猪捷申病毒Swine/CH/IMH/03分离株细胞适应性和致病性研究

为确定猪捷申病毒(PTV)Swine/CH/IMH/03株的易感细胞系及其致病性,本实验将该病毒接种不同细胞,通过细胞病变、电子显微镜观察和PCR技术进行鉴定。并进一步将该病毒回归本动物鉴定其致病性。试验结果表明,PTV Swine/CH/IMH/03对IB-RS-2和PK-15细胞系敏感,能够在IB-RS-2和PK-15细胞系中增殖。致病性试验结果显示,Swine/CH/IMH/03分离株能够导致实验猪出现神经症状,剖检可见脑、肾、脾、肠道发生出血,病理组织学检查可见脑实质内胶质细胞浸润、肾脏间质小血管淤血、脾淋巴细胞减少、小肠伴有出血及淋巴细胞浸润等病理变化,对仔猪具有强致病性。     

牛传染性鼻气管炎病毒LN01/08株灭活疫苗的研制

为研制牛传染性鼻气管炎病毒(IBRV)灭活疫苗,本研究以国内分离鉴定的IBRV LN01/08株为种毒,优化病毒增殖条件,获得病毒滴度达108.0TCID50/mL,将其灭活制备疫苗.为比较不同免疫佐剂的效果,分别以矿物质白油和Montanide ISA206佐剂配制灭活疫苗,进行牛体免疫试验.临床观察和血清中和抗体检测结果表明,Montanide ISA206佐剂乳化的疫苗在降低副反应和增强免疫效果方面优于矿物质白油佐剂.应用Montanide ISA206佐剂制备3批灭活疫苗,并对其安全性和免疫保护效果进行测定.结果表明该疫苗安全可靠,对强毒攻击可产生较好的抵抗力,攻毒保护率达80%.疫苗在2℃～8℃保存12个月后仍能保持良好的免疫效果.     

猪捷申病毒3D蛋白的原核高效表达及其反应活性

根据猪捷申病毒（PTV）Swine／CH／IMH／03株核苷酸序列，设计了1对特异性引物，以全长基因组重组质粒pSK—PTVFL为模板扩增了3D基因，并将扩增产物定向克隆到原核表达载体pET30a（＋）中，阳性质粒转化BL21（DE3），阳性菌经0．3mmol／L IPTG诱导后，进行Western—blotting检测。结果显示，3D蛋白获得了高效表达，表达量占菌体总蛋白的66．1％，且重组蛋白能与PTV Swine／CH／IMH／03株阳性血清发生特异性反应。表明，3D蛋白能在大肠杆菌中高效表达，并具有良好的免疫原性，这为开发相应的鉴别诊断技术奠定了基础。     

猪圆环病毒2型灭活疫苗佐剂选择

用水包油佐剂Merckinade SDA 25、HS1010和双向佐剂Montanide ISA 206三种不同佐剂分别制成猪圆环病毒2型灭活疫苗，对三种疫苗的稳定性、黏度、免疫仔猪后的安全性及效力进行了比较。试验结果表明，以3 000 r/min离心15 min，三种疫苗均无水析出；三种疫苗的黏度均低于200 cP；三种疫苗按2.0 mL/头的剂量接种21日龄健康易感猪，试验猪体温、采食、饮水、精神状况均正常，均无不良临床反应，剖检后各脏器均无病理变化；加强免疫后21d，各组试验猪采血，检测猪圆环病毒2型抗体效价，结果表明，佐剂MontanideISA206相较MerckinadeSDA25可诱导更高水平抗体效价。攻毒试验结果表明免疫组可以得到100%的保护。根据以上试验结果，确定猪圆环病毒2型灭活疫苗使用Montanide ISA 206。     

猪捷申病毒HB株的分离与全基因序列分析

为分析河北省某规模化猪场猪捷申病毒(PTV)的遗传变异和进化关系,作者成功分离到PTV HB株,对其进行了毒力测定和动物回归试验,设计引物进行了全基因序列测定,并与国内外42株PTV全基因序列进行了同源性比较。结果表明猪捷申病毒HB株病毒滴度为10-6.4 TCID50.mL-1,健康仔猪接毒28d后均出现明显猪捷申病症状,该毒株全基因组序列长度为7 090bp。系统进化树分析表明,猪捷申病毒HB株(JQ664746)属于PTV-8型血清型,和国内分离的PTV-jilin/2003株(GQ293092)同源性最高,从而为进一步研究该毒株的遗传变异提供参考。     

