禽多杀性巴氏杆菌外膜蛋白H基因重组质粒在鸡体内免疫效果的研究

为检测禽多杀性巴氏杆菌(P.multocida)外膜蛋白H基因的免疫效果,本研究利用PCR技术扩增禽P.multocida的omph基因片段,并克隆于pcDNA3.1(+)中构建重组质粒pOMPH,经体外表达检测后进行动物免疫,实验动物共分4组:pOMPH组、弱毒疫苗组、pcDNA3.1(+)空载体组和PBS对照组。免疫后对各组鸡进行免疫指标检测,并于三免后两周进行攻毒。结果显示,pOMPH免疫组和弱毒疫苗免疫组血清抗体水平持续上升,与两对照组相比差异极显著(p<0.01),并且pOMPH免疫组血清抗体水平明显高于弱毒疫苗组(p<0.05)。淋巴细胞增殖试验结果也表明,pOMPH和弱毒疫苗组的刺激值(SI值)极显著高于两对照组(p<0.01),并且重组质粒组高于弱毒疫苗组。免疫鸡外周血淋巴细胞产生的IFN-γ极显著高于两对照组(p<0.01),并且pOMPH组高于弱毒疫苗组(p<0.05)。攻毒后pOMPH组和弱毒疫苗组的保护率分别为66.7%和73.3%,表明pOMPH虽然可以为免疫鸡提供一定的保护,但效果不如商品化的弱毒活疫苗,还需采取措施进一步增强其免疫原性。     

禽脑脊髓炎油乳剂灭活疫苗与弱毒活疫苗免疫效果的比较研究

采用28日龄SPF鸡,对禽脑脊髓炎油乳剂灭活疫苗及弱毒活疫苗进行了免疫效果评估。抗体检测结果表明,疫苗免疫后21日,油乳剂灭活疫苗免疫组抗体水平及抗体转阳率优于弱毒活疫苗免疫组。攻毒保护试验结果表明,禽脑脊髓炎油乳剂灭活苗及弱毒活疫苗均具有良好的免疫保护性,对禽脑脊髓炎病毒强毒VR株的保护率分别为100%、90%。综上所述,灭活疫苗和活疫苗均可有效预防禽脑脊髓炎。     

禽多杀性巴氏杆菌基因组表达文库的构建及其免疫效果

以限制性内切酶Sau3A I酶切禽多杀性巴氏杆菌基因组DNA,回收500～3 000 bp的DNA片段,连接真核表达裁体pcDNA3.1(+),电转化于大肠杆菌感受态细胞TG1中,构建禽多杀性巴氏杆菌的基因组表达文库.将文库随机分为5个子文库(子文库Ⅰ-子文库Ⅴ),分别提取各子文库重组质粒,以pcDNA3.1(+)和PBS为对照进行动物试验,每组16只BALB/c小鼠,各子文库组和pcDNA3.1(+)组以100 μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μL 1×PBS,各组均免疫3次,每次间隔2周.间接ELISA检测免疫小鼠的血清抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况,三免2周后检测脾淋巴细胞IFN-γ的分泌情况.强毒攻击,计算小鼠的相对保护率.结果显示,动物免疫后子文库Ⅰ组质粒免疫的小鼠血清抗体水平及淋巴细胞增殖水平和IFN-γ分泌水平均持续上升,明显高于其他各组(P＜0.05).动物攻毒试验表明,各子文库组的重组质粒均可为免疫小鼠提供一定的保护,其中第Ⅰ组的保护效果最好,说明子文库Ⅰ组中含有较好的保护性抗原基因,这为进一步筛选相关的免疫原基因和研制新型疫苗奠定了的基础.     

