中华绒螯蟹嗜水气单胞菌口服疫苗的制备及其免疫效应研究
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摘要
本文研究了含或不含β-葡聚糖佐剂的嗜水气单胞菌全细胞口服疫苗对中华绒螯蟹免疫功能及抗病力的影响、嗜水气单胞菌微胶囊口服疫苗制备工艺和嗜水气单胞菌微胶囊口服疫苗对中华绒螯蟹免疫功能及抗病力的影响。
将嗜水气单胞菌全细胞和含β-葡聚糖佐剂的全细胞口服疫苗(佐剂与疫苗的比例为1:1)的冻干粉分别以0(对照)1、3、6 g/kg的剂量添加到基础饲料中口服免疫中华绒螯蟹7天。在停喂试验组饲料后0、1、2、3周,分别采样进行一系列免疫学指标的测定;并在停喂试验组饲料后第2周,以1.2×107 cfu/kg体重的剂量,体腔注射致病性嗜水气单胞菌CL99920菌株,记录接种10d后中华绒螯蟹的累积死亡率。结果显示,适量剂量的嗜水气单胞菌全细胞疫苗能显著提高THC与HC,显著增强血细胞吞噬活性与呼吸爆发活性,显著提升血清酚氧化酶(PO)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、溶菌酶(LSZ)活性和肝胰腺POD、ACP、ALP、LSZ活性(P<0.05),能明显增强中华绒螯蟹对致病性嗜水气单胞菌感染的免疫保护力,最高免疫保护率57.14%。适量剂量的含β-葡聚糖佐剂的嗜水气单胞菌全细胞疫苗能显著提高THC,显著增强血细胞吞噬活性与呼吸爆发活性,显著提升血清PO、SOD、ACP、ALP、LSZ活性和肝胰腺POD、SOD、ACP、LSZ活性(P<0.05),能明显增强中华绒螯蟹对致病性嗜水气单胞菌感染的免疫保护力,最高免疫保护率为57.14%。结果表明,β-葡聚糖对嗜水气单胞菌全细胞疫苗具有弱的佐剂效应。
以生物相容性良好的海藻酸钠为壁材,嗜水气单胞菌灭活疫苗为囊心,采用乳化法制备微胶囊。通过单因素试验和正交试验,确定了嗜水气单胞菌微胶囊化的较佳工艺条件:海藻酸钠浓度为2%,菌胶比例为1:8,乳化时间为15 min,搅拌速度为800 rpm,包封率为71.34%。以该工艺制备的微胶囊具有良好的耐酸性和肠溶性。
将嗜水气单胞菌微胶囊口服疫苗的冻干粉分别以0(对照)1、3、6 g/kg的剂量添加到基础饲料,采用与试验一相同的试验方法。结果显示,适量剂量的疫苗能显著提高THC,显著增强血细胞吞噬活性与呼吸爆发活性,显著提升血清PO、POD、SOD、ACP、ALP、LSZ活性和肝胰腺POD、SOD、ALP、LSZ活性,能明显增强中华绒螯蟹对致病性嗜水气单胞菌感染的免疫保护力,最高免疫保护率为71.43%。结果表明,微胶囊疫苗的效果优于全细胞疫苗和加β-葡聚糖的全细胞疫苗,可以在生产中试用,建议使用3 g/kg饲料的剂量。
Effects of Aeromonas hydrophila whole-cell oral vaccine with or withoutβ-glucan adjuvant on the immunological function and disease resistance of Chinese mitten crab Eriocheir sinensis, preparation of microcapsules of A. hydrophila and effects on immuno- logical function of E. sinensis were studiesd in the paper. The results are summarized as follows:
Experiment one: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of A. hydrophila whole-cell vaccine, hydrophila whole-cell vaccine andβ-glucan (1:1) dosed at 0, 1 3 or 6 g/kg dry diet for 1 week, respectively. The total haemocyte count (THC), different haemocyte count (DHC), the phagocytic activities, respiratory burst (release of superoxide anion) activities of haemocytes, the phenoloxidase (PO), nitric oxide synthase (NOS) activities and total antioxidative competencein (T-AOC) in serum, the peroxidase (POD), superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (ALP) and lysozyme (LSZ) activities in serum or hepatopancreas were examined at 0, 1, 2, and 3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results were as follows: (1) The THC and HC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, POD, SOD, ACP, ALP and LSZ activities in serum, the POD, ACP, ALP and LSZ activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine by certain dose were significantly higher than control group at many sampling time points (P<0.05). However, no significant differences were observed in the SGC and GC, the T-AOC, NOS activities in serum, and the of SOD activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine compared with control group (P>0.05). Furthermore, the crabs fed with the A. hydrophila whole-cell vaccine were the more resistant. The best immune protection rate was 57.14% at the group of 3 g/kg dry diet. (2) The THC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, SOD, ACP, ALP, LSZ, and NOS activities in serum, the POD, SOD, ACP and LSZ activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine withβ-glucan adjuvant by certain dose were significantly higher than control group at many sampling time points (P<0.05). However, no significant differences were observed in the DHC, the POD activities, T-AOC in serum, and the of ALP activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine withβ-glucan adjuvant compared with control group (P>0.05). Furthermore, the crabs fed with the A. hydrophila whole-cell vaccine withβ-glucan adjuvant were the more resistant. The best immune protection rate was 57.14% at the group of 3 g/kg dry diet.
