Seroconversion of puppies to canine parvovirus and canine distemper virus: a comparison of two combination vaccines

Sixty puppies were randomly assigned to receive one of two commercially available combination vaccines, and responses to the canine parvovirus and canine distemper virus components of the vaccines were determined by measuring serum antibody titers. The percentage of puppies that seroconverted to canine parvovirus was significantly higher and the mean time for seroconversion was significantly shorter for puppies that received one of the vaccines than for puppies that received the other vaccine. Percentages of puppies that seroconverted to canine distemper virus were not significantly different.     

Dog response to inactivated canine parvovirus and feline panleukopenia virus vaccines

Inactivated canine parvovirus (CPV) and inactivated feline panleukopenia virus (FPV) vaccines were evaluated in dogs. Maximal serologic response occurred within 1-2 weeks after vaccination. Antibody titers then declined rapidly to low levels that persisted at least 20 weeks. Immunity to CPV, defined as complete resistance to infection, was correlated with serum antibody titer and did not persist longer than 6 weeks after vaccination with inactivated virus. However, protection against generalized infection was demonstrated 20 weeks after vaccination. In unvaccinated dogs, viremia and generalized infection occurred after oronasal challenge with virulent CPV. In contrast, viral replication was restricted to the intestinal tract and gut-associated lymphoid tissue of vaccinated dogs. Canine parvovirus was inactivated by formalin, beta-propiolactone (BPL), and binary ethylenimine (BEI) in serum-free media; inactivation kinetics were determined. Formalin resulted in a greater loss of viral HA than either BEI of BPL, and antigenicity was correspondingly reduced.     

Antigen quantification as in vitro alternative for potency testing of inactivated viral poultry vaccines

Routine batch control of licensed inactivated viral vaccines for poultry usually includes a potency assay as a measure of vaccine efficacy. Potency assays often consist of vaccination-challenge experiments in the target species or in laboratory animals. Instead of measuring the protection of vaccinated animals against virulent pathogens, the serological response after vaccination can be quantified for some vaccines. In vitro antigen quantification assays would be attractive alternatives for the current potency assays because the time and costs involved could be greatly reduced and animal use could be avoided. Such in vitro assays will only be acceptable when the correlation between results and efficacy or potency has been demonstrated convincingly. The results of our studies on antigen quantification assays indicate that, in principle, quantification of viral antigens from inactivated oil-adjuvanted vaccines is feasible and reproducible using specially developed antigen capture ELISAs in combination with specific software for statistical analysis of the ELISA data. We have developed methods to quantify the haemagglutination-neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV), the viral protein 3 (VP3) of the infectious bursal disease virus (IBDV), and the spike-1 (S1) protein of the infectious bronchitis virus (IBV). Vaccination experiments with inactivated ND vaccines indicate that the in vitro quantified HN- or F-proteins of NDV are reliable indicators of the serological response after vaccination.     

Antigen quantification as in vitro alternative for potency testing of inactivated viral poultry vaccines

Abstract

Routine batch control of licensed inactivated viral vaccines for poultry usually includes a potency assay as a measure of vaccine efficacy. Potency assays often consist of vaccination‐challenge experiments in the target species or in laboratory animals. Instead of measuring the protection of vaccinated animals against virulent pathogens, the serological response after vaccination can be quantified for some vaccines. In vitro antigen quantification assays would be attractive alternatives for the current potency assays because the time and costs involved could be greatly reduced and animal use could be avoided. Such in vitro assays will only be acceptable when the correlation between results and efficacy or potency has been demonstrated convincingly.

The results of our studies on antigen quantification assays indicate that, in principle, quantification of viral antigens from inactivated oil‐adjuvanted vaccines is feasible and reproducible using specially developed antigen capture ELISAs in combination with specific software for statistical analysis of the ELISA data. We have developed methods to quantify the haemagglutination‐neuraminidase (HN) and fusion (F) proteins of Newcastle disease virus (NDV), the viral protein 3 (VP3) of the infectious bursal disease virus (IBDV), and the spike‐1 (S1) protein of the infectious bronchitis virus (IBV). Vaccination experiments with inactivated ND vaccines indicate that the in vitro quantified HN‐ or F‐proteins of NDV are reliable indicators of the serological response after vaccination.     