鸡传染性法氏囊病免疫复合物疫苗的安全性和免疫效力试验

用鸡法氏囊病免疫复合疫苗(IBDV-Icx)和常规IBD活疫苗分别免疫1日龄商品鸡和SPF鸡,进行疫苗的安全性及免疫效力比较.免疫后8d检测,IBDV-Icx免疫组鸡的法氏囊未见明显萎缩,而传统弱毒疫苗免疫组鸡的法氏囊萎缩明显;免疫后28d各组用IBDV标准强毒株进行攻击,IBDV-Icx免疫组鸡的攻毒保护率均为10/10,常规IBD活疫苗对照组分别为8/10及9/10.免疫后3个月,IBDV-Icx免疫组血清抗体可达AGP1∶32,攻击强毒仍为10/10保护,而常规IBD活疫苗免疫后2个月抗体AGP为0,攻毒保护率为1/10,免疫后3个月时攻毒10/10发病.     

中药疫苗稀释剂BC-6对猪伪狂犬病弱毒疫苗免疫效果的研究

为探究以人参茎叶皂甙为主要成分的疫苗稀释剂BC-6对弱毒疫苗的免疫增强作用,本研究用BC-6稀释伪狂犬病(PR)弱毒疫苗免疫小鼠,检测其血清特异性伪狂犬病毒(PRV)gB抗体水平、脾淋巴细胞增殖活性和细胞因子的表达水平以及对免疫小鼠的攻毒保护率。结果显示和生理盐水稀释剂比较,BC-6显著增强了疫苗诱导的血清gB抗体应答,显著促进了刀豆蛋白(Con A)、脂多糖(LPS)和PRV抗原诱导的淋巴细胞增殖活性和淋巴细胞表达IFN-γ、IL-12、IL-5和IL-10 mRNA的水平,显著提高免疫小鼠感染PR野毒后存活率。BC-6对gB抗体的促进作用高于目前国内市场上的其它6种商品疫苗稀释剂,和其中的4种稀释剂比较有显著性差异。本研究为BC-6作为疫苗稀释剂临床应用提供了实验依据。     

猪乙型脑炎(HW1株)细胞灭活疫苗免疫抗体水平分析

为评估猪乙型脑炎(HW1株)细胞灭活疫苗的免疫原性,用商品化的乙型脑炎鼠脑疫苗、乙型脑炎弱毒疫苗(SA14-14-2株)以及自制乙型脑炎细胞灭活疫苗(HW1株)分别免疫仔猪,通过抗体水平监测试验、中和抗体试验、小鼠攻毒保护试验分别检测了其特异性抗体消长情况、中和抗体效价产生情况及疫苗对小鼠的攻毒保护率。结果显示,3种疫苗均能产生中和抗体,乙型脑炎细胞灭活疫苗免疫组中和抗体水平要高于鼠脑疫苗和弱毒疫苗免疫组,乙型脑炎细胞灭活疫苗和弱毒疫苗免疫组对小鼠的攻毒保护率高于鼠脑疫苗免疫组,但两者之间差异不显著。抗体消长情况显示,至8周观测期结束,鼠脑疫苗免疫组的抗体阳性率为80%,乙型脑炎细胞灭活疫苗和弱毒疫苗免疫组抗体阳性率为100%。结果表明猪乙型脑炎(HW1株)细胞灭活疫苗的免疫原性优于鼠脑灭活疫苗和弱毒疫苗。     

1日龄中国黄羽肉鸡新城疫与传染性支气管炎二联弱毒活疫苗免疫保护试验

对1日龄黄羽肉鸡免疫新城疫与传染性支气管炎二联弱毒活疫苗——威支灵(即新威灵+H120),在21日龄进行新城疫强毒攻毒,与a疫苗(Mass株+La Sota克隆株)(保护率为86.7%)及c疫苗(LaSota+H120)(保护率为90%)免疫组相比,威支灵免疫组表现出了较高的保护力(保护率为96.7%),未免疫攻毒组的死亡率为40%。新城疫病毒HI检测结果表明强毒攻毒后的鸡群的新城疫HI抗体升高,同样证明了攻毒的有效性。     

