2个品系豚鼠对口蹄疫疫苗的免疫反应

为了评价不同豚鼠品系对O-A型口蹄疫（FMD）疫苗的免疫应答,用经过剂量筛选的O-A型FMD双价疫苗免疫2个品系豚鼠,以间接血凝法、ELISA法和流式细胞法,测定豚鼠血清IgG、IgG1、IgG2及持续期IgG,以及T淋巴细胞增殖率及细胞因子IFN-γ、IL-4。结果显示,O-A型FMD疫苗对2个品系豚鼠均能诱导出特异性IgG抗体,其主要成分是IgG2亚类。Zmu-1：DHP豚鼠产生的IgG维持时间较长。DHP豚鼠T淋巴细胞增殖率和细胞因子分泌量显著高于Zmu-1：DHP豚鼠。表明Zmu-1：DHP豚鼠较适于制作FMD病毒感染模型,DHP豚鼠适于研究T、B淋巴细胞及生物制品的免疫功能。豚鼠对FMDV的敏感性主要由细胞免疫所决定,而体液免疫的作用较小。     

Emergence of antigenic variants with in serotype A foot and mouth disease virus in India and evaluation of a new vaccine candidate panel

Emergence of genetically and antigenically divergent lineages/genotypes and poor intergenotypic antigenic coverage is a major concern in serotype A foot-and-mouth-disease virus (FMDV) in India. In 2009, to cover antigenic diversity emerged in serotype A virus field isolates, IND40/2000 was selected as the new vaccine strain for incorporation in the trivalent FMD vaccine formulation used in India. Although current vaccine strain (IND40/2000) covers most isolates antigenically, a few VP3(59)-deletion group isolates showed low r-value in routine vaccine matching exercise. The VP3(59)-deletion group within genotype 18 emerged first in late part of 2002 and in 2007 causing outbreaks along with non-deletion isolates of the same genotype. In case of emergence or re-emergence of more antigenically divergent isolates in future, a need for a new vaccine candidate to cover maximum isolates of both deletion and non-deletion group may arise. Four alternate candidate vaccine strains (IND281/2003, IND195/2007, IND360/2007 and IND123/2008) were selected based on set criteria and antigenic relationships with field isolates sampled between 2002 and 2009 were analyzed using a micro-neutralization test. Phylogenetic analysis based on capsid region of serotype A isolates revealed existence of two broad distinct clusters (VP3(59)-deletion and non-deletion group) within genotype 18. The VP3(59)-deletion group has diversified genetically with time giving rise to three different sub-lineages (clade18a, 18b and 18c). The present study indicates that the virus candidates IND281/2003 (VP3(59)-deletion group) and IND195/2007 (non-deletion group) can be used as an adjunct or alternative strain to currently used vaccine strain IND40/2000 in case of emergence of more antigenically divergent isolates in future.     

Evaluation of a monoclonal antibody-based approach for the selection of foot-and-mouth disease (FMD) vaccine strains

Foot-and-mouth disease (FMD) virus exists as seven serotypes within which are numerous variants necessitating careful selection of vaccine strains. Currently, a serological assay system based on the use of polyclonal vaccine antisera is widely used for this selection. However, inherent variability in the matching antisera used makes the tests poorly reproducible and difficult to interpret. In this study, we have explored the possibility of replacing or supplementing the polyclonal antibody (PAb)-based method with one based on use of monoclonal antibodies (MAb). Panels of MAbs raised against two serotype O vaccine strains were examined for reactivity with 22 field viruses, isolated over a 10-year period between 1991 and 2001. Antigenic site 2 was found to comprise more than one epitope. The sequence variation in capsid protein VP2 harbouring antigenic site 2 was analysed and the amino acid residues at positions 79 and 134 appeared to greatly influence the binding of site 2 MAbs. Prediction of antigenic match based on MAb reactivity did not correlate closely with the results of a PAb-based "gold-standard" method and it was concluded that a wider panel of MAbs are needed that recognise all protective epitopes present on the surface of FMD virus together with a better understanding of those epitopes which are important in conferring protection.     

