffusion tests showed that specific antibodies of SW in the rabbit body was produced.5. The gas chromatography analysismethord was established to detect SW in rabbit serum, after rabbit serums were dealed with adding acetone, ultrasound and centrifugal purification, freeze-drying and silylation derivatization. The linear range of this method was 0.000 08 g/L ~ 2.5 g/L. The correlation coefficient wasγ= 0.999 6, the recovery of standard addition was 89.81% ~ 96.23% in the 0.001 g/L ~ 1.0 g/L 4 levels. The relative standard deviation was 2.46% ~ 4.35%. This method is suitable for detecting swainsonine in rabbit serum.6. Rabbits were immuned with SW-BSA vaccined twice, the serum antibody titer changes were tested by indirect ELISA assay and attacking toxic with Oxytropis kansuensis (10 g/kg/d) when the antibody titers were in higher. At the same time, the serum biochemical indicators were detected.The results showed that the time of clinical toxic symptom of the infection immune rabbits was delayed 30 d than the control group, the concentrations of SW in serum were delayed for 21 d to rise at higher level in the immunized and poisoning group compared with poisoning control group, the activity level of AST, ALP, LDH, BUN in serum were delayed for 31 d, the descending activity ofα-mannosidase was delayed for 28 d compared with poisoning control group. The pathological changes of tissues and organs was acute toxicity, hypoxia-ischemia and vacuolar degeneration.7. SW-BSA vaccine was used twice to rabbits, the serum antibody titer changes were tested by indirect ELISA assay and attacking toxic with Oxytropis kansuensis (10 g/kg/d) when the antibody titers were in higher level. At the same time, the serum biochemical indicators were detected. The results showed that the goats of infection control group presented the typical toxic symptoms in the 15th~20th day after attacking toxin, while the goats of immunized and poisoning in the 50th ~ 55th day, the time was delayed 30 d~40 d. The biochemical indicators in serum of control group changed rapidly after attacking toxic while immune group were progressive. In which SW concentration, the activity of AST, ALP, LDH, BUN,α-mannosidase respectively were delayed for 27 d, 24 d, 27 d, 30 d, 24 d, 30 d to reach a higher or lower point. The pathological changes of tissues and organs was the same acute toxicity, hypoxia-ischemia and vacuolar degeneration as rabbits.These results show that the artificial synthetic antigen SW-BSA vaccine has a protective effect to rabbits and goats. It can delay the locoweed poisoning time of animals 30 d ~ 40 d and provide a basis for the SW-BSA vaccine clinical trial. |
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