BALB/c鼠口服免疫猪水肿病大肠杆菌基因突变菌株的免疫效果

为了研究猪水肿病(ED)大肠杆菌SLT-Ⅱe基因突变菌株作为口服疫苗的免疫效果,本实验用已构建的猪ED大肠杆菌SLT-Ⅱe基因突变菌株口服免疫BALB/c小鼠,检测其血清中的IgG抗体及粪便和肠黏液中的slgA抗体水平,并进行淋巴细胞增殖检测及攻毒保护实验.结果表明该基因突变菌株具有良好的免疫性,能诱导小鼠体内产生IgG和sIgA抗体,并且能引起T淋巴细胞增殖反应.攻毒保护实验结果显示,口服免疫突变菌株能对小鼠提供良好的保护,保护率为75%(15/20).本研究结果证明,该大肠杆菌基因突变菌株在小鼠体内能激发体液免疫和细胞免疫反应,可作为猪ED口服疫苗的候选菌株.     

鸡传染性法氏囊病毒病毒样颗粒免疫原性研究

对鸡传染性法氏囊病病毒(IBDV)病毒样颗粒(VLPs)的免疫原性进行了探讨,并将其与鸡传染性法氏囊病活疫苗进行比较。分别用VLPs及VLPs+Poly IC对14日龄非免鸡进行免疫,并用IBDV B87株弱毒商品疫苗作为阳性对照,同时设置空杆粒蛋白组作阴性对照及PBS对照。免疫前进行颈静脉采血,首免后每隔1周进行3次采血,通过间接ELISA抗体水平检测、中和试验和淋巴细胞增殖试验来进行分析比较。抗体水平检测结果显示,首免后第7天,在鸡的体内可检测到IBDV特异性抗体的组别为:VLPs组、VLPs+Poly IC组和B87株组,且在加强免疫后,抗体水平明显增高(P0.05)。首免后第14天,在3组鸡的体内均检测到高水平的抗IBDV中和抗体,且呈快速增长趋势。淋巴细胞增殖试验结果显示,VLPs组、VLPs+Poly IC组和B87株组鸡体内淋巴细胞增殖动态明显高于接种PBS和空杆粒蛋白组的鸡(P0.01),并且在加强免疫后明显提高(P0.05)。动物攻毒保护试验结果显示,攻毒后第2天PBS组和空杆粒蛋白组的鸡均表现出IBD的典型临床症状和病理变化且在攻毒后第6天全部死亡,VLPs组鸡的存活率为88.7%;VLPs+Poly IC组鸡存活率为80%;B87疫苗组鸡存活率为88.7%。器官指数分析结果显示,3组与空白组相比差异不显著(P0.05)。本试验制备的鸡传染性法氏囊病病毒样颗粒疫苗能够诱导机体产生较高水平的体液和细胞免疫应答,具有良好的应用前景。     

禽多杀性巴氏杆菌ompa DNA疫苗在小鼠体内免疫效果试验

应用PCR技术扩增获得禽多杀性巴氏杆菌的ompa基因片段,克隆到pUCm-T载体,再亚克隆到真核表达质粒载体pCDNA3.1(+)上,构建重组质粒pcA,体外转染Vero细胞,RT-PCR和间接免疫荧光试验检测其转录表达情况。动物免疫分为3组:pCDNA3.1(+)组、PBS对照组和pcA组,每组16只BALB/c小鼠,pCDNA3.1(+)组和pcA组以100μg/只的剂量肌注免疫,PBS组每只小鼠肌注100μL 1×PBS,各组均免疫3次,每次间隔2周。间接ELISA检测免疫后小鼠血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况,三免2周后检测脾淋巴细胞IFN-γ分泌情况。强毒攻击,计算小鼠存活数目及保护率。结果显示,间接免疫荧光试验和RT-PCR检测结果均表明pcA可在体外培养的Vero细胞中表达目的蛋白。动物免疫后,pcA组免疫小鼠血清抗体水平持续上升,与pCDNA3.1(+)组和PBS组相比差异极为显著(P<0.01)。经提取的禽多杀性巴氏杆菌总外膜蛋白(Omps)刺激后,pcA组的刺激值(SI值)与pCDNA3.1(+)组及PBS免疫组相比均差异显著(P<0.05)。脾细胞产生的IFN-...     

