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1.
ELISA法检测犬腹泻粪样中的犬冠状病毒   总被引：2，自引：0，他引：2  
用ＦＥ细胞增殖犬冠状病毒（ＣＣＶ）参考株，分别免疫家兔和ＢＡＬＢ／ｃ小鼠制备ＣＣＶ多抗和单抗，建立了夹心ＥＬＩＳＡ及Ｄｏｔ－ＥＬＩＳＡ诊断方法。在检测的８４例犬腹泻粪样中，多抗、单抗夹心法显示ＣＣＶ阳性１６例，Ｄｏｔ－ＥＬＩＳＡ阳性１３例，后１３例包括在前１６例中。从８４例腹泻犬粪样中随机取３８例作ＣＣＶ、犬细小病毒（ＣＰＶ）双项检测，ＣＣＶ阳性１６例，ＣＰＶ阳性６例，ＣＣＶ、ＣＰＶ混合感染４例。结果显示，在南京地区流行的犬腹泻中，ＣＣＶ感染比例有超过ＣＰＶ的趋势。  相似文献   

2.
OBJECTIVE: To estimate the frequency of serum antibodies (IgG and IgM) to canine coronavirus (CCV) in the Australian dog population and evaluate the role of CCV as a causative agent of gastroenteritis. DESIGN: A serological survey of antibodies to CCV among different dog populations. PROCEDURE: The development and characterisation of an indirect ELISA for the detection of antibodies (IgG and IgM) to CCV was undertaken. Sera collected from both diarrhoeal and non-diarrhoeal dogs from various populations throughout Australia were tested for these antibodies to CCV. RESULTS: Serum samples (1396) collected from 1984 to 1998 were tested for the presence of IgG antibodies to CCV. Samples were divided into two categories on the basis of the number of dogs housed together. The groups were either an open population containing dogs housed as groups of three or less, or kennel populations. Sera from 15.8% of the open population and 40.8% of kennelled dogs were positive for CCV antibodies. The prevalence of antibodies varied from zero to 76% in kennelled dogs. About 23% of 128 dogs positive for IgG antibodies to CCV were also positive for IgM antibodies to CCV, indicating recent CCV infection. Of those dogs that were presented with clinical signs of gastroenteritis such as diarrhoea and vomiting (n = 29), 85% were positive in the IgM ELISA and 85.7% in the IgG ELISA for antibodies to CCV. In comparison, for those dogs presented without any history of gastroenteritis only 15% were positive for IgM and 30% positive for IgG. CONCLUSION: Serological evidence indicates that infection with CCV in dogs is widespread throughout the Australian mainland. The prevalence of antibodies varies greatly among different populations, with an average of 40.8% positive in kennelled populations and 15.8% in the open population.  相似文献   

3.
From the reasons that canine coronavirus (CCV) grows more efficiently than feline coronavirus in a cell culture and they are mutually related in their antigenicities, an enzyme-linked immunosorbent assay (ELISA) using CCV-infected feline kidney (CRFK) cells as substrate antigens was developed for detection of anti-coronavirus antibodies in cats. It was indispensable for generating coronavirus-specific ELISA antibody activities that the sample was applied to the mock-infected, normal CRFK cells in parallel with the CCV-infected cells and then the optical density values given by the mock-infected cell antigen were subtracted from those given by the virus-infected cell antigen. On the basis of ELISA antibody titers obtained in sera from the cats experimentally infected with CCV and from the spontaneous feline infectious peritonitis (FIP) cases, the ELISA described in the present study was found to be applicable as a simple and easy serologic test which was able to detect anti-coronavirus antibodies as efficiently as the indirect immunofluorescence assay with homologous FIP virus.  相似文献   

