鸡球虫早熟株免疫效力及EtATPase基因特性研究
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摘要
1975年Jeffers创立了球虫早熟株选育技术，与母株相比，球虫早熟株具有独特的生物学特性：潜在期明显缩短，最后一代裂殖体缺失或缺陷，致病力和繁殖能力明显降低，保持良好的免疫原性，具有较好的遗传稳定性。国外已研制出商品化的鸡球虫早熟弱毒活疫苗Paracox和Livacox，在大规模试验和田间试验中都表明具有良好的免疫效果。本论文以所在实验室早期选育出的堆型艾美耳球虫（Eimeria acervulina）、巨型艾美耳球虫（E. maxima）、柔嫩艾美耳球虫（E.tenella）3个早熟株为对象，对其药物敏感性、免疫剂量、早熟株疫苗的免疫效力、ATPase基因等进行了研究，为球虫早熟株疫苗的开发提供依据。
1.3个球虫早熟株的药物敏感性研究
为评价堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株对常用抗球虫药物的敏感性，选用地克珠利、氯苯胍、癸氧喹酯、尼卡巴嗪、二硝托胺、马杜米星、拉沙洛西、盐霉素等8种药物进行试验，以最适抗球虫活性百分率（POAA）、病变记分减少率（RLS）、相对卵囊产量（ROP）、抗球虫指数（ACI）为试验指标进行综合评定。结果显示，堆型艾美耳球虫早熟株和母株药物敏感性一致，均对二硝托胺不敏感，对盐霉素轻度敏感，对地克珠利、氯苯胍、癸氧喹酯、尼卡巴嗪、马杜米星、拉沙洛西等6种药物敏感，证明遗传背景相同的虫株，其药物敏感性也相同；巨型艾美耳球虫早熟株和柔嫩艾美耳球虫早熟株对8种药物均相对敏感。结果表明，3个球虫早熟株对常用抗球虫药物均较敏感，可用于球虫弱毒活疫苗的研制。
2.3个球虫早熟株的免疫剂量研究
为确定堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株合适的免疫剂量，比较堆型艾美耳球虫、巨型艾美耳球虫早熟株与母株的免疫效果，每个虫株设置7个免疫剂量组（每羽鸡口服孢子化卵囊分别为100、200、400、600、800、1000、2000个），进行二次免疫（7日龄首免、14日龄二免），二免后7天（21日龄）以母株进行攻虫，攻虫后7天结束试验，以增重、肠道病变记分、卵囊减少率为试验指标，并选择2～4个免疫效果较好的剂量组进行重复试验。结果显示，堆型艾美耳球虫早熟株600～1000免疫组、巨型艾美耳球虫早熟株200～800免疫组、柔嫩艾美耳球虫早熟株600～2000免疫组的综合免疫保护效果较好，其卵囊减少率分别超过90%、95%和60%，堆型艾美耳球虫、巨型艾美耳球虫早熟株与母株的综合免疫保护效果相当；经重复试验，确定堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株推荐的免疫剂量分别为600、200、600个卵囊/羽。研究结果为配置鸡球虫三价弱毒疫苗奠定了基础。
3.早熟株三价弱毒活疫苗的免疫效力初步研究
按照堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株按600、200、600比例配成三价活疫苗，以0.25、0.5、1.0、1.5和2.0头份/羽免疫4日龄雏鸡，设商品化球虫疫苗Coccivac（1头份/羽）组和不免疫组作为对照。免疫后第4～24d进行粪便卵囊OPG计数；检测免疫后第1、7、14、21d鸡只的抗体水平、脾脏淋巴细胞数量以及鸡只增重情况；免疫后第14和21d，各组分别取10只鸡用亲本毒株混合卵囊进行攻虫试验。研究发现，早熟株三价苗免疫期间增重情况，各免疫组鸡只在免疫后7d和14d出现增重减缓的趋势，免疫21d后出现补偿性增长，相对增重率与空白对照组相当。免疫期间OPG变化情况，各免疫组共出现3个排卵高峰，其中最大的一次出现在免疫后11～12d。免疫期间抗体水平变化，在免疫后14d抗体水平迅速升高且明显高于空白对照组，免疫后21d抗体水平有所下降，但任维持在较高水平。免疫后鸡脾脏T淋巴细胞数量变化，1.0倍免疫组脾脏T淋巴细胞的增殖能力于14d明显高于健康对照组，21d达到峰值，28d维持在较高水平。攻虫试验结果发现，无论是免疫后14d还是21d，所研制的球虫弱毒活疫苗均可获得比进口疫苗Coccivac稍好的免疫效果。
4.柔嫩艾美耳球虫ATPase基因特性研究
对艾美耳球虫ATPase基因进行扩增、表达量分析、细胞定位、体外抑制等研究。研究发现，保守性分析，分别从堆型艾美耳球虫、巨型艾美耳球虫和柔嫩艾美耳球虫3株早熟株中都能扩增出ATPase，并且同源性分析发现其序列相似性高达100%，表明该基因在不同种球虫存在保守性。表达量分析，ATPase基因在三株早熟株相对于母株表达量都下调；EtATPase基因在未孢子化卵囊、孢子化卵囊、子孢子和第二代裂殖子4个发育阶段都能表达，尤其在孢子化卵囊中转录拷贝数最高；EtATPase基因表达的融合蛋白是包涵体蛋白，分子量大约是22kDa，Western-blot分析发现具有良好的抗原性。利用EtATPase重组蛋白制备的多克隆抗体和间接免疫荧光技术发现，EtATPase定位于子孢子的折光体上。体外抑制试验发现，EtATPase对球虫子孢子入侵细胞无明显的抑制作用。
The precocious line of Eimeria was first selected by Jeffers in1975. Compared with its parentsrtain, the precocious line has a reduced prepatend period for the missing or defect of the last generationschizont, markedly less pathogenicity, and much lower reproductive potential, but retained goodimmunogenicity and genetic stability. The attenuated live anti-coccidial vaccines, Paracox and Livacox,have been developed and widely used for the control of coccidiosis. Immunization with the attenuatedvaccines had