Maternally and naturally acquired antibodies to Mannheimia haemolytica and Pasteurella multocida in beef calves

The dynamics and duration of maternally derived antibodies as well as the onset of acquired immunity against Mannheimia haemolytica and Pasteurella multocida in range-pastured beef calves were investigated. Two groups of unvaccinated cattle were used in this study. Serum antibody responses were measured by enzyme-linked immunoassay for antibodies of the IgG1, IgG2 and IgM isotypes binding M. haemolytica whole cells (WC) or leukotoxin (LKT) and P. multocida outer membrane proteins (OMPs). Comparisons of mean antibody responses to M. haemolytica LKT and WC and P. multocida OMPs were made within each group. Maternally derived antibodies against M. haemolytica and P. multocida reached lowest levels at 30-90 days after birth. Calves began production of antibodies against M. haemolytica and P. multocida between 60 and 90 days of age in both groups. Based on the results of this study, in beef herds vaccinated against M. haemolytica and/or P. multocida, it may be best to vaccinate calves around 3 months of age. In contrast, beef calves from unvaccinated herds might benefit from vaccination at 4 months of age.     

禽多杀性巴氏杆菌血清型与外膜蛋白型相关性研究

为了解禽多杀性巴氏杆菌(Pasteurella multocida,Pm)分离菌株的荚膜血清型、菌体血清型与外膜蛋白型之间的相关性,首先对自行分离的10个菌株采用间接血凝试验和琼脂扩散试验进行鉴定(均为A:1);然后采用超声波破碎、高速离心和十二烷基肌氨酸钠提取外膜蛋白,通过SDS-PAGE电泳的方法对上述10个菌株与C48-1(A:1)、X73(A:1)、P1059(A:3)、CU(A:3,4)等一起进行外膜蛋白(Outer membrane proteins,OMP)分型研究。结果表明:14个禽多杀性巴氏杆菌菌株以2个主要蛋白OmpH和OmpA的差异为依据分为3种主要OMP型;依据次要蛋白的差异,OMP-1,3菌株进一步分为OMP型1.1,1.2和3.1,3.2,其中OMP型1.1有6个菌株,OMP型1.2有5个菌株;OMP型2有1株(YZ7031);P1059为OMP型3.1;CU为OMP型3.2;另外,血清型为A:1的12个菌株中有11个菌株外膜蛋白型均为OMP-1型,血清型为A:3、A:3,4的菌株外膜蛋白型属于3型。说明禽多杀性巴氏杆菌血清型与特定的外膜蛋白型具有很强的相关性。     

Immunization with outer membrane proteins of Pasteurella multocida (6:B) provides protection in mice

The immunoprotective efficacy of Pasteurella multocida (6:B) outer membrane proteins (OMPs) was examined in the mouse model. Bacterial OMPs were extracted using sarkosyl method and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. Prototype vaccines were prepared using OMPs with adjuvants including dioleoyl phosphatidyl choline-based liposome and Montanide ISA206 water-in oil-in water emulsion. Antibody response to the vaccine was monitored using indirect enzyme linked immunosorbent assay. The results of the study showed that immunized mice had high titre with both the formulations. The vaccinated mice were able to survive a live virulent bacterial challenge. Based on the findings of the study it can be inferred that OMPs are important determinants of immunoprotection hence can serve as vaccine candidates against haemorrhagic septicaemia.     

