狂犬病 犬肠炎 犬瘟热 犬腺病毒病犬副流感五联弱毒冻干疫苗的研究

从国外引进的犬瘟热病毒（ＣＤＶ）、犬细小病毒（ＣＰＶ）、犬腺病毒－２型（ＣＡＶ２）和犬副流感病毒病毒（ＣＰＩＶ）四联弱毒样品中，分离筛选出增殖性良好的ＣＰＶ０ＸＮ１，ＣＡＶ２－ＸＮ３和ＣＰＩＶ－ＸＮ４株。另外还通过离体异种细胞交叉传代，获得的ＣＤＶ－ＸＮ１１１２弱毒株；从狂犬病毒株疫苗样品中筛选出ＥＲＡ８３６株。经试验证明这５株病毒在５代以内可作为制苗用种毒。采用静置和旋转培养法高滴度扩增这５株弱     

犬2型腺病毒对犬的口服免疫研究

对未经犬腺病毒免疫的两月龄左右杂交狼犬，随机分为６组每组５只，另设５只对照，用作者分离鉴定和致弱驯化的犬喉气管炎病毒的第６０代ＤＫ细胞培养物（ＳＹＣＡＶ－２－ＤＫ６０）对犬进行免疫试验，在免疫第４周采血分离血清，测定犬腺病毒的血凝抑制抗体（ＨＩ）效价，结果３０只试验犬全部产生了ＨＩ抗体，表明ＳＹＣＡＶ－２可通过口服或饵料的途径对犬实施免疫，为犬口服联合疫苗以及犬２型腺病毒为勒体的口服狂犬 －腺病毒     

犬瘟热疫苗弱毒的复壮与免疫试验

将一株犬瘟热疫苗株病毒通过Ｖｅｒｏ细胞２０代（ＣＤＶ／Ｒ－２０）后又通过敏感犬的腹腔巨噬细胞，获得一株免疫原性更高的弱毒株（ＣＤＶ／Ｒ－２０／８）。接种同样剂量１０４ＴＣＩＤ５０的ＣＤＶ／Ｒ－２０／８和ＣＤＶ／Ｒ－２０的犬，产生出血清中和（ＳＮ）抗体价的几何平均数分别为１：５４和１：２６。前者为后者的２倍多。接种１０５ＴＣＩＤ５０的ＣＤＶ／Ｒ－２０／８能完全保护犬（１０／１０）抵抗ＣＤＶ强毒的攻击，接种１０４ＴＣＩＤ５０的保护９／１０犬，而对照犬５只全部发病，其中４只死亡。犬体内ＳＮ价可持续一年之久。冻干培养物在４和－３０℃中可分别保存９个月和１２个月而效力不减。     

犬场蚊子与犬细小病毒

２０００年４月，应用套式ＰＣＲ技术从犬场蚊子的血液样品检测出犬细小病毒。将分离到的犬细小病毒ＶＰ２－Ｙ３４基因片段克隆到ＰＭＤ１８－Ｔ载全，其病毒基因组ＣＰＶ－ＰＶ２－Ｙ３４序列测定结果显示与作者在ＧＥＮＥＢＡＮＫ发表的肺病变犬细小病毒ＣＰＶ－ＨＮ－１株有９９％同源性。     

犬冠状病毒超免疫血清的制备及应用

采用犬冠状病毒（ＣＣＶ）弱毒株和强毒株多次免疫健康犬，采集超免疫血清，其ＣＣＶ血清中和（ＳＮ）抗体效价高达１：２１０。试验结果表明，该超免疫血清安全、特异，无毒副作用，每只幼犬注射１．５ｍＬ／ｋｇ体重的该血清即可使９０％以上的犬获得１０￣１５ｄ的被动免疫保护；对１１０只患ＣＣＶ性肠炎病犬的临床治疗结果表明，该超免疫血清治疗效果可靠，其治愈率达９４％。     