犬GM-CSF基因对犬细小病毒VP2 DNA疫苗的免疫增强作用

犬细小病毒编码的VP2蛋白是该病毒重要的结构蛋白和抗原蛋白.利用VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应.为进一步提高VP2 DNA疫苗的免疫活性,本实验利用犬粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因作为生物佐剂研究其对犬细小病毒VP2 DNA疫苗的免疫增强作用.首先通过RT-PCR方法从犬淋巴细胞中扩增GM-CSF基因,并将其插入到pcDNA3.1栽体上,分别构建该基因的两个分泌型真核表达载体,即非融合表达载体pcDNA-cGMCSF和与Myc His融合的表达栽体pcDNA-cGMCSF/MH.用pcDNA-cGMCSF/MH载体转染HEK293T细胞以确定GM-CSF基因能否在真核细胞中进行分泌表达.然后用本室构建的VP2基因表达栽体单免疫小鼠,用VP2表达载体与pcDNA-cGMCSF共免疫小鼠(pcDNA3.1空载体作为阴性对照).免疫后用ELISA方法检测不同时间小鼠血清的抗体水平.用MTT法检测小鼠免疫后35 d时淋巴细胞的增殖活性,同时用ELISA试剂盒检测小鼠淋巴细胞γ干扰素的表达水平.结果表明,本试验构建的表达载体能够介导重组GM-CSF在真核细胞中进行分泌表达.免疫实验表明,利用GM-CSF基因与VP2基因共免疫小鼠,抗体的水平明显高于VP2基因单免疫组(P＜0.01).共免疫组小鼠淋巴细胞的刺激指数和γ干扰素的表达水平均明显高于单免疫组(P＜0.05).由此可见,GM-CSF表达载体可明显提高CPV VP2 DNA疫苗的免疫应答水平.     

犬细小病毒抗体间接ELISA检测方法的建立

利用本实验室建立的昆虫细胞/杆状病毒表达系统表达犬细小病毒病毒样颗粒（CPV-VLPs）,采用硫酸铵沉淀、蔗糖密度梯度离心对表达的CPV-VLPs进行纯化,用电子显微镜、SDS-PAGE及Western-blotting方法检测纯化效果。以纯化的CPV-VLPs作为包被抗原建立CPV间接ELISA检测方法,对各反应条件进行优化并分析其特异性、敏感性、重复性。结果显示,CPV-VLPs经过纯化后纯度可达到95%以上;优化的ELISA最佳工作条件为：纯化抗原包被浓度为5.0mg/L,4℃包被过夜;1%BSA,37℃封闭2h;待检血清1∶40稀释,37℃孵育1.5h;HRP标记的酶标二抗1∶20 000稀释,37℃孵育1h;TMB室温避光显色30min;确定的血清阴性阳性临界值D450nm值为0.264。该方法可特异性检测犬细小病毒阳性血清,与犬瘟热、犬传染性肝炎、犬冠状病毒病、狂犬病阳性血清均不发生反应。该方法的敏感性为1∶640,批内重复试验变异系数小于6%,批间重复试验变异系数小于8%。42份临床血清样本的检测结果表明,与血凝抑制试验的符合率为90.48%。     

Latex agglutination test for canine parvovirus

Canine parvovirus (CPV) was detected in faeces from dogs with diarrhoea by a specific slide agglutination test using latex particles coated with anti-CPV monoclonal antibody (LA-anti-CPV). The agglutination of LA-anti-CPV with CPV on a glass slide was evident macroscopically within 2 min. The sensitivity of the latex agglutination (LA) test was similar to that of the hemagglutination test. The LA test is available for the rapid diagnosis of CPV infection at an animal hospital.     

Anti-endotoxin immunotherapy for canine parvovirus endotoxaemia

The morbidity and mortality associated with canine parvovirus disease (CPV) is caused, in part, by endotoxin (LPS). Equine anti-endotoxin hyperimmune plasma (Anti-LPS) was administered to 89 CPV patients in addition to conventional therapy. In Anti-LPS treated CPV patients mortality was lower (16-8 per cent, 15/89) than in controls that received conventional therapy alone (66-7 per cent, 24/36, P < 0–0005). The hospitalization period of survivors was reduced from 8-5 ± 4-0 days (controls) to 5-2 ± 2-0 days (Anti-LPS treated group). These results suggest that an anti-endotoxin specific therapy should be incorporated into the treatment regimen of CPV, and possibly, other canine enteric disorders, known to produce endotoxaemia.     

犬细小病毒环介导等温扩增快速检测方法的初步建立

为建立犬细小病毒(CPV)环介导等温扩增(LAMP)检测方法,实现CPV的早期快速诊断,本研究根据GenBank登录的CPV VP2基因序列,在其序列保守区域设计LAMP引物,利用CPV基因组DNA为模板进行扩增。结果表明:LAMP方法检测灵敏度达到10-1TCID50/mL;并且与其它细小病毒等无特异性扩增,表现出良好的特异性。与PCR技术相比,LAMP法操作更加简单方便,更适合基层和实验室的快速检测。     