禽大肠杆菌病免疫保护机理的研究

以禽病原性大肠杆菌O18、O78分离株制成超声波裂解铝佐剂灭活苗免疫14日龄鸡，以相同或不同外膜蛋白型（Outer membrane protein pattern,OMP型）的O18、O78分离株攻毒。结果表明：O78血清相同和不同OMP型分离株间能获得最大保护；O18血清型相同OMP型分离株间获得最大保护，而不同OMP型分离株间不能保护；上述两个血清型的分离株间不论OMP型是否相同，均缺乏保护。以间接ELISA试验、间接血凝试验分别测定了试验鸡临攻毒前针对大肠杆菌OMPs和脂多糖(Lipopolysaccharide,LPS）的抗体。结果表明：免疫组鸡血清上述两种抗体明显高于攻毒对照组；在免疫组，存活鸡临攻毒前血清中上述两种抗体滴度恒高于死亡鸡，但除3个组外，多数组差异不显著。攻毒对照组这一关系不稳定。结果说明：禽大肠杆菌疫苗的免疫保护，主要与O血清型有关，部分与OMP型有关，如O18分离株，免疫保护性抗原含OMPs,LPS等多个抗原表位。     

Pasteurella multocida and bovine respiratory disease

Pasteurella multocida is a pathogenic Gram-negative bacterium that has been classified into three subspecies, five capsular serogroups and 16 serotypes. P. multocida serogroup A isolates are bovine nasopharyngeal commensals, bovine pathogens and common isolates from bovine respiratory disease (BRD), both enzootic calf pneumonia of young dairy calves and shipping fever of weaned, stressed beef cattle. P. multocida A:3 is the most common serotype isolated from BRD, and these isolates have limited heterogeneity based on outer membrane protein (OMP) profiles and ribotyping. Development of P. multocida-induced pneumonia is associated with environmental and stress factors such as shipping, co-mingling, and overcrowding as well as concurrent or predisposing viral or bacterial infections. Lung lesions consist of an acute to subacute bronchopneumonia that may or may not have an associated pleuritis. Numerous virulence or potential virulence factors have been described for bovine respiratory isolates including adherence and colonization factors, iron-regulated and acquisition proteins, extracellular enzymes such as neuraminidase, lipopolysaccharide, polysaccharide capsule and a variety of OMPs. Immunity of cattle against respiratory pasteurellosis is poorly understood; however, high serum antibodies to OMPs appear to be important for enhancing resistance to the bacterium. Currently available P. multocida vaccines for use in cattle are predominately traditional bacterins and a live streptomycin-dependent mutant. The field efficacy of these vaccines is not well documented in the literature.     

Pasteurella multocida lipopolysaccharide: the long and the short of it

Pasteurella multocida is a capsulated, gram-negative cocco-bacillus that can cause serious disease in a wide range of mammals and birds. P. multocida strains are classified into 16 serovars based on lipopolysaccharide (LPS) antigens. LPS is an essential virulence factor of P. multocida; mutants expressing severely truncated LPS are completely attenuated in chickens. LPS is also a major immunogen of P. multocida and protection against infections caused by P. multocida is generally considered to be serovar specific. In this review we summarize current knowledge of the structure and genetics of LPS assembly of P. multocida strains belonging to five different serovars. These include strains belonging to serovars 1 and 3, the most common serovars found in the poultry industry, and strains belonging serovars 2 and 5, the serovars associated with bovine haemorrhagic septicaemia outbreaks. A number of the serovars are genetically related; serovars 1 and 14 share the same LPS outer core biosynthesis locus, but due to a mutation within the phosphocholine biosynthesis gene, pcgA, the serovar 14 strain produces a truncated LPS structure. Similarly serovars 2 and 5 share an identical LPS outer core locus and express near-identical LPS structures. However, due to a single point mutation in the phosphoethanolamine (PEtn) transferase gene, lpt_3, the serovar 2 strain does not elaborate a PEtn residue on heptose II. Knowledge of the genetic basis for the LPS structures expressed by P. multocida will facilitate the development of rapid molecular methods for typing and diagnosis and will be essential for a rational approach to vaccine formulation.     