4.
本研究旨在克隆犬冠状病毒（canine coronavirus,CCV）N基因,体外表达N蛋白,并制备抗该蛋白质的多克隆抗体,用于CCV的诊断及其抗原的检测。参考GenBank中CCV的N基因序列（登录号：KY063618.2）,选择CCV流行毒株的N基因,通过对该基因密码子进行优化和基因合成,最后选择一段有效基因构建重组表达质粒pET-B2M-N,将成功构建的重组质粒转化大肠杆菌DH5α感受态细胞,挑取阳性克隆提取质粒,转化大肠杆菌BL21（DE3）感受态细胞,通过0.5 mmol/L IPTG 30 ℃进行诱导表达。结果表明,优化诱导条件后成功表达出大小约为49 ku的重组蛋白。将重组蛋白与弗氏佐剂按一定比例混合,每隔2周免疫G767、G768两只日本大耳白兔数次,用间接ELISA检测G768抗体效价可达1∶512 000,选用G768抗体进行抗体纯化,纯化后浓度可达10 mg/mL,用间接ELISA、Western blotting和间接免疫荧光试验对N蛋白纯化后制备的兔多克隆抗体进行检测分析,表明表达的重组N蛋白免疫原性良好,制备的多克隆抗体具有良好的反应原性。本研究为犬冠状病毒抗原抗体检测以及靶标CCV诊断试剂盒的建立奠定了一定的基础。  相似文献   

5.
An enzyme-linked immunosorbant assay (ELISA) was developed to measure antibody to Bordetella bronchiseptica in dogs. The ELISA test was more rapid and sensitive and required 50 to 150 times less antigen than the amount of antigen required for the conventional tube agglutination test. A survey of 50 canine serum samples using ELISA suggested that 8% of all sera had titers greater than 1:64, 56% had titers of 1:8 to 1:64, and 36% had titers of less than 1:8. The mean titer of survey sera was 1:46 and the median titer was 1:16. Serum antibody responses in dogs inoculated with a commercially available bacterin were compared with responses in dogs inoculated with experimental endotoxin depleted bacterin.  相似文献   

6.
In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.  相似文献   

7.
In order to determine if canine coronavirus (CCV) could be transmitted to pigs, two dogs were inoculated orally with virulent CCV. After 24 h, the dogs were moved to an isolation room that contained three three-day-old pigs. A wire mesh fence, allowing close contact between the animals, separated the dogs from the pigs. The dogs and pigs were observed for 14 days for clinical signs of disease. Samples of blood were obtained from dogs and pigs immediately before the dogs were inoculated with virus and 14 and 28 days later. The dogs developed mild clinical signs of an infection, but the pigs remained normal throughout the observation period. The dogs shed CCV for eight days after exposure. All three pigs developed neutralizing antibodies against CCV and transmissible gastroenteritis virus by 14 days after they were exposed to the dogs.  相似文献   

8.
犬冠状病毒YS1株人工感染犬试验   总被引：5，自引：0，他引：5  
使用从腹泻犬体内分离获得的犬冠状病毒ＹＳ１第５代ＣＲＦＫ细胞培养物,以不同剂量人工感染５０只２￣３月龄健康幼犬 ,１４ｄ内计有１６只犬发病,其中１４只耐过,２只死亡,发病率为３２％,死亡率为１２．５％,患犬粪便经电镜检查和ＲＴ－ＰＣＲ检测,前者见有ＣＣＶ样病毒,后者扩增出ＣＣＶ特异核酸片段。４８只试验犬血清对该病毒的ＳＮ抗体效价随接毒量的不同分别平均为１：３１０、１：２４５、１：１８５、１：１１５  相似文献   