犬冠状病毒的分离鉴定及其纤突蛋白基因的克隆与序列比较

以犬胎原代细胞，采用同步接种、带毒传代和添加新细胞的方法，从１例腹泻犬脾脏和１例临床健康犬肠内容物中分离出２株犬冠状病毒（ＣＣＶＹＳ１，ＣＣＶ ＣＩ１）。该２株病毒可使ＣＲＦＫ细胞出现典型的细胞融合病变，与从美国引进的ＣＣＶＮＬ－１８参考株形成的细胞病变相似；电镜负染和超薄切片观察，分离毒株细胞培养物中均含有典型的冠状病毒粒子，并形成胞浆内包涵体；经理化学、生物学鉴定，濉有ＣＣＶ的特性；间接免疫荧     

西宁地区黑白花奶牛,猪及藏犬红细胞免疫功能初探

应用红细胞Ｃ＿３ｂ受体（ＲＢＣ－Ｃ＿３ｂＲ）花环和红细胞免疫复合物（ＲＢＣ－ＩＣ）花环试验对西宁地区饲养的３５例黑白花奶牛，２０例８０日龄左右的荣昌猪及６例藏犬的红细胞免疫功能，分别进行了检测。结果证明被测三种动物的红细胞膜上均具有Ｃ＿３ｂＲ，并测得ＲＢＣ－Ｃ＿３ｂＲ及ＲＢＣ－ＩＣ的花环百分率分别为：牛９．３４±６．１４％及６．４９±３．１４％，猪４．３５±１．０８％及２．３５±１．１％，藏犬３．５９±０．４８％及１．３４±０．３３％。从而证明红细胞免疫系统（ＲＣＩＳ）亦适用于黑白花奶牛、荣昌猪及藏犬等动物。文中还对黑白花奶牛血清中红细胞免疫粘附（ＲＣＩＡ）促进因子和抑制因子进行了探讨。     

检测犬冠状病毒中和抗体的方法与应用

在２４孔组织培养板上应用从美国引进的犬冠病毒（ＣＣＶ）参考株ＮＬ－１８与猫肾传代细胞（ＣＲＦＫ）采用固定病毒（１００ＴＣＩＤ５０）稀释血清法建立了ＣＣＶ中和试验,运用该方法测定了１４２条群养犬和３５条散养犬的中和抗体水平,以１：４作为阳性血肖的判定标准,群养犬与散养犬的血清阳性率分别为１００％和８２．９％,测定了母犬的血清与乳汁,所产仔犬脐带血的ＣＣＶ抗体效价,证实母犬可以通过胎盘及乳汁将抗体转移     

动物细胞系的染色体组型分析和致癌／致瘤性

为确定供生物制品和生物工程产品生产所用动物传代细胞系有无致癌／致瘤性,对研制生产犬、貉、狐用五联病毒,即犬狂犬病病毒（ＲＶ）、犬瘟热病毒（ＣＤＶ）、犬细小病毒（ＣＤＶ）、犬腺病毒２型（ＣＡＶ２）和犬副流感病毒（ＣＰＩＶ）的活疫苗以及猫、狮、虎、豹用三联病毒,即泛白细胞减少症病毒（ＦＰＬＶ）、猫疱疹病毒１型（ＦＨＶ１）和猫杯状病毒（ＦＣＶ）的活疫苗,所用猫肾细胞系（Ｆ８１,ＣＲＦＫ）、犬肾细胞系（ＭＤＣＫ）、非洲绿猴肾细胞系（Ｖｅｒｏ,Ｖｅｒｏ２）、恒河猴肾细胞系（ＭＡ１０４）和叙利亚金…     