H9N2亚型禽流感疫苗株的筛选

为了筛选免疫原性更好的H9N2亚型禽流感病毒疫苗株。本研究在分析H9N2病毒HA基因遗传进化规律基础上,对预选的毒株进行血凝效价(HA)、鸡胚半数感染量(EID50)测定及交叉血凝抑制试验,筛选出疫苗用毒株进行免疫攻毒保护试验。结果显示,CK/AH/AJ3/15株HA效价达11 log2、EID50值为9.17 log10/0.1 mL,有很好的繁殖能力;对当前流行株的HI反应效价比疫苗株F/98高2-3个滴度;以CK/AH/AJ3/15株制作灭活疫苗,整体保护率达到97.5%,比F/98疫苗株的85%明显提高。表明CK/AH/AJ3/15株可以作为H9N2亚型禽流感新型疫苗株的候选株。     

The pathogenesis of Pasteurella multocida serotype A:3,4 infection in turkeys: a comparison of two vaccine strains and a field isolate

The pathogenesis of avian pasteurellosis caused by two vaccine strains, M-9 and Clemson University (CU), and a highly virulent field isolate, 86-1913, of Pasteurella multocida (serotype A:3,4) was studied in 7-week-old turkeys inoculated by an oculo-nasal-oral technique. Turkeys inoculated with strain CU and isolate 86-1913 developed severe progressive bacteremia that began at 4 hours postinoculation (PI) and peaked at 16-20 hours PI. Turkeys inoculated with strain CU and isolate 86-1913 had significantly higher concentrations of bacteria in blood and tissues, and greater histologic lesion scores for necrosis, heterophil infiltrates, and intralesional bacteria than turkeys inoculated with strain M-9. Immunohistochemical staining specific for P. multocida demonstrated numerous extracellular bacteria in tissues from turkeys inoculated with strain CU and isolate 86-1913. The mortality for turkeys inoculated with isolate 86-1913 was significantly higher than for turkeys receiving the two vaccine strains.     

Testing two Lactobacillus plantarum and Lactobacillus acidophilus strains for their suitability as a lipoid probiotic

Two strains of lactobacilli (Lactobacillus acidophilus T-135 and Lactobacillus plantarum 4/97) were selected in order to study their inhibitory properties against frequent udder pathogens (Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Salmonella enteritidis and Bacillus pumilus), their production of organic acids as well as their ability to survive on the teat skin, the teat duct mucosa and in a lipoid emulsion. Both strains inhibited the tested pathogenic microbes and survived on the investigated surfaces and in an emulsion for more than 6 hours and 11 days, respectively.     

口蹄疫灭活疫苗效力检验替代方法研究进展

目前,口蹄疫灭活疫苗的效力检验方法是采用本动物进行免疫攻毒试验,此方法需要大量的非免疫本动物,存在着动物来源困难,试验成本高、周期长等问题。建立与本动物具有一定统计学关系的口蹄疫灭活疫苗效力检验的本动物替代方法是国内外亟待解决的一个问题。过去数十年中,口蹄疫灭活疫苗结合其他疫苗的研究情况,主要进行了以下几种替代方法的研究:试验动物替代法、血清学替代法、疫苗抗原定量法。但到现阶段仍没有一个像本动物攻毒试验那样确定统一的标准替代方法。为了更深入的了解口蹄疫灭活疫苗效力替代方法的研究现状以及今后的发展方向,本文结合传统口蹄疫灭活疫苗效力检测方法,针对以上几种替代方法的研究进展展开探讨。     

Determination of prevalence,serological diversity,and virulence of Dichelobacter nodosus in ovine footrot with identification of its predominant serotype as a potential vaccine candidate in J&K,India