猪瘟活疫苗效力检验替代方法的研究

目前,我国猪瘟活疫苗效力检验最常用的方法是测定猪瘟疫苗中病毒的兔体感染量(RID),但是用该方法易受到检验兔品种、个体和饲养环境的影响。为了探索一种不依赖动物的疫苗效力检验新方法,本研究将待检猪瘟活疫苗系列稀释后分别接种易感细胞,使用抗原捕获ELISA、RT-nest PCR和RT-PCR三种方法检测不同稀释度疫苗中活病毒在感染细胞后增殖的子代病毒,计算疫苗病毒的最高细胞感染剂量,建立了猪瘟疫苗病毒的细胞感染量(CID)的效力检验方法。结果显示:疫苗中活病毒粒子越多,CID就越高;对不同类型猪瘟疫苗的检测结果表明,该方法适用于传代细胞源和细胞源猪瘟活疫苗的效力效检。使用CID和RID两种检测方法同时对12批猪睾丸传代细胞源(传代细胞源)和3批牛睾丸原代细胞源(细胞源)猪瘟活疫苗进行了比对效检,结果表明,用CID测定方法检验,12批猪瘟活疫苗(传代细胞源)每头份均含105CID,3批猪瘟活疫苗(细胞源)每头份含量均为104CID;用兔子感染体温测定法,12批猪瘟活疫苗(传代细胞源)每头份含量在2.6×104~3.0×104RID之间,3批猪瘟活疫苗(细胞源)每头份含7.0×103~8.0×103RID。试验证明:本实验室建立的CID检测结果与现有质量标准RID的结果存在良好的相关性,能够较好反映疫苗中活病毒的含量,有望成为RID的替代方法。     

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.     

Comparative trial of the canine parvovirus, canine distemper virus and canine adenovirus type 2 fractions of two commercially available modified live vaccines

The results of vaccinating two groups of puppies with commercial vaccines, both of which claimed to provide adequate protection with a final vaccination at 10 weeks of age, were compared. Groups of 19 and 20 puppies with similar titres of maternally derived antibodies against canine parvovirus (cpv), canine distemper virus (cdv) and canine adenovirus type 2 (cav-2) at four weeks of age were vaccinated at six and 10 weeks of age and their responses to each vaccination were measured by comparing the titres against cpv, cdv and cav-2 in the serum samples taken immediately before the vaccination and four weeks later. After the vaccination at six weeks of age, all 19 of the puppies in group 1 had responded to cpv and cdv, and 14 had responded to cav-2; in group 2, 17 of the 20 had responded to cpv, 19 to cdv and 15 to cav-2. In both groups the puppies that did not respond to the first vaccination had responded serologically to cpv, cdv and cav-2 at 10 weeks of age.     

犬细小病毒病防治难点与对策

犬细小病毒病是由犬细小病毒(CPV)引起犬的一种接触性、急性、致死性传染病,特征为出血性肠炎或非化脓性心肌炎。本病无明显发病季节,各年龄段犬均可发生,发病率50%～100%,死亡率10%～50%。2～4月龄幼犬感染率     

Stray dogs as sentinels for canine parvovirus

An attempt to determine the prevalence of canine parvovirus in the stray dog population of Franklin County, Ohio (U.S.A.) was made by sampling dogs during the first 6 months of 1981. Serum and fecal samples, which were collected from 209 strays at time of euthanasia, were tested by serum hemagglutination inhibition (HI) and fecal hemagglutination (HA) techniques to determine canine parvovirus experience (seropositive) or fecal virus shedding, respectively. Sera collected from 93 strays for an unrelated study conducted in 1979 were used as the comparison group. All of the 1979 sera were HI negative (< 1:80) whereas, 139 of 201 (69.2%) sera suitable for testing from the 1981 group of strays were HI positive (? 1:80). The fecal HA results from the 1981 group revealed 26 of the 209 (12.4%) dogs were shedding parvovirus at time of euthanasia (HA titers ? 1:64). Of these 26 of the 209 (12.4%) dogs were shedding parvovirus at time of euthanasia were found to be seropositive. These results indicate that the stray population of Franklin County, Ohio, was not exposed in 1979, but by 1981 had experienced and for the most part, had recovered from canine parvovirus as indicated by a 69.2% seropositive dog population with 12.4% active virus shedders. The stray dog population, if sampled regularly, could thus serve as a sentinel for canine parvovirus activity in a community.     

Surveillance activity for canine parvovirus in Italy

Recent identification of unusual canine parvovirus (CPV) mutants in cats and dogs suggests that CPV type 2 (CPV-2), which emerged suddenly in the late 1970s, is undergoing continual genetic and antigenic variations. A peculiarity of parvoviruses is that single-nucleotide substitutions may determine drastic phenotypic changes. The effects of either natural or artificial mutations on CPV phenotypic properties have been largely investigated, and this sets up CPV as an interesting model to study virus evolution. By monitoring the evolution of CPV-2 in Italy, we observed the onset and quick spread of a Glu-426 mutant, antigenically different from the pre-existing variants that were partially displaced within a few years of the initial identification of the new mutant. The identification of CPV-2 variants raises several questions concerning their impact on the efficacy of the current CPV-2 vaccines, based on the original CPV-2 strain that no longer exists in the field.     