猪捷申病毒SD株的分离与鉴定

从山东某疑似感染猪捷申病毒(Porcine teschovirus,PTV)的猪群中分离到1株病毒。经一步法RT-PCR扩增得到PTV部分VP1基因序列。经序列测定,与Gen Bank上的毒株序列进行比对,并做了该病毒的致病性试验。结果显示,该分离株与参照的猪捷申病毒相似性为100%,并对猪具有一定的致病性。本试验成功分离获得1株PTV山东地方流行毒株,命名为SD株。     

猪血凝性脑脊髓炎病毒不同佐剂灭活疫苗免疫BALB/c小鼠的比较试验

用血凝/血凝抑制试验（HA/HI）检测自制的猪血凝性脑脊髓炎病毒（HEV）氢氧化铝胶灭活苗和蜂胶佐剂灭活苗免疫BALB/c小鼠后的抗体效价,依据小鼠抗体效价比较不同免疫佐剂的效果。结果表明,用氢氧化铝胶佐剂配制的疫苗可刺激接种动物产生快速免疫应答反应,抗体产生效价高、维持时间长,其效果优于其它佐剂,有望成为HEV灭活疫苗的候选佐剂。     

大肠杆菌不同佐剂灭活苗血清抗体与卵黄抗体消长规律的研究

笔者用分离得到的猪源致病性大肠杆菌制成灭活疫苗免疫产蛋鸡,定期检测蛋鸡血清和卵黄的抗体效价。试验结果表明,免疫蛋鸡血清中和卵黄中抗体的消长规律相似,同一时期的血清抗体比卵黄抗体较高。同时比较3种不同佐剂疫苗产生的效果,结果表明,氟氏佐剂和氢氧化铝佐剂疫苗产生的抗体效价较白油佐剂疫苗产生的抗体效价高,但氟氏佐剂疫苗和白油佐剂疫苗较氢氧化铝佐剂疫苗抗体持续时间长。综合比较,氟氏佐剂较其他2种佐剂产生的抗体效价高、持续时间长。     

猪繁殖与呼吸综合征病毒NADC30(SY株)不同佐剂灭活疫苗免疫效果的研究

为研究利用不同佐剂制备的猪繁殖与呼吸综合征病毒NADC30(NADC30-PRRSV)SY株灭活疫苗的免疫效果,本研究对由ISA201VG和ISA70VG佐剂分别制备的2种NADC30-PRRSV SY株疫苗的免疫效果进行比较研究。本研究选用35日龄PRRSV抗体和抗原均为阴性的健康仔猪15头,随机分为3组:ISA201VG免疫组、ISA70VG免疫组和未免疫攻毒组。免疫组首免107.0TCID50/头,间隔21 d后以相同剂量进行二次免疫,采用ELISA法检测PRRSV血清抗体,结果显示,免疫后第35 d,ISA70VG免疫组5头试验猪PRRSV抗体阳转率达100%,ISA201VG免疫组5头试验猪PRRSV抗体阳转率达40%。二免后14 d采用NADC30-PRRSV SY株攻击试验猪,攻毒剂量为1×10~(5.0)TCID_(50)/mL,检测各组猪体温度化,结果显示,ISA70VG免疫组试验猪从攻毒后第8 d起体温超过40℃,持续6 d;ISA201VG免疫组5头试验猪只有1头体温超过40℃;未免疫攻毒对照组5头猪攻毒后第11 d~13 d体温升至40℃持续3 d。各组猪增重检测显示,ISA70VG佐剂免疫组与未免疫感染对照组相比较,体重呈现负增长且体重减少均在5%以上,而ISA201VG与未免疫感染组相比较,体重呈现正增长且增长均在7%以上。对PRRSV感染后两个实验组肺组织病理观察均可见肺脏呈局限性间质性肺炎和淤血,并伴有血栓、部分肺泡隔增宽,支气管周围有淋巴细胞浸润伴有间质性肺炎,但ISA70VG组比ISA201VG组组织病变更加严重。本实验表明ISA201VG佐剂效果更好,同时首次证实类NADC30-PRRSV(SY株)也能导致严重抗体依赖增强效应,不益作为疫苗株。     

Montanide IMS 1313 N VG PR nanoparticle adjuvant enhances antigen-specific immune responses to profilin following mucosal vaccination against Eimeria acervulina