The aim of this study was to determine the prevalence, serological diversity, and virulence of Dichelobacter nodosus in footrot lesions of sheep and identification of its predominant serotype as a potential vaccine candidate. The overall prevalence of footrot in sheep was 16.19%, and ranged from 13.69 to 19.71%, respectively. A total of 759 flocks with 22,698 sheep were investigated for footrot and 2374 clinical samples were collected from naturally infected sheep exhibiting footrot lesions. Of the 2374 samples collected, 1446 (60.90%) were positive for D. nodosus by polymerase chain reaction (PCR). These positive samples when subjected to serogroup-specific multiplex PCR, 1337 (92.46%) samples carried serogroup B, 247 (17.08%) possessed serogroup E, 86 (5.94%) serogroup I, and one (0.069%) serogroup G of D. nodosus. While mixed infection of serogroups B and E was detected in 127 (8.78%), B and I in 46 (3.18%) and B, E, and I in 26 (1.79%) samples, respectively. The serogroup B of D. nodosus was the predominant (92.47%) serogroup affecting sheep population with footrot followed by serogroup E (19.91%) and serogroup I (4.57%), respectively. Virulent status of D. nodosus strains were confirmed by presence of virulence-specific integrase A (intA) gene and the production of thermostable proteases. The intA gene was detected in 709 (72.79%) samples while gelatin gel test carried out on 246 representative isolates all positive for intA gene produced thermostable proteases, confirming their virulence nature. The PCR-restriction fragment length polymorphism (PCR-RFLP) of whole fimA gene of serogroup B revealed the predominance of serotype B5 (82.97%) of serogroup B. This information suggests that serotype B5 is the predominant serotype of D. nodosus associated with severe footrot lesions in sheep in Jammu & Kashmir (J&K), India. Hence, this serotype can be a potential vaccine candidate for the effective control and treatment of ovine footrot.

     

Assessment of attenuated Salmonella vaccine strains in controlling experimental Salmonella Typhimurium infection in chickens

Salmonella hold considerable promise as vaccine delivery vectors for heterologous antigens in chickens. Such vaccines have the potential additional benefit of also controlling Salmonella infection in immunized birds. As a way of selecting attenuated strains with optimal immunogenic potential as antigen delivery vectors, this study screened 20 novel Salmonella Typhimurium vaccine strains, differing in mutations associated with delayed antigen synthesis and delayed attenuation, for their efficacy in controlling colonization by virulent Salmonella Typhimurium, as well as for their persistence in the intestine and the spleen. Marked differences were observed between strains in these characteristics, which provide the basis for selection for further study as vaccine vectors.     

猪口蹄疫O型灭活疫苗PD50效检攻毒方式探索

用10 000 TCID50的OR/80株F13细胞毒或用10 000 LD50的OR/80株乳鼠组织毒(ORMF8),通过肌肉注射途径、蹄踵球部或趾间皮内注射途径接种O型口蹄疫抗体阴性架子猪进行攻毒试验,三种途径均不能使试验猪产生规律性发病.     

Genomic assessment of suitability of pigs for inclusion in the Pied Danish Pig conservation program – A case study

ABSTRACT

Breed assignment was performed for pigs putatively belonging to Pied Danish Pigs, an endangered Danish conservation breed. DNA array data were available for the candidates who had insufficient pedigree information. So were data for a reference population of animals from Danish conservation breeds as well as data from a range of Northern European pig breeds, and wild boar. Breed proportions were estimated using the program ADMIXTURE. Half the candidates were found to have at least 95% of their ancestry from Pied Danish Pigs. Several candidates showed varying degrees of admixture with other breeds, most notably Gloucester Old Spot. The same was observed for several of the Pied Danish Pig reference animals. The analyzes identified suitable breeding candidates and prevented unwanted recent admixture from spreading in the breeding population.     