been proved to be effective and is increasingly playing an important role in the control ofcoccidiosis in the poultry industry. The precocious lines of Eimeria acervulina, E. maxima and E.tenella have been selected successfully and their biological characteristics have been studied in our lab.The objective of the present study was to study the drug sensitivity and optimal immunizing dose of thethree precocious lines, and to evaluate the anti-coccidial efficacy of trivalent vaccine. The results fromthis study would help to develop the live, attenuated anticoccidial vaccine.
1. Sensitivity of three precocious lines to eight anticoccidial drug
In order to test sensitivity of precocious line of E.maxima, E.acervulina and E.tenella toanticoccidial drugs, eight anticoccidial drugs, including of diclazuril, robenidine, dinitolmide,decoquinate, nicarbazin, maduramicin, salinomycin and lasalocidt, were selected to use in the study.Based on the percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS),relative oocyst production (ROP) and anticoccidial index (ACI), the results showed that the sensitivityof the precocious line and parent strain of E.acervulina were basically similar. The two strains ofE.acervulina were completely sensitive to diclazuril, robenidine, decoquinate, nicarbazin, maduramicinand lasalocid, partly sensitive to salinomycin and not sensitive to dinitolmide.the precocious line ofE.maxima was completely sensitive to eight anticoccidial drugs, the precocious line of E. tenella waspartly sensitive to salinomycin and completely sensitive to diclazuril, robenidine, dinitolmide,decoquinate, nicarbazin, maduramicin and lasalocidt. This study will provide experimental bases fordevelopment and application of the precocious lines vaccines of coccidia.
2. Optimal immunization dose of three precocious lines
In order to test optimal immunizing doses of precocious lines of E. acervulina, E.maxima andE.tenella, dosages of100,200,400,600,800,1000and2000sporulated oocysts were administeredorally to7-day-old chickens for two times at a7-day interval.21-day-old chickens were challenged withthe sporulated oocysts of the parent strains. Weight gain, intestinal lesion scores and oocysts inhibitionratio were used to evaluate the immunizing effectiveness.2-4optimal immunizing doses obtained fromthe first trial was repeated. The results from the first trial showed that the600-1000E. acervulina,200-800E. maxima and600-2000E. tenella could induce sufficient immunological protection, whichthe reduction of oocysts ratios were over90%、95%and60%, respectively. The results from therepeated trial indicated that the optimal immunizing dose of the precocious line of E. acervulina, E.maxima and E. tenella was600,200and600ooysts/bird, respectively.
