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1.
A monoclonal antibody (mAb) directed against the equine arteritis virus (EAV) nucleocapsid (N) protein was used for indirect enzyme-linked immunosorbent assays (ELISAs) using viral antigen from different sources. The same mAb was labelled with fluorescein isothiocyanate for direct immunofluorescence tests (DIFTs). The N-specific mAb appeared to be suitable for the detection in both ELISA and DIFT of different EAV strains and field isolates from semen and tissue samples after passage in lines of RK-13, Vero and fetal equine kidney cells. The ELISA described is an easy and fast method which can be used in most cases to replace the microneutralization test to prove the EAV specificity of the cytopathic effect of cell cultures. The DIFT, however, is more sensitive than both the ELISA and the microneutralization test because EAV antigen can be detected even in cell cultures without or with very weak cytopathic effect.  相似文献   

2.
A potent ELISA antigen was prepared from equine arteritis virus (EAV) by differential centrifugation of EAV-infected cell culture fluid, followed by solubilization of the preparation by Triton X-100 treatment. Using this antigen and a mouse monoclonal antibody against the G(L) protein of EAV, a reliable blocking ELISA (bELISA) was developed for the detection of EAV antibodies in equine sera. The bELISA was evaluated using a total of 837 test serum samples. The relative sensitivity (n = 320) of the bELISA compared to the serum neutralization (SN) test was 99.4%. The bELISA appears to be a highly specific test, the specificity of which did not appear to be adversely affected by previous exposure of horses to non-EAV-containing biologicals. Of 119 serum samples, 21 from horses without any history of exposure to EAV and 98 from racetrack Thoroughbreds, 118 were negative in the SN test and bELISA. One sample was SN-negative but suspicious with the bELISA. Based on testing 465 SN-negative field samples and 52 SN-negative samples from experimental horses, and excluding any sera giving a suspicious reaction, the relative specificity of the bELISA was 97.7%. Samples should be examined undiluted and diluted 1/10 in the bELISA because the testing of sera of high neutralizing antibody titer may be affected by a prozone-like phenomenon. The bELISA is a more rapid and cost-efficient test than the SN test for the detection of EAV antibodies in equine sera.  相似文献   

3.
An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of serum antibodies to equine arteritis virus (EAV). Results from this assay produced a good correlation with results from virus neutralisation tests in horses which had not been regularly vaccinated with commercially available mammalian tissue culture-derived viral vaccines. Vaccination of some horses with tissue culture-derived vaccines induced the formation of antibodies to bovine serum. These antibodies reacted with the bovine protein contaminants in the EAV ELISA antigen, producing false-positive results. Non-viral protein contaminants were found to be closely associated with EAV in that they co-purified with the virus during gradient centrifugation.  相似文献   

4.
From 105 field cases of diarrhea in neonatal or young foals, rotavirus was detected by electron microscopy (EM) and/or by enzyme-linked immunosorbent assay (ELISA) in the feces of 65 foals on 16 different premises. ELISA was performed with Rotazyme test kits developed by Abbot and Company for the detection of rotaviruses. Twenty-four field isolates from the feces of diarrheic foals with equine rotavirus infection as ascertained by EM were placed in MA-104 cell cultures after pretreatment of the viral suspension with 10 micrograms ml-1 of trypsin and incorporation of 0.5 micrograms ml-1 or 1 microgram ml-1 of trypsin in Earle's minimal essential medium (MEM), 2% lactalbumen hydrolysate, and antibiotics. The isolates that replicated in cell culture produced varying degrees of cytopathic effect. After the 24 isolates had been transferred 5 or 7 times in cell culture, viral particles were observed in 17 by EM, and 22 had positive ELISA tests as determined by visual color chart and spectrophotometric readings. Concentrated tissue-cultured viral antigen of 9 isolates fixed complement using Nebraska calf diarrhea rotavirus calf antiserum while four isolates gave negative results. The same 13 tissue-cultured viral suspensions failed to fix complement using reovirus antiserum. The 9th passages of two isolates (EID1 and EID2) yielded titers of 10(4.45) ml-1 TCID50 and of 10(4.95) ml-1 TCID50, respectively, as measured by cytopathic effect. After 13 tissue-cultured passages, 2 other isolates, EID3 and EID4, each had titers of 10(6.2) ml-1 TCID50 and of 10(5.95) ml-1 TCID, respectively. Cytoplasmic or intranuclear inclusions were not seen in any cells of the MA-104 infected cell cultures. Small, but distinct, plaques in MA-104 cell cultures were produced by the EID1 isolate. Polyacrylamide gel electrophoresis tests of EID1 and EID2 isolates at the 9th cell passage and EID3 and EID4 isolates at the 13th cell passage each showed that the RNA genome had 11 segments with a migrating pattern that was identical for each isolate and characteristic of rotaviruses. These 4 equine tissue-cultured isolates when tested by ELISA, utilizing a monoclonal antibody serum pool that cross-reacted with many rotavirus isolates, each gave positive values comparable to rotavirus antigen controls.  相似文献   