鸡白痢沙门菌减毒候选疫苗株的环境应激评价及主要生物学特性测定

为评估鸡白痢沙门菌减毒候选疫苗株对环境应激及消毒剂的抵抗力,本研究对S6702△rfaG△csgA、S6702△rfaL△csgA、S6702△rfaG△csgA△ompR和S6702△rfaL△csgA△ompR等4种鸡白痢沙门菌双基因缺失株和三基因缺失株进行了生长曲线、生物被膜形成能力、药敏试验、环境应激试验及消毒剂的灭活效果的测定。结果显示,4株缺失株均丧失生物被膜形成能力并保留了与亲本株相似的药物敏感性。多基因缺失株对pH为4.0和8.0的培养条件较野生菌株和单基因缺失株更为敏感,呈现微耐酸和不耐碱的特性;所有菌株对54℃热处理和紫外线处理均敏感。消毒剂对实验菌株的杀灭效果显示,野生菌株和各基因缺失株均对0.01%的聚维酮碘溶液敏感,基因缺失株相比野生菌株过硫酸氢钾复合物更为敏感。因此,鸡白痢沙门菌减毒候选疫苗株对环境应激的抵抗力均有所降低,本研究为该菌后续候选疫苗株的进一步筛选奠定了基础。     

北京地区牛口蹄疫母源抗体水平及其疫苗免疫效果的调查研究

用液相阻断ELISA(LPB-ELISA)方法对北京地区主要牛场进行口蹄疫(FMD)母源抗体、免前和免后抗体水平的调查，以探寻牛体内FMD抗体消长规律，制定合理的免疫程序，从而指导北京地区FMD的免疫。结果发现母源抗体水平在5日龄前小于1：32，5日龄开始上升，高水平抗体一直维持到75日龄，90日龄开始回落；加强免疫后抗体滴度大幅度升高，抗体效价离散度降低；用FMD疫苗免疫青年牛1次，21d和51d的抗体平均值都为1：256；成年母牛的分别为1：256和1：248；育成牛的分别为1：248和1：240。说明牛FMD首免的适宜日期为90日龄；适时的加强免疫有利于牛群抵抗FMD；FMD疫苗对育成牛、青年牛和成年母牛都有理想的免疫效果，对青年牛的免疫效果最好。     

国内现有猪口蹄疫O型灭活疫苗对2010年流行毒株的抵抗力

在2010年上半年,猪O型口蹄疫不断发生,作为国内几个生产厂家的猪口蹄疫现用疫苗种毒的提供者,我组进行了猪源流行毒对该种毒灭活疫苗免疫猪的攻毒保护试验及观察。结果如下:1)、猪源流行毒致猪发病速度慢。猪源流行毒接种猪后,致猪发病的时间在3～5天,而疫苗株致猪发病时间是接种后2～3天;2)、该疫苗毒株能有效抵抗猪源流行毒的攻击,PD50大于6;3)、免疫保护和ELISA抗体水平没有线性相关性;4)、免疫保护与140S抗原量有相关性。     

Pathogenesis of two strains of avian paramyxovirus serotype 2, Yucaipa and Bangor, in chickens and turkeys

Nine serologic types of avian paramyxovirus (APMV) have been recognized. Newcastle disease virus (APMV-1) is the most extensively characterized virus, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two strains of APMV-2, Yucaipa and Bangor, in 9-day-old embryonated chicken eggs, 1-day-old specific-pathogen-free (SPF) chicks, and 4-wk-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was more than 168 hr for both strains, and their intracerebral pathogenicity index (ICPI) was zero, indicating that these viruses are nonpathogenic in chickens. When inoculated intracerebrally in 1-day-old chicks, neither strain caused disease or replicated detectably in the brain. This suggests that the zero ICPI value of APMV-2 reflects the inability of the virus to grow in neural cells. Groups of twelve 4-wk-old SPF chickens and turkeys were inoculated oculonasally with either strain, and three birds per group were euthanatized on days 2, 4, 6, and 14 postinoculation for analysis. There were no overt clinical signs of illnesses, although all birds seroconverted by day 6. The viruses were isolated predominantly from the respiratory and alimentary tracts. Immunohistochemistry studies also showed the presence of a large amount of viral antigens in epithelial linings of respiratory and alimentary tracts. There also was evidence of systemic spread even though the cleavage site of the viral fusion glycoprotein does not contain the canonical furin protease cleavage site.     