猪生殖与呼吸综合征病毒(PRRSV)CH-1a株非结构基因的分子克隆及其基因特征的研究

本研究对猪生殖－呼吸道综合征病毒（ＰＲＲＳＶ）的非结构基因,包括ＯＲＦ１基因、５’端非编码区基因（Ｎｏｎ－ＣｏｄｉｎｇＲｉｇｏｎ,ＮＣＲ）和３’端非编码区基因（ＮＣＲ）分别进行了分子克隆和测序,并对其基因特征作了研究分析。结果表明,ＰＲＲＳＶ－ＣＨ－１ａ株ＯＲＦ１ａ起始密码子上游是由保守序列５’ＵＵＡＡＣＣ３’连接的５’ＮＣＲ,在该区附近的约４３个碱基有较高的保守性,其余核苷酸的变异较大。ＯＲＦ１ａ编码的多聚蛋白又可切割生成六个非结构蛋白（Ｎｓｐ１ａ、Ｎｓｐ１β、Ｎｓｐ２－５）,其中Ｎｓｐ２可能具有型特异性,在不同基因型的ＰＲＲＳＶ分离株之间表现出较大的变异,和ＶＲ２３３２株的ＮｓＰ２的编码基因相比有８６％的同源性,同ＬＶ株只有４５％的同源性;ＯＲＦ１ｂ所编码的聚合蛋白经切割后形成四个非结构蛋白（ＲｄＲｐ、ＣＰ２－４）,同 ＯＲＦ１ａ所编码的蛋白相比较为保守,但是,在ＣＰ４的Ｃ端仍有较大的变异,其编码区和ＶＲ２３３２株相比有８８．７％的同源性,而和ＬＶ株只有５３．４％的同源性。ＣＨ－１ａ株的ＯＲＦ１ａ－ＯＲＦ１ｂ的衔接区为核糖体移码区,在所有的ＰＲＲＳＶ分离株中高度保守,具有保守的５’ＵＵＵＡＡＡＣ３’序列和     

犬冠状病毒YS1株细胞培养弱毒的实验免疫研究

应用纯化后的犬冠状病毒 (CCV)YS1株细胞培养致弱的弱毒 (CCVYS1V60 ) ,经口鼻和肌肉接种CCV ,SN抗体小于 1∶2的易感犬作安全试验 ,结果未见任何CCV临床症状与病理解剖学改变 ;用不同剂量的该CCV弱毒分组免疫CCV易感犬 ,经SN抗体测定与用CCV强毒攻毒试验 ,结果SN抗体达 1∶60以上的犬 90 %以上可获得免疫保护 ;应用该CCV弱毒分别免疫母源抗体达 1∶4和 1∶8的 2组试验犬 ,第 1次免疫 1 4d后SN抗体均未见升高 ,追加 2～ 3次免疫后 ,SN抗体才逐渐上升至 1∶60以上的免疫保护水平 ;用CCV易感犬和猫肾传代细胞 (CRFK)分别将该CCV细胞培养弱毒连续传 5代和 2 0代 ,结果对犬仍然安全 ,免疫原性也未见下降 ;与犬瘟热病毒 (CDV)、犬传染性肝炎病毒 (ICHV)和犬细小病毒(CPV)弱毒作免疫互扰试验 ,结果与各弱毒的单独免疫结果未见差异 ;该毒的免疫期在 1年以上 ,- 2 0℃冻结保存的保存期为 9个月。     

犬冠状病毒核酸探针的制备及其基因序列的测定与比较

采用RT-PCR方法扩增犬冠状病毒(CCV)YS1、C11和NL-18株5′端部分S基因序列,以随机插入DNA法对CCV YS1株纯化的PCR产物标记32P同位素,制备核酸探针,并与3株CCV反转录产物杂交.用平端连接法将3株CCV PCR产物克隆于pUC19 Sma I位点或pGEM-T载体中,经PCR鉴定为正确重组质粒.以双脱氧末端终止法测定了重组质粒的cDNA核苷酸序列,并用DNASIS计算机软件进行多重比较分析,绘制系统树.结果表明,所制备的核酸探针可与3株CCV反转录产物杂交,其核苷酸序列与多株CCV相应序列的同源性高达91.9%～99.1%,为CCV的保守区.由此说明,制备的核酸探针可用于犬CCV感染的分子流行病学研究.     

A short incubation serum neutralization test for bovine viral diarrhea virus.

A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.     

Detection of antibodies to avian pneumovirus by a micro-indirect immunofluorescent antibody test.

