狂犬病病毒中和抗体ELISA技术的建立

将狂犬病病毒(RV)糖蛋白(G蛋白)中和抗原表位串联表达的重组蛋白作为抗原,建立了检测RV中和抗体的间接ELISA技术。结果表明,最佳抗原包被量为2μg/孔,被检血清最佳稀释倍数为1:200。该方法与快速荧光灶抑制试验(RFFIT)的阳性符合率为88%,阴性符合率为96%。特异性试验表明,该抗原不与犬腺病毒I型、犬细小病毒、犬瘟热病毒、犬副流感病毒和犬冠状病毒阳性血清发生交叉反应,具有良好的特异性。板内和板间重复性试验的平均变异系数分别为2.7%和4.2%,具有良好的重复性,为动物RV中和抗体检测提供了简单快捷的检测方法。     

Characterization of canine adenovirus type 1 isolated from American black bears

Viruses recovered from tissues taken at necropsy from American black bears were examined by use of immunofluorescence with polyclonal and monoclonal antibodies, virus neutralization with monoclonal antibodies, and restriction endonuclease analyses of the viral genomes. With these techniques, viruses were determined to be canine adenovirus type 1. Seronegative dogs that were inoculated with the virus had clinical signs typical of infectious canine hepatitis, suggesting that the virus, which was virulent for bears, was not a vaccinal strain, but a wild strain of canine adenovirus type 1.     

Respiratory disease outbreak in a veterinary hospital associated with canine parainfluenza virus infection

A cluster of canine parainfluenza virus infections was identified in a veterinary referral hospital. While hospital-associated outbreaks of canine parainfluenza virus infection have not been previously reported, veterinary hospitals possess some of the same risk factors that may be present in traditional high-risk sites such as kennels. Hospital-associated transmission of canine respiratory pathogens, therefore, must be considered.     

Compatibility of a bivalent modified-live vaccine against Bordetella bronchiseptica and CPiV, and a trivalent modified-live vaccine against CPV, CDV and CAV-2

Eight puppies (group 1) were vaccinated once with a bivalent modified-live vaccine against infectious tracheobronchitis by the intranasal route and at the same time with an injectable trivalent vaccine against canine parvovirus, canine distemper virus and canine adenovirus; a second group of eight puppies (group 2) was vaccinated only with the intranasal bivalent vaccine, and a further eight puppies (group 3) were vaccinated only with the injectable trivalent vaccine. Three weeks later they were all challenged with wildtype Bordetella bronchiseptica and canine parainfluenza virus by the aerosol route, and their antibody responses to the five vaccine organisms were determined. Oronasal swabs were taken regularly before and after the challenge for the isolation of bacteria and viruses, and the puppies were observed for clinical signs for three weeks after the challenge. There were no significant differences in the puppies' titres against canine parvovirus, canine distemper virus and canine adenovirus type 2 between the groups vaccinated with or without the bivalent intranasal vaccine. After the challenge the mean clinical scores of the two groups vaccinated with the intranasal vaccine were nearly 90 per cent lower (P=0.001) than the mean score of the group vaccinated with only the trivalent injectable vaccine, and the puppies in this group all became culture-positive for B bronchiseptica and canine parainfluenza virus. There were only small differences between the rates of isolation of B bronchiseptica from groups 1, 2 and 3, but significantly lower yields of canine parainfluenza virus were isolated from groups 1 and 2 than from group 3.     

犬副流感病毒的分离鉴定及其F基因的遗传分析

14份疑似患副流感病毒病犬鼻咽拭子,处理后同步接种vero细胞,分离病毒。通过PCR检测、红细胞吸附试验和动物回归试验,确定1株为副流感病毒,命名为CPIV-CC。该株副流感病毒具有吸附豚鼠红细胞能力,能使vero细胞出现细胞病变。F基因与其他的犬副流感病毒F基因同源性高达97%以上。     

Differentiation of canine coronavirus and porcine transmissible gastroenteritis virus by neutralization with canine,porcine and feline sera