3. Protective efficacy of the trivalent attenuated live coccidiosis vaccine
The trivalent coccidiosis vaccine was prepared based on the optimal immunizing doses of theprecocious lines of E. maxima, E. acervulina and E. tenella, containing200,600and600oocysts ofeach line, respectively, per dose.0.25,0.5,1.0,1.5and2.0dose of vaccine were administered to4-day-old chicks, respectively. The protective efficacy of this vaccine was evaluated as comparison withthe commercial vaccine, Coccivac (1dose per chicken) and the unvaccinated birds. The oocyst per gramfecal (OPG) was accounted at4-24days after vaccination. The antibody level, the capability of splenicT lymphocyte proliferation and the weigh gains were investigated at the day of-1,7,14and21aftervaccination. On the day of14and21after vaccination, ten birds of each group was challenged with themixed oocysts of the parent and virulent strains of each precocious lines used in this vaccine,respectively. The results demonstrated that the reduction in weigh gains of the vaccinated chickens wasobserved at7and14days after immunization, while the weigh gains were similar at21days afterimmunization between the vaccinated chickens and unvaccinated ones. Three peaks of oocystsproduction were observed and the highest peak occurred at the day11-12after immunization. Theantibody level of the vaccinated group at the day of14after immunization was significant higher thanthe unvaccinated one, and maintained at the high level at the day of21after immunization. Thecapability of splenic T lymphocyte proliferation of immunized chickens was significantly higher thanthe he unvaccinated ones at the day of14after immunization, reached the highest at21days afterimmunization and maintained at the high level at the day of28after immunization. Results from the twochallenged trials showed that the protective efficacy of this trivalent coccidiosis vaccine was superior tothe Coccivac vaccine.
4Identification and partial characterization of ATPase gene
The results showed that the ATPase gene can be amplified from three precocious lines of E.maxima, E. acervulina and E. tenella. Homology analysis revealed that the sequence similarity were upto100%, indicating that the ATPase were conserved genes in different species of Eimeria. Real-timequantitative PCR analysis revealed that the EtATPase gene was expressed at lower levels in the threeprecocious line than their parent strains, and at higher levels in sporozoites than in the otherdevelopmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites).The fusion protein of EtATPase gene is an inclusion body protein with the molecular weight of22kDa.Western-blot analysis showed that this protein had good antigenicity. Indirect immunofluorescencetechnique with specific antiserum generated against the recombinant protein localized the EtATPaseprotein in the refractive body of E. tenella sporozoites. Invasion inhibition assay showed that EtATPasemay not play an important role in invasion of the sporoziotes in the host.