This study investigated protection against Eimeria acervulina (E. acervulina) following vaccination of chickens with an Eimeria recombinant profilin in conjunction with different adjuvants, or by changing the route of administration of the adjuvants. Day-old broilers were immunized twice with profilin emulsified in Montanide IMS 1313 N VG PR adjuvant (oral, nasal, or ocular routes), Montanide ISA 71 VG adjuvant (subcutaneous route), or Freund's adjuvant (subcutaneous route) and orally challenged with virulent E. acervulina parasites. Birds orally immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus ISA 71 VG, had increased body weight gains compared with animals nasally or ocularly immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus Freund's adjuvant. All adjuvant formulations, except for IMS 1313 N VG PR given by the nasal or ocular routes, decreased fecal parasite excretion and/or reduced intestinal lesions, compared with non-vaccinated and infected controls. Compared with animals vaccinated with profilin plus Freund's adjuvant, chickens immunized with profilin plus IMS 1313 N VG PR or ISA 71 VG showed higher post-infection intestinal levels of profilin-reactive IgY and secretary IgA antibodies. Finally, immunization with profilin in combination with ISA 71 VG was consistently better than profilin plus IMS 1313 N VG PR or Freund's adjuvant for increasing the percentages of CD4(+), CD8(+), BU1(+), TCR1(+), and TCR2(+) intestinal lymphocytes. These results indicate that experimental immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 VG adjuvants increases protective mucosal immunity against E. acervulina infection.     

猪圆环病毒2型灭活疫苗免疫佐剂的比较试验

以猪圆环病毒2型(PCV2)遗传标记毒株为毒种,经细胞培养传代,优化了病毒增殖条件,获得较高滴度的病毒培养物用于PCV2灭活疫苗的研制。为了比较不同免疫佐剂的效果,本试验对国产矿物质白油和铝胶两种佐剂以及赛比克公司提供的MONTANIDETMISA206、ISA15VG、IMS1315VG3种佐剂配制的灭活疫苗进行了猪体免疫试验。通过临床观察、血清抗体效价测定,确认ISA15VG佐剂配制的疫苗在增强免疫效果方面优于其它佐剂。按5种佐剂免疫效果排序为ISA15VG、IMS1315VG、铝胶、ISA206、国产白油。ISA15VG佐剂属于水包油剂型,可刺激接种动物产生快速免疫应答反应,抗体产生效价高、持续时间长,有可能成为新型PCV2灭活疫苗佐剂的候选。     

Stimulation of serum antibody formation in pigs by oral, subcutaneous or combined administration of live porcine rotaviruses or intramuscular inoculation of inactivated porcine rotaviruses in an oil adjuvant]

Only live vaccines prepared from attenuated strains have been used for the specific prophylaxis of rotavirus infections in pigs. These vaccines are administered to sows per os or parenterally to increase the content of antibodies in the blood serum, colostrum and milk, and in this way to provide for the better passive protection of suckling piglets through the maternal antibodies, or to induce the active immunity by pig vaccination. The data on the efficiency of live vaccines administered in both ways differ as to their ability to stimulate significantly increases in the actual levels of antibodies in sows and also as to the possibility of inducing the protection of vaccinated pigs from virulent virus infection. The objective of our trials was to compare the intensity of antibody response evoked by pig vaccination with live virus if the virus was implanted in different ways, and by vaccination with inactivated virus emulsified in oil adjuvant. The live vaccine consisted of a suspension of porcine rotavirus, strain OSU/6, cultivated in MA-104 culture medium with the content of 10(7) TKID50.ml-1, the inactivated vaccine was the identical virus suspension inactivated by an addition of 0.2% formaldehyde during 24 hours at a temperature of 37 degrees C, emulsified in oil adjuvant by means of an ULTRATURAX equipment at a 4:1 ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

鸡毒支原体两种油佐剂灭活疫苗的安全与效力比较试验

本研究选择ISA 775 VG佐剂和Marcol-52白油佐剂制备鸡毒支原体灭活疫苗（R株）,对其物理性状、安全性和效力进行比较试验.结果表明：两种佐剂制备的疫苗物理性状良好;ISA 775 VG佐剂疫苗粘度比Marcol-52白油佐剂疫苗低;Marcol-52白油佐剂疫苗安全性优于ISA 775 VG佐剂疫苗;气囊损伤保护率ISA 775 VG佐剂疫苗为100％,Marcol-52白油佐剂疫苗组为79.81％.     