Monospecific antisera were prepared in rabbits against canine coronavirus (CCV) and transmissible gastroenteritis virus of pigs (TGEV), and in 24 pigs and 3 cats against TGEV alone. Neutralizing antibody titres were higher for the immunizing than the heterologous virus, although cross-neutralization usually was detected. This confirmed that CCV and TGEV are distinct, but antigenically related coronaviruses. In sera from 41 dogs, CCV-neutralizing titres were on average 2.7 fold higher than TGEV-neutralizing titres, suggesting that CCV was the causal agent. Sera from 29 cats in colonies with feline infectious peritonitis (FIP) and known to contain TGEV-neutralizing antibody, were found to have titres 12.3 fold higher against CCV. The FIP virus (FIPV) is probably more closely related to CCV than TGEV as judged by antigens involved in virus neutralization.Antisera to two isolates of bovine coronavirus, three isolates of haemagglutinating encephalomyelitis virus, seven strains of avian infectious bronchitis virus and the 229E strain of human coronavirus all failed to neutralize CCV and TGEV. Thus CCV, TGEV and probably FIPV fall into a group of antigenically related agents, separable from other members of the family Coronaviridae, by both virus neutralization and immunofluorescence tests.     

Natural infection of captive coyote pups with a herpesvirus antigenically related to canine herpesvirus

Herpesviruses were isolated from captive coyote pups with ocular discharge and hepatomegaly. The viruses were shown to be antigenically related to canine herpesvirus on the basis of specific virus neutralization with canine herpesvirus antiserum. The epizootiology of the outbreak suggested that the herpesvirus was acquired by indirect contact with guard dogs being cared for by the same animal technicians who cared for the coyotes.     

Mass mortality in seals caused by a newly discovered morbillivirus

During a recent disease outbreak among harbour seals (Phoca vitulina) in the North and Baltic seas, more than 17,000 animals have died. The clinical symptoms and pathological findings were similar to those of distemper in dogs. Based on a seroepizootiological study, using a canine distemper virus (CDV) neutralization assay, it was shown that CDV or a closely related morbillivirus (phocid distemper virus-PDV) was the primary cause of the disease. The virus was isolated in cell culture from the organs of dead seals and characterized as a morbillivirus by serology (immunofluorescence neutralization and enzyme-linked immunosorbent assays) and by negative contrast electron microscopy. Experimental infection of SPF dogs resulted in the development of mild clinical signs of distemper and CDV-neutralizing antibodies. The disease was reproduced in seals by experimental inoculation of organ material from animals that had died during the outbreak. However, seals that had been vaccinated with experimental inactivated CDV vaccines were protected against this challenge. This fulfilled the last of Koch's postulates, confirming that the morbillivirus isolated from the seal organs, was the primary cause of the disease outbreak. The recent demonstration of the presence of a similar virus in Lake Baikal seals (Phoca sibirica), which infected these Siberian seals 1 year before the northwestern European seals were infected, raises new questions about the origin of this infectious disease in pinnipeds.     

In vitro antiviral efficacy of ribavirin against feline calicivirus, feline viral rhinotracheitis virus, and canine parainfluenza virus.

Ribavirin had marked in vitro activity against feline calcivirus, strain 255, and canine parainfluenza virus, but showed only slight antiviral effect on feline viral rhinotracheitis virus. Antiviral activity was manifested by partial to complete suppression of viral cytopathic effect and of viral replication, depending on concentration of ribavirin in the culture medium and dosage of viral inoculum. Concentrations of ribavirin as small as 3.2 microgram/ml and 1.0 microgram/ml showed some activity against feline calcivirus and canine parainfluenza virus, respectively.     

Isolation and identification of a canine coronavirus strain from giant pandas (Ailuropoda melanoleuca)

Two giant pandas (Ailuropoda melanoleuca) died of unknown causes in a Chinese zoo. The clinical disease profile suggested that the pandas may have suffered a viral infection. Therefore, a series of detection including virus isolation, electron microscopy, cytobiological assay, serum neutralization and RT-PCR were used to identify the virus. It was determined that the isolated virus was a canine coronavirus (CCV), on the basis of coronavirus, neutralization by canine anti-CCV serum, and 84.3% to 100% amino acid sequence similarity with CCV. The results suggest that the affected pandas had been infected with CCV.     