A micro-indirect immunofluorescent antibody (micro-IFA) test with a 96-well, flat-bottomed microplate was developed for measuring avian pneumovirus (APV) antibodies. Two Japanese APV strains (MM-1, 8597/CV94) isolated at different places and times and Vero cells were used for antigen preparation in this test. The test results were compared with those of a serum neutralization (SN) test. By the micro-IFA test, specific immunofluorescent antigens were observed in the cytoplasm of cells infected with either strain, and the antibody titers of antisera to these strains were quite similar. In most cases, the results were obtained within 3 hr. Antibody titers between the micro-IFA and SN tests were highly correlated, with correlation coefficients of 0.873 (MM-1 strain) and 0.889 (8597/CV94 strain). We also investigated APV antibody status in two farms for a period of about 2 yr by the micro-IFA test and revealed that APV infections were repeated within these farms. On the basis of these results, we conclude that our micro-IFA test is useful for routine serologic surveys of APV infections, particularly when a large number of samples are to be treated, because this test was time and labor saving relative to SN tests or conventional IFA tests utilizing embryo tracheal organs or coverslip cell cultures.     

应用琼脂免疫扩散试验检测猪伪狂犬病抗体的研究

建立了ＡＧＩＤ用于猪伪狂犬病（Ｐｓｅｕｄｏｒａｂｉｅｓ,Ｐｒ）的诊断。对９８份被俭血清的ＡＧＩＤ结果与ＳＮ结果比较,当ＳＮ滴度大于１：８时两者的阳性检出符合率为１００％,ＳＮ滴度小于或等于１：８时,两者的阳性检出符合率为８７．５％,ＡＧＩＤ和ＳＮ总的阳性符合率为９４．４％,结果表明,ＡＧＩＤ对Ｐｒ可进行特异性诊断,流行病学调查和免疫动态监测。     

Evaluation of serologic methods for the detection of antibodies to encephalomyocarditis virus in swine fetal thoracic fluids.

Hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) tests were compared to the serum neutralization (SN) test to evaluate their ability to detect antibodies to encephalomyocarditis virus (EMCV). Swine fetal thoracic fluids of known EMCV SN antibody titers (200 samples greater than or equal to 1:2, 100 samples less than 1:2) were selected from a collection of field cases. The thoracic fluids were tested for EMCV antibodies by HI and AGID, and the results were compared to those of the SN test. Of 200 SN antibody-positive samples, 183 (91.5%) and 173 (86.5%) were positive in HI and AGID tests, respectively. Of 100 SN-negative samples, 81 (81%) and 94 (94%) were negative in HI and AGID tests, respectively. Agreement between the tests was analyzed by calculating Kappa values. The values were 0.73 between SN and HI tests and 0.77 between SN and AGID tests, indicating very good to excellent agreement for HI and AGID tests with the SN test. Of 200 SN-positive samples, 19 samples with low SN titers (1:2-1:16) were further tested by Western immunoblotting, and all were confirmed as positive. Interpretation of the present results suggests that both HI and AGID tests can be used as alternatives to the SN test.     

Immune potency of lyophilized, killed vaccine for equine viral arteritis and its protection against abortion in pregnant mares

Immune potency test was conducted in horses by inoculating a killed vaccine for equine viral arteritis (EVA) which had been freeze-dried and contained aluminum hydroxide adjuvant. Serum neutralizing (SN) antibody to equine arteritis virus (EAV) was detected at maximal titers of 1:80 to 1:640, 1 to 2 weeks after 2-dose vaccination of 6 female horses. However, 6 pregnant mares inoculated with the vaccine which had been kept in storage for 1 year at 4°C produced much higher titers ranging from 1:320 to 1:1280. A maximal mean titer of 1:199.5 occurred in the 1st and 2nd week after 2-dose inoculation with the nonpreserved vaccine, whereas a maximal mean titer of 1:794.3 occurred in the 2nd week using the preserved vaccine. The horses showed no systemic or local adverse reactions clinically or hematologically after vaccination. Four of the 6 vaccinated pregnant mares were exposed to the Bucyrus strain of EAV but resisted challenge exposure, while 3 nonvaccinated control pregnant mares revealed acute EVA causing abortion and death. Isolation of EAV was positive from the body tissues of the aborted and dead fetuses and their dams, but was negative from the vaccinated mares. No significant rise of SN antibody titers was detected in the vaccinated mares following challenge exposure, suggesting that the vaccine can protect against EAV infection in pregnant mares and prevent abortion or death.     