Comparison of three adjuvants used to produce polyclonal antibodies to veterinary drugs

Two commercially available adjuvants, Gerbu LQ 3000 and Montanide ISA 50V, were assessed as potential replacements for Freund's adjuvant by evaluating their efficacy in the production of polyclonal antibodies to veterinary drugs in rabbits. The aim was to find an adjuvant that could produce a similar (or enhanced) immune response in the host animal without the undesirable side effects associated with Freund's complete and incomplete adjuvant. The assessment involved the examination of each injection site and the characterisation of the resultant antibodies with regards to antibody titre and sensitivity. It was found that the rabbits immunised with Gerbu adjuvant produced some of the most sensitive antibodies. However, titres were relatively low and adverse effects at injection sites were relatively common. Montanide adjuvant produced no adverse effects and the related antibodies were found to be of adequate sensitivity when compared to those from rabbits immunised with Freund's. It was concluded that Montanide ISA 50V could be considered as a suitable replacement to Freund's for the production of polyclonal antibodies, to low molecular weight compounds in rabbits.     

鸡黄病毒灭活疫苗的研制及免疫效果研究

鸡黄病毒FQ—C1株经SPF鸡胚连续传40代后,检测其尿囊液毒的ELD50达到1×10^-4.33／0．1mL。将该病毒第40代SPF鸡胚液毒灭活后制备成油乳剂灭活疫苗。SPF雏鸡和蛋鸡安全性试验表明,该疫苗的安全性好,无不良影响。以该疫苗一次单剂量（1mI／只）免疫开产蛋鸡,28d后攻以鸡黄病毒强毒测定疫苗的保护效果,结果显示疫苗免疫组较未免疫组蛋鸡的产蛋率高出75．8％,产蛋保护率达93．2％以上。试验表明,本研究制备的鸡黄病毒灭活疫苗安全性好,且具有良好的免疫原性,免疫后可有效抵抗鸡黄病毒所致的产蛋骤降。     

猪圆环病毒2型灭活疫苗种毒株的筛选

为筛选满足猪圆环病毒2型灭活疫苗制苗要求的毒株,选择河南省8个地区的8株猪圆环病毒2型分离株进行抗原性分析,将各分离株培养后用不同滴度的病毒原液和其分别稀释至同一滴度(4.233×10⁴拷贝/μL)的病毒液分别制成油佐剂灭活疫苗,免疫小鼠后采血测定其抗体动态水平,以进行免疫效力评价。结果显示,8株圆环病毒2型分离株中8#和12#分离株原液制备的疫苗免疫后诱导产生的抗体水平较高,二免后1周抗体水平即高于1∶800,达到猪圆环病毒2型灭活疫苗的国家质量标准要求,3周时达到高峰,比对照商品疫苗诱导产生的抗体水平更高;其分别稀释130倍和463倍后制备的疫苗诱导产生的抗体水平在二免后3周也能达到1∶800。提示,猪圆环病毒2型8#和12#分离株毒价高,免疫效力良好,是理想的疫苗候选株。本研究丰富了猪圆环病毒2型灭活疫苗毒株的种毒资源,为研制高质量的猪圆环病毒2型疫苗奠定了基础。     

猪丹毒丝菌灭活疫苗免疫佐剂的筛选

旨在筛选适宜于猪丹毒丝菌灭活疫苗的佐剂。以分离鉴定出的猪丹毒丝菌1a型HG-1株灭活菌体为抗原分别配制矿物油佐剂疫苗（简称矿物油疫苗）、氢氧化铝胶佐剂疫苗（简称铝胶疫苗）、ISA201双相油乳佐剂疫苗（简称ISA201疫苗）、GEL02水溶性聚合物佐剂疫苗（简称GEL疫苗）、IMS1313水溶性纳米佐剂疫苗（简称IMS1313疫苗）共5种佐剂的灭活疫苗。小鼠免疫保护试验结果表明,二免14 d后使用约为4 LD₅₀的HG-1株对小鼠进行腹腔攻毒,矿物油疫苗和GEL疫苗的保护率分别为100%（7/7）和71%（5/7）,其他三种佐剂疫苗的保护率均为14%（1/7）。本研究进一步选择铝胶疫苗和GEL疫苗进行猪体对比试验;仔猪安全性试验结果表明,两种佐剂疫苗的副反应均较小;免疫保护试验结果表明,两次免疫后使用约为16 LD₁₀₀的HG-1株对免疫仔猪进行耳缘静脉攻毒,两种佐剂疫苗的保护率分别为60%（3/5）和100%（5/5）。本研究最终选择GEL佐剂作为开发猪丹毒灭活疫苗的最适佐剂。