9.
A monoclonal antibody against a plasma membrane antigen of Trypanosoma rhodesiense was used for the detection of T brucei group-specific circulating antigen in 24 adult local dogs experimentally infected with T brucei brucei strain 8/18. Ten of the dogs were splenectomised and the remainder non-splenectomised (intact). Five dogs each from the splenectomised and intact groups were inoculated intravenously with try-panosomes. The infected dogs developed trypanosomiasis between days 4 and 8 after infection. The circulating antigens were detected as early as six days after infection and remained high until two weeks after treatment, when the circulating antigen declined. The detection of the antigens showed the existence of infection unlike the antibody test. The treatment of the infected dogs with diminazene aceturate (Berenil; Hoechst) at a dose of 7-0 mg/kg on day 21 after infection cleared all the parasites but elevated the circulating antigen levels. The antigen capture enzyme-linked immunosorbent assay is a useful diagnostic tool for complementing parasitological diagnosis, for detecting infection in the field and for ascertaining the efficacy of trypanocidal drugs.  相似文献   

10.
Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.  相似文献   

11.
Simple methods for measuring the levels of serum antibody against canine distemper virus (CDV) would assist in the effective vaccination of dogs. To develop an enzyme-linked immunosorbent assay (ELISA) specific for CDV, we expressed hydrophilic extra-viral domain (HEVD) protein of the A75/17-CDV H gene in a pET 28a plasmid-based Escherichia (E.) coli vector system. Expression was confirmed by dot and Western blotting. We proposed that detection of E. coli-expressed H protein might be conformation-dependent because intensities of the reactions observed with these two methods varied. The H gene HEVD protein was further purified and used as an antigen for an ELISA. Samples from dogs with undetectable to high anti-CDV antibody titers were analyzed using this HEVD-specific ELISA and a commercial CDV antibody detection kit (ImmunoComb). Levels of HEVD antigenicity measured with the assays and immunochromatography correlated. These data indicated that the HEDV protein may be used as antigen to develop techniques for detecting antibodies against CDV.  相似文献   

12.
Canine angiostrongylosis is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We developed a sandwich-ELISA based on a monoclonal antibody (mAb Av 56/1/2) and on polyclonal rabbit antibodies directed against Angiostrongylus vasorum adult excretory/secretory - antigen for the detection of circulating serum antigen of A. vasorum. The sensitivity of the test was 95.7% (78.1-99.9, 95% CI) as determined with sera of 23 dogs naturally infected with A. vasorum. The specificity was 94.0% (83.5-98.7, 95% CI) using 50 dog sera (control group) submitted for reasons other than parasitic infections. Potential cross-reactions were investigated with sera of a group of totally 61 dogs with proven infections with Dirofilaria immitis (n=23), Crenosoma vulpis (n=14), Ancylostoma caninum (n=4) or Toxocara canis (n=20). No significant difference was observed concerning the proportion of positive reactions between the control group and the group with proven helminth infections other than A. vasorum. In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In contrast, in dogs with a single treatment with imidacloprid/moxidectin at four or 32 dpi, no circulating antigen was observed, while in dogs treated at 88-92 dpi, OD values decreased within 13-34 days. The specific detection of circulating A. vasorum antigen by ELISA represents a valid alternative for reliable diagnosis and for follow-up investigations after anthelmintic treatment. Moreover, the test can be used for mass screening in large epidemiological investigations.  相似文献   

13.
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。  相似文献   

14.
Canine hepatozoonosis is a tick-borne protozoal disease caused in the Old World and South America by Hepatozoon canis. An enzyme-linked immunosorbent assay (ELISA) using purified H. canis gamont antigen was applied for the detection of antibodies reactive with H. canis. Evaluation of the ELISA with sera from naturally infected parasitemic dogs indicated that it was sensitive (86%), specific (97%), and comparable to the indirect fluorescent antibody test (IFAT) for the detection of H. canis antibodies. A variable degree of serologic cross-reactivity was found between sera from H. americanum-infected dogs and the H. canis antigen. Dogs experimentally infected with H. canis seroconverted 1-4 weeks post-infection (PI). Antibody levels peaked at 7-9 weeks PI and gradually declined thereafter remaining above the cut-off value until the conclusion of the study 7 months PI. The ELISA will be valuable for serological evaluation of dogs suspected of exposure to H. canis and for epidemiological studies.  相似文献   

