猪细小病毒病弱毒疫苗免疫效力评价及临床免疫试验

为制定猪细小病毒病弱毒疫苗效力检验的标准,用3批猪细小病毒病弱毒疫苗进行接种猪与接种豚鼠的平行试验;同时进行临床免疫试验。3批疫苗接种豚鼠和猪后定期进行PPVHI抗体检测;猪于免疫后攻毒,并进行病毒分离。免疫母猪在怀孕早期进行强毒攻击,40d扑杀进行病毒分离。用3批疫苗免疫后备母猪统计产仔成绩。结果显示,豚鼠接种后21d、猪接种后7d全部产生抗体反应。免疫攻毒的猪均未从血浆和内脏中分离到病毒,而从对照猪分离到病毒;怀孕母猪强毒攻击后扑杀,胎儿病毒分离均为阴性,而对照猪胎儿病毒分离为阳性;统计数据表明免疫猪的产仔成绩比未免疫猪高,平均每窝多产活仔1.85头,少产死胎木乃伊胎0.65头。结果表明,当免疫豚鼠PPV HI≥64时,免疫猪能抵抗PPV强毒攻击,两者呈正相关;免疫母猪的攻毒试验表明免疫母猪能抵抗PPV经胎盘感染;临床免疫试验证明疫苗具有良好的免疫原性和安全性。     

Comparison of inoculation regimes for the experimental production of swine erysipelas arthritis: II Serological findings in a gel diffusion precipitin test and enzyme-linked immunosorbent assay

The serological response of pigs to Erysipelothrix rhusiopathiae inoculation was monitored by a gel diffusion precipitin test (GDPT) using a crude, serotype-specific, autoclaved antigen and an enzyme-linked immunosorbent assay (ELISA) using a heat-extracted, alcohol precipitated and molecular seived antigen previously shown to react with serum from pigs infected with serotypes 1 or 2. All pigs receiving 3 or 5 weekly intravenous inoculations of either a highly virulent (VRS 229) or a lowly virulent isolate (VRS 252) produced GDPT-reactive antibody within 3 weeks, but only 44% were still reactive at 8 to 9.5 weeks. The ELISA response was significantly higher in pigs inoculated with the highly virulent strain, and was similar in pigs receiving 3 or 5 doses of either strain. In a dose-response trial, after 3 doses of VRS 229, GDPT reactivity occurred earlier and was stronger in pigs given higher doses of E. rhusiopathiae, but the response peaked 3 to 5 weeks after the start of challenge and was short lived. GDPT reactivity correlated with dose, but not with the severity of arthritis. The ELISA demonstrated specific IgG antibody was present by 2 weeks, and persisted to at least 11 weeks. The ELISA reactivity was significantly higher in pigs with arthritis than in pigs that received low doses and were not arthritic. Within groups of pigs with arthritis a significant, dose dependent, linear ELISA response developed but did not correlate with the presence or degree of arthritis at slaughter. Non-arthritic pigs had similar low ELISA responses to uninoculated controls.     

一例猪细小病毒病的诊断

从取自某猪场表现初产母猪流产胎儿的肾脏、脾脏、肠系膜淋巴结组织,进行研磨后接种猪原代肾细胞,成功分离到一株病毒。该病毒的豚鼠红细胞血凝活性为27,用针对猪细小病毒结构蛋白VP2的特异性引物对分离病毒进行扩增,将扩增结果进行克隆测序,结果经BLAST分析后,证实分离到一株猪细小病毒。     

猪细小病毒灭活疫苗安全性试验及佐剂的筛选

本研究以猪细小病毒(PPV)现地分离株(BQ)第30代细胞培养毒株作为灭活疫苗研究用种毒,通过优化病毒增殖条件和培养方法,获得较高滴度的病毒传代细胞培养物用于PPV灭活疫苗的制备.PPV细胞培养毒株分别用甲醛和β-丙内酯进行灭活,对2种灭活剂灭活的病毒液分别用国产铝胶佐剂、进口矿物质白油佐剂以及法国赛比克公司的MONTANIDETM ISA 206、ISA15AVG、IMS251CVG 3种佐剂制备10种灭活疫苗,然后分别进行10日龄乳鼠、60日龄仔猪、不同妊娠阶段母猪的安全性试验及成年豚鼠的免疫效果对比试验.通过比较不同灭活疫苗的安全性和对成年豚鼠的免疫效果,初步确认甲醛灭活的病毒液与佐剂ISA15AVG的组合为PPV灭活疫苗的最佳灭活剂和佐剂组合.     