Observations on the use of an inactivated canine parvovirus vaccine

Data are presented on studies of field and experimental use of a formalin-inactivated canine parvovirus vaccine. There was an absolute correlation between a single successful vaccination and subsequent protection against clinical disease. Unsuccessful vaccinations were consistently associated with the presence of maternal antibody at the time of vaccination. The vaccine induced an antibody response within two days and anamnestic responses within 24 hours. It is suggested that a single successful vaccination probably protects against clinical parvovirus disease for life.     

Breed-related risk factors for canine parvovirus enteritis

Case records of 305 dogs with canine parvovirus (CPV) enteritis, seen at the Veterinary Hospital of the University of Pennsylvania from July 1, 1981 to Aug 31, 1982, were selected on the basis of admitting diagnoses or signs of diarrhea and vomiting. The case records were subdivided into 3 diagnostic categories, based on final diagnoses and laboratory test results. There were 96 dogs with definite CPV enteritis, 139 with possible CPV enteritis, and 70 with unlikely CPV enteritis. These cases were then stratified by animal's age (less than or equal to 6 months or greater than 6 months) and specific hospital service (medicine or emergency). A control group was selected from all canine case records from the Veterinary Hospital of the University of Pennsylvania for conditions other than the criteria used in selecting the case group. Approximately 2 hospital patients were selected for each CPV enteritis case by frequency matching for hospital service and age. The proportion of dogs with definite CPV enteritis that had each of the clinical signs that were studied was greater than that of dogs in the other CPV enteritis diagnostic categories. The overall survival rate for dogs with definite CPV enteritis was 64.0%; survival was not associated with any given clinical sign of disease. Odds ratios (OR) for the risk of CPV enteritis were calculated for breeds with 3 or more dogs with definite CPV enteritis. The Doberman Pinschers (OR = 3.1), Rottweilers (OR = 6.0), and English Springer Spaniels (OR = 8.1) had a significantly increased risk of CPV enteritis.(ABSTRACT TRUNCATED AT 250 WORDS)     

白细胞介素-12对犬细小病毒VP2 DNA疫苗的免疫增强作用

犬细小病毒编码的VP2蛋白是该病毒重要的结构蛋白和抗原蛋白。利用VP2基因制备的DNA疫苗能够刺激机体产生免疫应答反应。为进一步提高VP2DNA疫苗的免疫应答水平,本研究在小鼠体内尝试了利用白细胞介素12（IL-12）基因表达载体提高VP2DNA疫苗的免疫应答水平。首先采用RT-PCR方法从小鼠脾淋巴细胞中分别扩增IL-12大亚基（P40）和小亚基（P35）cDNA基因;然后在真核表达载体pcDNA3.1A上通过引入内部核糖体进入位点（IRES）序列,分别将P40基因和P35基因插入到IRES序列的上下游,构建成IL-12（P40和P35双亚基）基因表达载体,pcDNA-P40-IRES-P35。将上述表达载体与本室构建的VP2表达载体通过磷酸钙方法转染HEK 293T细胞进行瞬时表达,以确定构建的表达载体能否介导相应基因在真核细胞中进行分泌表达。然后用VP2载体单免疫和VP2载体和IL-12载体共免疫方法对小鼠进行免疫（用pcDNA3.1A作为对照）。免疫后在特定时间通过ELISA方法检测小鼠血清抗VP2蛋白的抗体水平,并通过淋巴细胞增殖实验检测免疫后35d小鼠脾脏淋巴细胞增殖反应。结果表明,扩增的小鼠IL-12P40和P35亚基基因与GenBank的参考序列基本一致。Western-blot检测结果表明,重组IL-12和VP2均能够在HEK293T细胞中进行分泌性表达。ELISA检测结果表明利用IL-2载体与VP2载体共免疫小鼠,其血清中抗VP2的抗体水平明显高于VP2载体单免疫组（P〈0.01）,抗体水平在第35天高达1：5120。淋巴细胞增殖试验结果表明,免疫小鼠的淋巴细胞刺激指数均明显高于对照组（P〈0.01）,VP2载体与IL2载体共免疫组的刺激指数明显高于VP2载体单免疫组（P〈0.05）。由此可见,在小鼠体内,IL-12基因表达载体可明显提高CPV VP2基因疫苗的免疫应答水平。