Experiment two: The sodium alginate which has good biology consistent was used as shell-material and the A. hydrophila whole-cell vaccine was used as core-material, then preparing microcapsulation with emulsification method. The optimal technological conditions of making the microcapsule were determined through single factor test and orthogonal experiment of L9 (34). The results were as follows: the concentration of sodium alginate was 2%, the emulsification time was 15 min, the stirring speed was 800 rpm, the proportion of A. hydrophila whole-cell vaccine and sodium alginate was 1:8. This method can prepare microcapsules which have good enteric solubility and acid resistance, embedding rate was 71.34%.
Experiment three: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of A. hydrophila microencapsulated vaccine dosed at 0, 1 3 or 6 g/kg dry diet for 1 week, respectively. The THC, DHC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, NOS activities and T-AOC in serum, the POD, SOD, ACP, ALP and LSZ activities in serum or hepatopancreas were examined at 0, 1, 2, and 3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of A. hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results were as follows: The THC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, POD, SOD, ACP, ALP, LSZ, and NOS activities in serum, the POD, SOD, ALP and LSZ activities in hepatopancreas of E. sinensis fed with the A. hydrophila microencapsulated vaccine by certain dose were significantly higher than control group at many sampling time points (P<0.05). However, no significant differences were observed in the DHC, the T-AOC in serum, and the of ACP activities in hepatopancreas of E. sinensis fed with the A. hydrophila microencapsulated vaccine compared with control group (P>0.05). Furthermore, the crabs fed with the A. hydrophila microencapsulated vaccine were the more resistant. The best immune protection rate was 71.43% at the group of 3 g/kg dry diet.
将嗜水气单胞菌全细胞和含β-葡聚糖佐剂的全细胞口服疫苗(佐剂与疫苗的比例为1:1)的冻干粉分别以0(对照)1、3、6 g/kg的剂量添加到基础饲料中口服免疫中华绒螯蟹7天。在停喂试验组饲料后0、1、2、3周,分别采样进行一系列免疫学指标的测定;并在停喂试验组饲料后第2周,以1.2×107 cfu/kg体重的剂量,体腔注射致病性嗜水气单胞菌CL99920菌株,记录接种10d后中华绒螯蟹的累积死亡率。结果显示,适量剂量的嗜水气单胞菌全细胞疫苗能显著提高THC与HC,显著增强血细胞吞噬活性与呼吸爆发活性,显著提升血清酚氧化酶(PO)、过氧化物酶(POD)、超氧化物歧化酶(SOD)、酸性磷酸酶(ACP)、碱性磷酸酶(ALP)、溶菌酶(LSZ)活性和肝胰腺POD、ACP、ALP、LSZ活性(P<0.05),能明显增强中华绒螯蟹对致病性嗜水气单胞菌感染的免疫保护力,最高免疫保护率57.14%。适量剂量的含β-葡聚糖佐剂的嗜水气单胞菌全细胞疫苗能显著提高THC,显著增强血细胞吞噬活性与呼吸爆发活性,显著提升血清PO、SOD、ACP、ALP、LSZ活性和肝胰腺POD、SOD、ACP、LSZ活性(P<0.05),能明显增强中华绒螯蟹对致病性嗜水气单胞菌感染的免疫保护力,最高免疫保护率为57.14%。结果表明,β-葡聚糖对嗜水气单胞菌全细胞疫苗具有弱的佐剂效应。
以生物相容性良好的海藻酸钠为壁材,嗜水气单胞菌灭活疫苗为囊心,采用乳化法制备微胶囊。通过单因素试验和正交试验,确定了嗜水气单胞菌微胶囊化的较佳工艺条件:海藻酸钠浓度为2%,菌胶比例为1:8,乳化时间为15 min,搅拌速度为800 rpm,包封率为71.34%。以该工艺制备的微胶囊具有良好的耐酸性和肠溶性。
将嗜水气单胞菌微胶囊口服疫苗的冻干粉分别以0(对照)1、3、6 g/kg的剂量添加到基础饲料,采用与试验一相同的试验方法。结果显示,适量剂量的疫苗能显著提高THC,显著增强血细胞吞噬活性与呼吸爆发活性,显著提升血清PO、POD、SOD、ACP、ALP、LSZ活性和肝胰腺POD、SOD、ALP、LSZ活性,能明显增强中华绒螯蟹对致病性嗜水气单胞菌感染的免疫保护力,最高免疫保护率为71.43%。结果表明,微胶囊疫苗的效果优于全细胞疫苗和加β-葡聚糖的全细胞疫苗,可以在生产中试用,建议使用3 g/kg饲料的剂量。
Effects of Aeromonas hydrophila whole-cell oral vaccine with or withoutβ-glucan adjuvant on the immunological function and disease resistance of Chinese mitten crab Eriocheir sinensis, preparation of microcapsules of A. hydrophila and effects on immuno- logical function of E. sinensis were studiesd in the paper. The results are summarized as follows:
Experiment one: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of A. hydrophila whole-cell vaccine, hydrophila whole-cell vaccine andβ-glucan (1:1) dosed at 0, 1 3 or 6 g/kg dry diet for 1 week, respectively. The total haemocyte count (THC), different haemocyte count (DHC), the phagocytic activities, respiratory burst (release of superoxide anion) activities of haemocytes, the phenoloxidase (PO), nitric oxide synthase (NOS) activities and total antioxidative competencein (T-AOC) in serum, the peroxidase (POD), superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (ALP) and lysozyme (LSZ) activities in serum or hepatopancreas were examined at 0, 1, 2, and 