15.
为了建立一种快速、敏感、特异的检测犬冠状病毒（ＣＣＶ）的方法,利用感染ＣＣＶ的犬肾传代细胞包被酶联反应板,以辣根过氧化物酶标记的金黄色葡萄球菌Ａ蛋白（ＨＲＰ－ＳＰＡ）作为第２抗体,建立了检测ＣＣＶ抗体的间接ＥＬＩＳＡ方法。抗原的包被量为每孔３×１０４个染毒细胞,酶标抗体的工作浓度为１∶４０。试验发现,包被的病毒抗原经甲醇固定３０ｍｉｎ后可以提高试验的敏感性,减少病毒抗原的使用量。与中和试验比较证实,２种抗体检测方法的测定结果呈正相关  相似文献   

16.
Complex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-, CCV- and rotavirus antigens in fecal samples were also developed. Both the serological and antigen-detection ELISAs were used to screen samples from dogs in The Netherlands, with or without a history of acute diarrhea. It was shown that the results of the respective serological ELISAs correlated well and that CPV was the major cause of virus-induced acute diarrhea in dogs in The Netherlands.  相似文献   

17.
为建立特异性和敏感性高的检验犬细粒棘球绦虫感染的方法。用细粒棘球绦虫(简称,Eg)成虫抗原分别免疫兔和绵羊,收集高免血清,纯化的高免抗体。依据抗体夹心ELISA工作原理,以兔抗体包被,检测感染Eg、不同犬带科绦虫的实验犬和空白犬粪样,绵羊抗体扑捉抗原,HRP标记兔抗绵羊IgG(1∶8 000)催化显色,用酶标仪测定OD 405nm吸光度,用以确定其特异性和敏感性。试验结果表明,敏感性为82.69%(43/52),特异性为85.88%(140/163);粪抗原在感染细粒棘球绦虫16d后可检出,最低抗原浓度为9.7ng/mL即犬感染5条成虫时可检测出阳性。该检测方法具有较好的特异性和灵敏性,为进一步研制检测细粒棘球绦虫虫体抗原ELISA检测试剂盒奠定了基础。  相似文献   

18.
An enzyme-linked immunosorbent assay (ELISA) for the diagnosis of canine herpesvirus (CHV) infection using antigen prepared by solubilizing infected cells was developed. The ELISA and two improved methods of serum neutralization test, the microplate serum neutralization test (MSNT) with complement and the 50% plaque reduction (PR) assay with complement, were compared for the results of antibody detection from a total of 557 field canine sera. Of 529 sample sera that were negative in the MSNT with complement, 119 were ELISA positive, and this result together with time course of serum antibody detection in a dog experimentally infected with CHV strongly suggested that the MSNT with complement is less sensitive for the detection of antibody in CHV infected dogs, especially those in early stages of infection. A correlation was found between the titers measured by the ELISA and 50% PR assay with complement, however, for field use, the ELISA is recommended as a highly sensitive test method of serodiagnosis of CHV infection adequate for dealing with a large number of samples with less demand on time and effort.  相似文献   

19.
Three serological tests, i.e. complement fixation test, indirect immunofluorescent assay, and enzyme-linked immunosorbent assay (ELISA) were compared for sensitivity in the detection and titration of murine cytomegalovirus antibody. The three tests were compared using sera from experimentally inoculated and naturally infected mice bled at intervals from 3 to 140 days postinfection. In the acute infection, complement fixation and indirect immunofluorescent assay tests were of comparable sensitivity for early detection of antibody, whereas the ELISA was less sensitive. In persistent infection, higher titers were recorded with ELISA. Since murine cytomegalovirus has been shown to exert significant effects on the immune response of infected mice, this antigen should be included routinely in viral antibody screening programs.  相似文献   

20.
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.  相似文献   

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