A monovalent attenuated serotype 2 bluetongue virus vaccine confers homologous protection in sheep

An outbreak of bluetongue caused by bluetongue virus serotype 2 virus in certain Mediterranean countries during 1999/2000, presented an opportunity to produce a monovalent type 2 vaccine. Since no data have been published previously on the protection conferred by the current live attenuated bluetongue vaccine strains used in the polyvalent vaccine, a challenge experiment was performed to determine the degree of homologous protection induced by the type 2 vaccine strain. The standard vaccine dose of 5 x 10(4) pfu of vaccine conferred 99.7% protection against clinical disease and no viraemia was detected in the vaccinates.     

根际促生菌筛选及其接种剂对箭薚豌豆生长影响的研究

通过测定分离自箭薚豌豆和玉米根际4株细菌的固氮酶活性、溶磷量及分泌生长素能力,将其制成植物根际接种剂,并结合半固体培养试验测定接种剂对箭薚豌豆生长的影响。结果表明,菌株J3-1、J1-15和Y16具备溶磷和分泌生长素能力,J1-15的溶磷能力最强,为548．9 mg／L,Y16分泌生长素能力最好,达17．8μg／mL,且菌株Y16具较强固氮能力,J3固氮酶活性为366．51 C2 H4 nmol／（mL·h）。与对照组相比,单一菌株制备的接种剂处理（Y16）可使箭薚豌豆地上生物量、地下生物量分别显著增加104．5％和254．1％（P＜0．05）,复合接种剂处理F（J3-1＋J1-15＋Y16＋J3）使箭薚豌豆地上和地下生物量分别增加76．1％和192．3％。综合各指标,复合接种剂处理效果明显优于单一接种剂,处理F（J3-1＋J1-15＋Y16＋J3）可使箭薚豌豆株高、根长、根表面积、根体积、根系活力,分别较对照增加29．4％,70．0％,174．0％,194．6％,38．3％。这主要是由于菌种间的互作效应造成的。     

口蹄疫疫苗效检模型动物测毒及口蹄疫种毒冻干试验

应用豚鼠作为口蹄疫疫苗效检模型动物检测口蹄疫病毒对模型动物的病原性.用体重400 g左右的豚鼠,将猪O型口蹄疫灭活疫苗效检攻毒毒株ORMF8经后肢蹠部皮内注射途径进行测毒.测毒结果表明,口蹄疫病毒可引起豚鼠出现典型发病,并产生明显病变,ORMF8种毒对豚鼠的毒价可达105.5ID50/0.2 mL.将乳鼠中和试验用种毒OMⅡ按一定比例加入5%蔗糖脱脂牛奶稳定剂进行冷冻真空干燥试验,3次冻干试验结果表明,种毒冻干后病毒含量有一定程度的下降,但下降程度不显著.     

Characterization of toxin from different strains of Pasteurella multocida serotype A and D

Chromosomal DNA from 13 different selected Pasteurella multocida spp. multocida strains of serotypes A and D were isolated and compared. All 10 toxigenic strains were recognized by a DNA probe which included the toxA gene coding for the Pasteurella multocida toxin (PMT). None of the three nontoxigenic strains reacted with the DNA probe. Toxin from the 10 toxigenic strains were isolated and compared. All were found to possess the biological characteristics previously described for the PMT isolated from P. multocida ssp. multocida NCTC 12178, including molecular mass of approx. 143 kDa and reactivity with a series of monoclonal antibodies. Toxin prepared from different toxigenic strains could not be differentiated immunologically by tandem crossed immunoelectrophoresis, Toxin, which was affinity purified from four of the strains and subsequently inactivated by formaldehyde, was cross-protective when used for vaccination of mice before challenge with PMT. It is concluded that the toxin from toxigenic strains of P. multocida ssp. multocida must be very similar, if not identical.