3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of Aeromonas hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results were as follows: (1) The THC and HC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, POD, SOD, ACP, ALP and LSZ activities in serum, the POD, ACP, ALP and LSZ activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine by certain dose were significantly higher than control group at many sampling time points (P<0.05). However, no significant differences were observed in the SGC and GC, the T-AOC, NOS activities in serum, and the of SOD activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine compared with control group (P>0.05). Furthermore, the crabs fed with the A. hydrophila whole-cell vaccine were the more resistant. The best immune protection rate was 57.14% at the group of 3 g/kg dry diet. (2) The THC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, SOD, ACP, ALP, LSZ, and NOS activities in serum, the POD, SOD, ACP and LSZ activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine withβ-glucan adjuvant by certain dose were significantly higher than control group at many sampling time points (P<0.05). However, no significant differences were observed in the DHC, the POD activities, T-AOC in serum, and the of ALP activities in hepatopancreas of E. sinensis fed with the A. hydrophila whole-cell vaccine withβ-glucan adjuvant compared with control group (P>0.05). Furthermore, the crabs fed with the A. hydrophila whole-cell vaccine withβ-glucan adjuvant were the more resistant. The best immune protection rate was 57.14% at the group of 3 g/kg dry diet.
Experiment two: The sodium alginate which has good biology consistent was used as shell-material and the A. hydrophila whole-cell vaccine was used as core-material, then preparing microcapsulation with emulsification method. The optimal technological conditions of making the microcapsule were determined through single factor test and orthogonal experiment of L9 (34). The results were as follows: the concentration of sodium alginate was 2%, the emulsification time was 15 min, the stirring speed was 800 rpm, the proportion of A. hydrophila whole-cell vaccine and sodium alginate was 1:8. This method can prepare microcapsules which have good enteric solubility and acid resistance, embedding rate was 71.34%.
Experiment three: The purpose of this study is to determine the variance of immune function of E. sinensis after being fed with the diets added lyophilized powder of A. hydrophila microencapsulated vaccine dosed at 0, 1 3 or 6 g/kg dry diet for 1 week, respectively. The THC, DHC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, NOS activities and T-AOC in serum, the POD, SOD, ACP, ALP and LSZ activities in serum or hepatopancreas were examined at 0, 1, 2, and 3 weeks after last administration with the diets of test groups. Crab were challenged at 1.2×107 colony forming units (cfu) /kg crab weight with a virulent strain of A. hydrophila (CL99920) of E. sinensis at 2 weeks after last administration with the diets of test groups, and mortalities were recorded over a 10-day period. The results were as follows: The THC, the phagocytic activities, respiratory burst activities of haemocytes, the PO, POD, SOD, ACP, ALP, LSZ, and NOS activities in serum, the POD, SOD, ALP and LSZ activities in hepatopancreas of E. sinensis fed with the A. hydrophila microencapsulated vaccine by certain dose were significantly higher than control group at many sampling time points (P<0.05). However, no significant differences were observed in the DHC, the T-AOC in serum, and the of ACP activities in hepatopancreas of E. sinensis fed with the A. hydrophila microencapsulated vaccine compared with control group (P>0.05). Furthermore, the crabs fed with the A. hydrophila microencapsulated vaccine were the more resistant. The best immune protection rate was 71.43% at the group of 3 g/kg dry diet.
引文
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