1.3个球虫早熟株的药物敏感性研究
为评价堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株对常用抗球虫药物的敏感性，选用地克珠利、氯苯胍、癸氧喹酯、尼卡巴嗪、二硝托胺、马杜米星、拉沙洛西、盐霉素等8种药物进行试验，以最适抗球虫活性百分率（POAA）、病变记分减少率（RLS）、相对卵囊产量（ROP）、抗球虫指数（ACI）为试验指标进行综合评定。结果显示，堆型艾美耳球虫早熟株和母株药物敏感性一致，均对二硝托胺不敏感，对盐霉素轻度敏感，对地克珠利、氯苯胍、癸氧喹酯、尼卡巴嗪、马杜米星、拉沙洛西等6种药物敏感，证明遗传背景相同的虫株，其药物敏感性也相同；巨型艾美耳球虫早熟株和柔嫩艾美耳球虫早熟株对8种药物均相对敏感。结果表明，3个球虫早熟株对常用抗球虫药物均较敏感，可用于球虫弱毒活疫苗的研制。
2.3个球虫早熟株的免疫剂量研究
为确定堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株合适的免疫剂量，比较堆型艾美耳球虫、巨型艾美耳球虫早熟株与母株的免疫效果，每个虫株设置7个免疫剂量组（每羽鸡口服孢子化卵囊分别为100、200、400、600、800、1000、2000个），进行二次免疫（7日龄首免、14日龄二免），二免后7天（21日龄）以母株进行攻虫，攻虫后7天结束试验，以增重、肠道病变记分、卵囊减少率为试验指标，并选择2～4个免疫效果较好的剂量组进行重复试验。结果显示，堆型艾美耳球虫早熟株600～1000免疫组、巨型艾美耳球虫早熟株200～800免疫组、柔嫩艾美耳球虫早熟株600～2000免疫组的综合免疫保护效果较好，其卵囊减少率分别超过90%、95%和60%，堆型艾美耳球虫、巨型艾美耳球虫早熟株与母株的综合免疫保护效果相当；经重复试验，确定堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株推荐的免疫剂量分别为600、200、600个卵囊/羽。研究结果为配置鸡球虫三价弱毒疫苗奠定了基础。
3.早熟株三价弱毒活疫苗的免疫效力初步研究
按照堆型艾美耳球虫、巨型艾美耳球虫、柔嫩艾美耳球虫3个早熟株按600、200、600比例配成三价活疫苗，以0.25、0.5、1.0、1.5和2.0头份/羽免疫4日龄雏鸡，设商品化球虫疫苗Coccivac（1头份/羽）组和不免疫组作为对照。免疫后第4～24d进行粪便卵囊OPG计数；检测免疫后第1、7、14、21d鸡只的抗体水平、脾脏淋巴细胞数量以及鸡只增重情况；免疫后第14和21d，各组分别取10只鸡用亲本毒株混合卵囊进行攻虫试验。研究发现，早熟株三价苗免疫期间增重情况，各免疫组鸡只在免疫后7d和14d出现增重减缓的趋势，免疫21d后出现补偿性增长，相对增重率与空白对照组相当。免疫期间OPG变化情况，各免疫组共出现3个排卵高峰，其中最大的一次出现在免疫后11～12d。免疫期间抗体水平变化，在免疫后14d抗体水平迅速升高且明显高于空白对照组，免疫后21d抗体水平有所下降，但任维持在较高水平。免疫后鸡脾脏T淋巴细胞数量变化，1.0倍免疫组脾脏T淋巴细胞的增殖能力于14d明显高于健康对照组，21d达到峰值，28d维持在较高水平。攻虫试验结果发现，无论是免疫后14d还是21d，所研制的球虫弱毒活疫苗均可获得比进口疫苗Coccivac稍好的免疫效果。
4.柔嫩艾美耳球虫ATPase基因特性研究
对艾美耳球虫ATPase基因进行扩增、表达量分析、细胞定位、体外抑制等研究。研究发现，保守性分析，分别从堆型艾美耳球虫、巨型艾美耳球虫和柔嫩艾美耳球虫3株早熟株中都能扩增出ATPase，并且同源性分析发现其序列相似性高达100%，表明该基因在不同种球虫存在保守性。表达量分析，ATPase基因在三株早熟株相对于母株表达量都下调；EtATPase基因在未孢子化卵囊、孢子化卵囊、子孢子和第二代裂殖子4个发育阶段都能表达，尤其在孢子化卵囊中转录拷贝数最高；EtATPase基因表达的融合蛋白是包涵体蛋白，分子量大约是22kDa，Western-blot分析发现具有良好的抗原性。利用EtATPase重组蛋白制备的多克隆抗体和间接免疫荧光技术发现，EtATPase定位于子孢子的折光体上。体外抑制试验发现，EtATPase对球虫子孢子入侵细胞无明显的抑制作用。
The precocious line of Eimeria was first selected by Jeffers in1975. Compared with its parentsrtain, the precocious line has a reduced prepatend period for the missing or defect of the last generationschizont, markedly less pathogenicity, and much lower reproductive potential, but retained goodimmunogenicity and genetic stability. The attenuated live anti-coccidial vaccines, Paracox and Livacox,have been developed and widely used for the control of coccidiosis. Immunization with the attenuatedvaccines had been proved to be effective and is increasingly playing an important role in the control ofcoccidiosis in the poultry industry. The precocious lines of Eimeria acervulina, E. maxima and E.tenella have been selected successfully and their biological characteristics have been studied in our lab.The objective of the present study was to study the drug sensitivity and optimal immunizing dose of thethree precocious lines, and to evaluate the anti-coccidial efficacy of trivalent vaccine. The results fromthis study would help to develop the live, attenuated anticoccidial vaccine.
