【摘要】 为评价口服免疫猪伪狂犬病毒三基因缺失重组弱毒株PRV-HNX-TK~-/gE~-/gG~-+PCV2 ORF2对母源抗体阳性保育猪的保护效果,本研究将该重组弱毒株通过口服和颈部肌肉注射两种不同途径免疫母源抗体阳性的断奶仔猪,通过ELISA方法测定血清PRV g B抗体、PCV2 Cap抗体和IFN-γ水平,利用固定病毒-稀释血清的中和试验方法检测PRV中和抗体水平;免疫结束后攻毒,记录各组猪临床表现和病理变化、测定其鼻拭子和肛拭子中病毒排毒时间、病毒载量等指标。结果显示,整个免疫试验过程中,口服组与注射组猪的PRV g B抗体均维持阳性、血清中PRV中和抗体效价均高于1∶4、IFN-γ含量在免疫后均能达到300 pg/mL以上,PCV2抗体阳性率则出现下降;而未免疫组猪在第7 d后g B抗体转阴,中和抗体效价低于1:4、IFN-γ含量维持在约250 pg/mL,PCV2抗体阳性率(母源抗体)持续到二免后28 d才开始下降。免疫后攻毒,检测结果显示,口服组与注射组比对照组猪提前2 d体温下降,提前7 d恢复采食,而对照组猪出现高温、打喷嚏、咳嗽、流鼻涕、食欲不振、精神沉郁和神经症状并持续至攻毒后第8 d,并于攻毒后第12 d开始采食;口服组与注射组猪排毒高峰在攻毒后第2 d～6 d,第12 d后均停止排毒,对照组猪排毒持续期大于14 d;脑组织病理切片观察可见,口服组与注射组猪脑组织中仅少量小胶质细胞和淋巴细胞增多,而对照组猪脑组织中出现胶质细胞结节、血管袖套现象、卫星现象、噬神经元现象等病理变化,对照组猪扁桃体隐窝腔内含有大量脱落的隐窝上皮细胞、白细胞和炎性渗出液,并有燕麦细胞增多。以上结果表明口服方式免疫该三基因缺失重组弱毒株能够使保育猪产生针对PRV的有效免疫应答,但并不能诱导高水平的PCV2抗体。本研究为PRV疫苗研发带来了新的方向,也为免疫途径提供了更多选择,同时首次证实了该缺失弱毒株口服和肌注均能提供相似的免疫保护效果。更多还原

【Abstract】 In order to evaluate the immune effect of porcine pseudorabies virus(PRV) three gene deletion attenuated strain(PRV-HNX-TK~-/gE~-/gG~-+PCV2 ORF2), maternal antibody-positive nursery pigs were vaccinated through oral and neck intramuscular immunization. Serum PRV g B antibody, PCV2 Cap antibody and IFN-γ levels were determined by ELISA, and PRV neutralizing antibody levels were detected by neutralization assay. After challenge, clinical manifestations and pathological changes were observed and recorded, and virus shedding in nasal swabs and rectal swabs was measured by fluorescence quantitative PCR.Duration of virus shedding from each group was also examined. Throughout the whole immunization experiment, the g B antibody in the oral and intramuscular groups remained positive, the PRV neutralizing antibody titer was higher than 1:4, and IFN-γ reached above 300 pg/mL, and PCV2 antibody positive rate decreased. While in the control group, g B antibody became negative after 35 days of age; the PRV neutralizing antibody titer was lower than 1∶4, and the levels of IFN-γ were maintained at around 250 pg/m L with no significant change. After the challenge, the clinical symptoms of the oral group and the injection group were alleviated from the fourth day, and the body temperature began to decrease 2 days earlier than the control group, and feeding began 7 days earlier than the control group. The control group showed symptoms such as elevated body temperature, sneezing, coughing, runny nose, loss of appetite, mental depression and neurological symptoms, which continued until the eighth day, and feeding began the twelfth day; virus shedding peak in each group ranged from day 2 to day 6, after day 12 two vaccinated groups stopped shedding;however, the duration of viral shedding in the control group was longer than 14 days. In the pathological section of the brain tissue,only a small amount of microglia and lymphocytes appeared in both oral group and the injection group; on the contrary, glial cell nodules, vascular cuff, satellite and neuronophagia phenomena were observed in the brain tissue of the control group. The tonsillar recess in the control group contained a large number of exfoliated crypt epithelial cells, white blood cells and inflammatory exudates and there was a growing oat cell. These results indicate that this recombinant attenuated strain orally immunized can produce an effective immune response against PRV, but does not induce high levels of PCV2 antibodies. This study provides a new direction for PRV vaccine development and more options for immune pathway.更多还原