猪细小病毒细胞适应株的培育及鉴定

从临床表现为皮炎消瘦症状的仔猪肝脏中分离到1株病毒,经聚合酶链式反应（PCR）证实为猪细小病毒（PPV）,采用仔猪原代肾细胞和传代ST细胞分离培养,经蚀斑克隆纯化,培育1株ST传代细胞培养适应毒株,命名为PPV-BQ2007株。免疫过氧化物酶单层细胞试验（IPMA）检测病毒抗原主要分布在细胞核及细胞质内。病毒感染细胞可被已知PPV阳性血清中和。免疫电镜可清晰见到聚集成团的大小不一的病毒粒子,近似圆形,无囊膜,直径大小约20nm-22nm。该分离株经ST细胞培养传代,能够产生典型的细胞病变,病毒滴度随代次显著增加,第30代后毒价达10^7 TCID50/mL以上,且毒价和血凝价稳定。测序结果表明PPV-BQ2007株VP2基因与NCBI公布的皮炎型毒株Kresse株的同源性最高,达99.8％,在系统进化分支上处于同一个分支。PPV-BQ2007株传代细胞培养适应株的成功培育和鉴定,为进一步开展该病毒流行病学、致病机理、疫苗免疫与诊断研究等奠定了良好基础。     

Studies on the multiplication of a porcine parvovirus

A porcine parvovirus has been characterized with regard to its replication in foetal porcine kidney cells and certain biophysical properties. Electron microscopy of infected cells at selected times postinfection revealed that porcine parvovirus replication took place within or near a series of granular intranuclear inclusions which may be contiguous with cellular heterochromatin. Developing virions were observed to aggregate into a nuclear-like amorphous mass which gradually disrupted as cellular integrity was lost. Purified virions were found to have a buoyant density in CsCl of 1.38 g/ml, while ‘empty’ particles has a buoyant density of 1.29 g/ml. The particle diameter was calculated to be approximately 22 nm.     

Interepizootic survival of porcine parvovirus

Porcine parvovirus (PPV) was transmitted by direct contact between experimentally infected and susceptible pigs at 1 and 2 weeks, but not at 4, 8, 16, or 25 weeks, after experimental infection. In contrast, PPV was found to remain infectious for at least 14 weeks in uncleaned rooms previously vacated by experimentally infected pigs. These findings suggest that facilities contaminated by secretions and excretions of infected pigs may provide the major means by which PPV survives between episodes of acute infection.     

Efficacy of porcine parvovirus vaccines

Three inactivated porcine parvovirus vaccines were tested for efficacy in 66 susceptible gilts. The gilts were challenged with virulent virus on the 40th day of gestation. All the vaccines provided excellent protection against fetal mortality despite insignificant serological responses to one of them. Good protection was obtained with two of the vaccines even when the dose was substantially reduced. Unvaccinated controls had very few viable fetuses.     

A slide latex agglutination test for the rapid detection of antibodies in serum against porcine parvovirus

A slide latex agglutination test (LAT) was developed and evaluated to detect serum antibodies against porcine parvovirus. Porcine parvovirus antigen was obtained by 10% PEG-6000 and 0.5 mol/l sodium chloride precipitation, and inactivated by 0.1% methanal. Two per cent suspensions of latex particles (0.5-0.8 microm) were coated by adding an equal volume of porcine parvovirus antigen at 0.34 microg/ml. Repeatability of latex agglutination test was evaluated with a panel of 100 sera using the same and different antigen lots. A good agreement between LAT and haemagglutination inhibit assay was observed. Because of convenience and speed of performance, this method would be used widely in clinic examination.     

猪细小病毒病油乳剂灭活疫苗保存期试验

将猪细小病毒病油乳剂灭活疫苗在2～8℃保存12～15个月后,进行了物理性状、无菌检验、安全检验及效力检验,检验结果符合<中华人民共和国兽用生物制品质量标准>.     

Comparison of the indirect haemagglutination and gel diffusion test for serotyping Haemophilus parasuis

The aim of this study was to compare the use of indirect haemagglutination (IHA) and gel diffusion (GD) tests for serotyping Haemophilus parasuis by the Kielstein-Rapp-Gabrielson (KRG) scheme. All 15 serovar reference strains, 72 Australian field isolates, nine Chinese field isolates, and seven isolates from seven experimentally infected pigs were evaluated with both tests. With the IHA test, 14 of the 15 reference strains were correctly serotyped-with serovar 10 failing to give a titre with serovar 10 antiserum. In the GD test, 13 reference strains were correctly serotyped-with antigen from serovars 7 and 8 failing to react with any antiserum. The IHA methodology serotyped a total of 45 of 81 field isolates while the GD methodology serotyped a total of 48 isolates. For 29 isolates, the GD and IHA methods gave discordant results. It was concluded that the IHA is a good additional test for the serotyping of H. parasuis by the KRG scheme if the GD methodology fails to provide a result or shows unusual cross-reactions.