Evaluation of the efficacy and duration of immunity of a canine combination vaccine against virulent parvovirus, infectious canine hepatitis virus, and distemper virus experimental challenges

The results of this study confirmed that dogs vaccinated subcutaneously with a commercially available multivalent vaccine containing modified-live canine distemper virus, canine adenovirus type 2, canine parvovirus type 2b, and canine parainfluenza virus antigens were protected against sequential experimental challenge 55 to 57 months after initial vaccination given at 7 to 8 weeks of age. All 10 vaccinates were protected against clinical diseases and mortality following parvovirus and infectious canine hepatitis experimental infections. All vaccinates were protected against mortality and 90% against clinical disease following distemper challenge. These data support at least a 4-year duration of immunity for these three "core" fractions in the combination vaccine.     

Diarrheal condition in dogs associated with viruses antigenically related to feline herpesvirus

Viruses with properties consistent with herpesvirus were isolated from dogs with diarrhea. The viruses were shown to be antigenically related to feline herpesvirus-1 (FHV-1) by virus neutralization tests. It was also observed that a canine herpesvirus (CHV) prototype, D004, and two field isolates from fatal CHV infections in 2-week-old and 6-week-old puppies were neutralized at a low level by antiserum to FHV-1. Reciprocal neutralization tests with CHV antiserum against FHV-1 were negative. These results indicated that viruses related to FHV-1 can infect the dog and that there appears to be uni-directional virus neutralization of CHV by FHV-1 antibody.     

H3亚型犬流感病毒的荧光定量PCR方法建立及其增殖特性研究

旨在建立H3亚型犬流感病毒(canine influenza virus,CIV)荧光定量PCR方法,利用该方法对病毒在犬肾细胞(MDCK细胞)上增殖特性进行研究.根据CIV的血凝素(HA)片段上的HA2基因保守区设计并合成特异性引物,构建CIV-HA重组质粒作为标准品,经过条件优化,建立H3亚型CIV荧光定量检测方法...     

犬瘟热病毒YD株的分离鉴定及其H基因序列分析

从疑似犬瘟热(CD)病犬的脏器中分离到1株病毒,经间接免疫荧光试验、RT-PCR鉴定、红细胞凝集试验和对不同动物致病性试验,证实该病毒为犬瘟热病毒(CDV)强毒株,命名为CDV YD株.对H基因序列的测定和分析表明,YD株H基因与国内强毒株更接近,核苷酸同源性为98.4％～99.7％,氨基酸同源性为97.8％～99.7...     

副流感病毒基因工程研究进展

副流感病毒是一类不分节段的单股负链RNA病毒,按基因型可分为1、2、3、4和5型,5个型之间有少量的交叉免疫,各型的生物学特性和结构类似。该病毒宿主谱广,可感染人、牛和犬等多种动物。副流感病毒具有许多优良的特性,可作为活病毒疫苗的候选载体,通过反向遗传操作系统可使其表达外源基因,研发新型疫苗。作者在综述副流感病毒病原学、基因组结构及主要蛋白质等生物学特征的基础上,着重介绍了副流感病毒作为病毒载体在基因工程方面的研究进展。     

Antigen requirements and specificity of enzyme-linked immunosorbent assay for detection of canine IgG against canine distemper viral antigens

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of specific immunoglobulin G (IgG) against canine distemper virus (CDV) antigens. Sucrose gradient separation of viral and cellular proteins was required to produce coating antigens for the ELISA. The specificity of the ELISA was demonstrated by blocking CDV-positive canine sera with CDV-specific antisera produced in goats and rabbits and adsorption of positive sera with CDV antigens. A comparison of the ELISA with the serum-neutralization technique for the detection of CDV antibodies was conducted. Anti-CDV IgG was detected in conventional dogs as early as 6 days after inoculation with a commercial vaccine to CDV. Paired sera from the immunized dogs were evaluated by both techniques and a statistically (P less than 0.01) significant agreement between the ELISA and the serum-neutralization technique was shown (r = 0.6121, n = 75).     