Evaluation of multiple immune parameters after vaccination with modified live or killed bovine viral diarrhea virus vaccines

The development of immunity to vaccine antigen was examined using three prime/boost strategies and the progression of immune activities was evaluated over the course of 8 weeks. Calves were vaccinated and multiple immune parameters were evaluated using several methods to assess humoral or cellular immunity from the same samples in parallel. The three vaccination protocols used were a killed vaccine followed by a killed boost (killed/killed), MLV vaccine and boost (MLV/MLV), or a MLV vaccine and killed boost (MLV/killed). All the vaccines used included modified live IBR/PI3 viruses to make the bystander context as similar as possible. The Singer strain of BVDV was used as the source antigen in the killed vaccine, and the NADL strain of BVDV was used in the MLV vaccine. Controls received a vaccine containing only MLV IBR/PI3. The assessment panel measured SN titers, as well as lymphocyte proliferation, cytokine mRNA expression, intracellular cytokine production, and released IFN-gamma after in vitro stimulation with three strains of BVDV virus. MLV/MLV and MLV/killed groups developed significant SN titers to the type 1 BVDV virus strains, Singer and NADL, and low crossover titers were also seen to the type 2 strain, 890 over the evaluation period. These two groups showed significant proliferation in response to the NADL virus as compared to controls. Multiple immune assessments were conducted simultaneously to attempt to provide a broader, more in depth evaluation of immune response to these BVDV vaccination protocols. We observed that the correlation among most of the assays conducted were weak; the correlation between SN titers and cellular proliferation assays demonstrated a moderate correlation.     

Detection of canine coronaviruses genotype I and II in raised Canidae animals in China

An RT-nPCR assay was used for testing fecal samples of dogs, foxes, raccoon dogs and minks for the presence of canine coronavirus (CCV). The animals were raised in homes, dog schools or farms. Seventy out of 81 healthy dog feces from three cities and 21 out of 48 diarrhea feces from pet dogs were positive for type II CCV. From a total of 61 healthy fox feces, 43 were positive for type II and 29 for type I CCV, out of which 25 were simultaneously positive for the two different genotypes. Among 24 raccoon dogs samples, 22 were CCV type II-positive, and from those 16 were additionally type I positive. No CCVs was detected from healthy mink feces. Sequence analysis found that ten type II CCVs fragments of M gene shared a high similarity with reference strain CCV 1-71 (96.5-99.5%), and four type I CCVs shared a high similarity (96.7%-98.1%) with a reported FCV-like CCV strain. The sequence of one particular M gene fragment was found to cluster between the type I and type II CCV branches in phylogenetic analysis, suggesting the existence of a novel strain. Our study confirmed that type II CCVs infection is very common in domestic dog, fox, and raccoon dog populations in China. This is also the first report on the co-existence of two CCV genotypes in healthy foxes and raccoon dogs.     

Evaluation of the radial immunodiffusion enzyme assay for the detection of antibodies to pseudorabies virus

The validity of radial immunodiffusion enzyme assay (RIDEA) as a diagnostic test for antibodies to pseudorabies virus (PRV) in porcine serum was determined. Serum samples from sows and offspring were tested for the presence of antibodies to PRV, using both the RIDEA and the PRV serum-neutralization (SN) test. Overall sensitivity and specificity of the RIDEA done on serums from the sows were 95.7% and 95.2%, respectively. This sensitivity compares with 97.3% sensitivity of the SN test of the same serums. In 658 swine serum samples from routine submissions to the University of Missouri-Columbia Veterinary Diagnostic Laboratory that were tested by the RIDEA, the calculated sensitivity and the specificity were 94.3% and 98.9%. The RIDEA and SN test were equally sensitive (99.0%) to detect antibodies resulting from infection with a field strain of virus. They had reduced sensitivity (RIDEA, 91.7%; SN test, 95.2%) in tests of serums from vaccinated sows. For the detection of passively transferred antibodies in young pigs, sensitivity of the RIDEA was 76.1%, and specificity was 100%. In all instances, RIDEA was 100% sensitive at SN titers of 1:16 or greater. In testing serum samples of swine after field virus infection, sensitivity and specificity of the RIDEA approximated those of the SN test. This reliability, together with its ease of performance, makes the RIDEA an ideal field test in programs to detect PRV-infected herds and in programs designed to free herds of PRV infection.