Protection of chickens by ribosomal vaccines from Pasteurella multocida: dependence on homologous lipopolysaccharide

Chickens were protected against fowl cholera by ribosomal vaccines prepared from noncapsulated Pasteurella multocida. Passive hemagglutination (PHA) titers to lipopolysaccharide (LPS) and the degree of protection conferred by ribosomal vaccines were diminished or abolished when ribosomes were chromatographed on an immunoadsorbent column. Addition of subimmunogenic amounts of serotype 1 (homologous) LPS to highly purified ribosomes resulted in vaccines that protected against challenge exposure and produced PHA titers to homologous LPS. Addition of serotype 5 LPS to highly purified ribosomes did not protect chickens against challenge exposure with serotype 1 P multocida, but produced PHA titers to serotype 5 LPS. Combinations of serotype 1 ribosomal RNA and serotype 1 (homologous) LPS did not protect chickens or produce PHA titers to LPS. Purified ribosomes from Brucella abortus, Aspergillus fumigatus, and chicken liver were combined with LPS from P multocida and were evaluated as vaccines. Brucella abortus and A fumigatus ribosomes combined with LPS protected chickens as well as did bacterin made from whole cells of P multocida. Chicken liver ribosomes combined with LPS did not provide protection. To determine whether a protein carrier would substitute for ribosomes, methylated bovine albumin (MBA) was combined with LPS and evaluated as a vaccine. A serologic response to LPS was induced by MBA-LPS vaccine, but the vaccine offered no better protection than when LPS was used alone as vaccine. Ribosome-LPS vaccines produced serologic responses to LPS that were at least 5-fold greater than those produced by MBA-LPS vaccine.     

Expression of iron-regulated outer membrane proteins by porcine strains of Pasteurella multocida.

The outer membrane protein (OMP) profiles of two strains of capsular type A Pasteurella multocida isolated from the lungs of pigs with enzootic pneumonia were studied. Sarkosyl extracted OMPs from P. multocida grown under iron-restricted and iron-replete conditions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Results showed that the iron-regulated outer membrane proteins (IROMPs) with molecular masses of 74 kDa, 94 kDa, 99 kDa and 109 kDa were expressed by strain A52, while 74 kDa, 82 kDa, 94 kDa and 99 kDa IROMPs were expressed by strain B80. Swine immune sera, obtained from pigs which were first immunized with a polyvalent P. multocida type A and type D bacterin and subsequently challenged with type A strain of P. multocida, contained antibodies against the IROMPs. These antibodies cross-reacted with the IROMPs expressed by avian strain P1059 of P. multocida. Convalescent-phase serum obtained from turkeys which survived fowl cholera, also cross-reacted with the IROMPs from porcine strains of P. multocida. These results suggested that IROMPs from porcine and avian strains of P. multocida may share common epitopes that were recognized by swine immune serum as well as turkey convalescent-phase serum.     

Incubation of Pasteurella haemolytica and Pasteurella multocida lipopolysaccharide with sheep lung surfactant

Lipopolysaccharides (LPS) were extracted from a serotype of each of 2 species of Pasteurella isolated from sheep with respiratory tract infections. Lipopolysaccharides from P haemolytica 82-25 (serotype 1A) or P multocida P-1573 (serotype 12) were mixed with sheep lung surfactant and were incubated for 6 hours at 37 C. After incubation, LPS-surfactant mixtures were centrifuged overnight in sucrose density gradients, and fractions were analyzed. Binding occurred between LPS and surfactant vesicles resulting in a stable complex with densities greater than those with the surfactant alone. The surfactant alone had a density of 1.052 to 1.060 g/ml. Diffuse bands of surfactant had a density of 1.075 to 1.092 when incubated with P haemolytica LPS and a density of 1.069 to 1.105 when incubated with P multocida LPS.