The indirect fluorescent antibody technique as a method for detecting antibodies in aborted fetuses.

In this investigation the indirect fluorescent antibody technique was used to titrate antibodies in bovine sera to parainfluenza 3, infectious bovine rhinotracheitis virus and bovine viral diarrhea virus. These results were compared to those determined on the same samples by hemagglutination inhibition for parainfluenza 3 virus and serum neutralization for bovine virus diarrhea and infectious bovine rhinotracheitis virus. The results of the serological methods agreed closely. The indirect fluorescent antibody technique is a rapid and sensitive method for detecting antibodies and the procedure lends itself to use in diagnostic laboratories. In addition to the above viruses the presence or absence of antibodies to bovine coronavirus and bovine adenovirus 3 were determined by the indirect fluorescent antibody technique in thoracic fluids from 100 aborted fetuses and 50 nonaborted fetuses. Results on these samples were not compared to hemagglutination inhibition or serum neutralization as the condition of fluid samples from aborted fetuses renders interpretation of such tests unreliable. Antibodies to one or more viruses were detected in 30 of the 100 aborted fetuses and in seven of the 50 nonaborted fetuses. Antibodies to more than one agent were detected in eleven of the 100 aborted and in one of the 50 nonaborted fetuses. Reasons for this occurrence and application of the test in determination of causes of abortion are discussed.     

Detection of IgM antibodies against a recombinant nucleocapsid protein of canine distemper virus in dog sera using a dot-blot assay

A dot-blot assay for the detection of IgM antibodies (ABs) against canine distemper virus (CDV) in canine serum is described. The diagnostic potential of this technique was evaluated by analysing sera from three test groups: (i) specific pathogen-free (SPF) beagle dogs experimentally infected with virulent CDV; (ii) SPF dogs immunized with a combined vaccine containing CDV, and (iii) SPF dogs immunized with a CDV-free vaccine. As antigen for the dot-blot assay we used the recombinant nucleocapsid protein (N protein) of the virulent A75/17 CDV strain. All 12 dogs of group 1, infected with virulent CDV, showed detectable CDV-specific IgM levels in their serum. All dogs of group 2 were also positive for anti-CDV IgM after the first immunization with the CDV-containing vaccine. The four dogs immunized with a CDV-free vaccine (group iii) remained negative throughout the course of the experiment. From these results, we conclude that the IgM detection test, which requires only a single serum sample, is a useful method for diagnosing current or recent CDV infection in CDV-infected or CDV-immunized dogs under experimental conditions.     

虎血清中犬副流感病毒的抗体流行病学调查研究

为查明犬副流感病毒 (CPIV)对虎的感染情况 ,应用CPIV细胞培养物为血凝抑制 (HI)抗原 ,对上海、桂林、哈尔滨、宜昌、郑州等地区未曾使用过任何含CPIV疫苗免疫的 38只虎的血清进行HI抗体检测。结果表明 ,有 2 5份血清CPIV的HI抗体效价为 1∶4～ 1∶32 ,有 1 3份抗体效价 <1∶2 ,血清中CPIV的HI抗体阳性率达 65 79%。说明上述地区的虎可能存在着CPIV的隐性感染     

Dysfunction of sense of smell caused by canine parainfluenza virus infection in dogs

Olfactory function of 5 dogs that were naturally infected with canine parainfluenza virus and of 4 dogs that were inoculated with the C958 strain of canine parainfluenza virus was evaluated. Except for one dog that was inoculated, the threshold for detection of benzaldehyde and/or eugenol was found to be excessively high during the course of the disease, as determined by electroencephalographic and behavioral olfactometry. In experimentally infected dogs, an increase in threshold developed in the absence of other clinical signs of disease. Changes were not observed in electro-olfactograms recorded throughout the study. Olfactory thresholds returned to normal after the disappearance of clinical signs of disease in the naturally infected dogs. Necropsies and histologic examinations performed during the course of the disease did not reveal abnormalities of the olfactory mucosa.