1. Sensitivity of three precocious lines to eight anticoccidial drug
In order to test sensitivity of precocious line of E.maxima, E.acervulina and E.tenella toanticoccidial drugs, eight anticoccidial drugs, including of diclazuril, robenidine, dinitolmide,decoquinate, nicarbazin, maduramicin, salinomycin and lasalocidt, were selected to use in the study.Based on the percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS),relative oocyst production (ROP) and anticoccidial index (ACI), the results showed that the sensitivityof the precocious line and parent strain of E.acervulina were basically similar. The two strains ofE.acervulina were completely sensitive to diclazuril, robenidine, decoquinate, nicarbazin, maduramicinand lasalocid, partly sensitive to salinomycin and not sensitive to dinitolmide.the precocious line ofE.maxima was completely sensitive to eight anticoccidial drugs, the precocious line of E. tenella waspartly sensitive to salinomycin and completely sensitive to diclazuril, robenidine, dinitolmide,decoquinate, nicarbazin, maduramicin and lasalocidt. This study will provide experimental bases fordevelopment and application of the precocious lines vaccines of coccidia.
2. Optimal immunization dose of three precocious lines
In order to test optimal immunizing doses of precocious lines of E. acervulina, E.maxima andE.tenella, dosages of100,200,400,600,800,1000and2000sporulated oocysts were administeredorally to7-day-old chickens for two times at a7-day interval.21-day-old chickens were challenged withthe sporulated oocysts of the parent strains. Weight gain, intestinal lesion scores and oocysts inhibitionratio were used to evaluate the immunizing effectiveness.2-4optimal immunizing doses obtained fromthe first trial was repeated. The results from the first trial showed that the600-1000E. acervulina,200-800E. maxima and600-2000E. tenella could induce sufficient immunological protection, whichthe reduction of oocysts ratios were over90%、95%and60%, respectively. The results from therepeated trial indicated that the optimal immunizing dose of the precocious line of E. acervulina, E.maxima and E. tenella was600,200and600ooysts/bird, respectively.
3. Protective efficacy of the trivalent attenuated live coccidiosis vaccine
The trivalent coccidiosis vaccine was prepared based on the optimal immunizing doses of theprecocious lines of E. maxima, E. acervulina and E. tenella, containing200,600and600oocysts ofeach line, respectively, per dose.0.25,0.5,1.0,1.5and2.0dose of vaccine were administered to4-day-old chicks, respectively. The protective efficacy of this vaccine was evaluated as comparison withthe commercial vaccine, Coccivac (1dose per chicken) and the unvaccinated birds. The oocyst per gramfecal (OPG) was accounted at4-24days after vaccination. The antibody level, the capability of splenicT lymphocyte proliferation and the weigh gains were investigated at the day of-1,7,14and21aftervaccination. On the day of14and21after vaccination, ten birds of each group was challenged with themixed oocysts of the parent and virulent strains of each precocious lines used in this vaccine,respectively. The results demonstrated that the reduction in weigh gains of the vaccinated chickens wasobserved at7and14days after immunization, while the weigh gains were similar at21days afterimmunization between the vaccinated chickens and unvaccinated ones. Three peaks of oocystsproduction were observed and the highest peak occurred at the day11-12after immunization. Theantibody level of the vaccinated group at the day of14after immunization was significant higher thanthe unvaccinated one, and maintained at the high level at the day of21after immunization. Thecapability of splenic T lymphocyte proliferation of immunized chickens was significantly higher thanthe he unvaccinated ones at the day of14after immunization, reached the highest at21days afterimmunization and maintained at the high level at the day of28after immunization. Results from the twochallenged trials showed that the protective efficacy of this trivalent coccidiosis vaccine was superior tothe Coccivac vaccine.
4Identification and partial characterization of ATPase gene
The results showed that the ATPase gene can be amplified from three precocious lines of E.maxima, E. acervulina and E. tenella. Homology analysis revealed that the sequence similarity were upto100%, indicating that the ATPase were conserved genes in different species of Eimeria. Real-timequantitative PCR analysis revealed that the EtATPase gene was expressed at lower levels in the threeprecocious line than their parent strains, and at higher levels in sporozoites than in the otherdevelopmental stages (unsporulated oocysts, sporulated oocysts, and second-generation merozoites).The fusion protein of EtATPase gene is an inclusion body protein with the molecular weight of22kDa.Western-blot analysis showed that this protein had good antigenicity. Indirect immunofluorescencetechnique with specific antiserum generated against the recombinant protein localized the EtATPaseprotein in the refractive body of E. tenella sporozoites. Invasion inhibition assay showed that EtATPasemay not play an important role in invasion of the sporoziotes in the host.
引文
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