5.
In vitro interference between equine herpesvirus types 1 and 2   总被引：2，自引：0，他引：2  
Interference between equine herpesvirus types 1 (EHV-1) and 2 (EHV-2) was studied in equine dermis (ED) monolayer cell cultures and equine lymphocyte cultures. Cell cultures were infected with EHV-2, and after a short incubation period, the cultures were superinfected with EHV-1. At various intervals, different measurements of EHV-1 expression in dually infected cultures, compared with those in cultures infected with EHV-1 alone, were studied. In dually infected ED cell cultures, the EHV-1 cytopathic effect, EHV-1 titer, and EHV-1 enzyme-linked immunosorbent assay antigen titer were maximally reduced to values of 40%, 58.5%, and 54.9%, respectively, at postsuperinfection hour (PSIH) 36. Values of these EHV-1 expressions were subsequently increased at PSIH 48. However, thymidine kinase activity was reduced to a maximum of 67.3% reduction at PSIH 48. In dually infected lymphocyte cultures, the EHV-1 titer, EHV-1 infective centers, EHV-1 enzyme-linked immunosorbent assay antigen titer, and thymidine kinase activity were maximally reduced to values of 77.4%, 78.7%, 98.3%, and 72.9%, respectively, at PSIH 24. These reductions of EHV-1 expressions were completely abrogated at PSIH 48 to 72. In both cell culture systems, a marked interference of EHV-1 by EHV-2 was observed; this was transient in the lymphocyte cultures, but was more prolonged in ED cell cultures. This interference appeared not to be interferon mediated. The multiplication of EHV-2 in the dually infected ED cell cultures appeared unaffected.  相似文献   

6.
Nineteen horses with no prior experience with equine arteritis virus (EAV) were inoculated IM with an avirulent live-virus vaccine against equine viral arteritis; the vaccinal virus had been passaged serially 131 times in primary cell cultures of equine kidney, 111 times in primary cell cultures of rabbit kidney, and 16 times in an equine dermis cell line (EAV HK-131/RK-111/ED-16). Three or 4 of the vaccinated horses each, along with appropriate nonvaccinated controls, were inoculated nasally with virulent EAV at each of months 3, 6, 9, 12, 18, and 24 after they were vaccinated. The following was concluded: Vaccination did not induce clinical signs of disease in any horse and, thus, seemed safe for use in the field. All vaccinated horses (n = 19) developed serum-neutralizing antibodies to EAV. Fourteen of the vaccinated horses were completely protected from clinical arteritis when exposed to large doses of virulent EAV. Four were partially protected, and one had little or no protection. Six of 13 nonvaccinated horses died of acute arteritis, and the remaining 7 horses experienced severe signs of disease, but survived the infection. All horses (n = 32), whether vaccinated or not, became infected when inoculated nasally with virulent EAV. Virus was recovered from 17 of the 19 vaccinated horses, and all 19 had a secondary humoral immune response. The duration and severity of thermal reaction and persistence of virus were more transitory in vaccinated horses than in the nonvaccinated controls. Protection afforded by this vaccine can persist for at least 24 months, the maximal time after horses were vaccinated that immunity was challenged in the present study.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

7.
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8.
Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1–2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4–8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4+ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.  相似文献   

9.
Serum samples from 72 stallions were examined for the occurrence of antibodies against equine arteritis virus, of which 41 animals (57%) were found to be positive. 32 of the seropositive stallions were then screened for persistent EAV infection, before and after the breeding season. Semen samples were investigated by RT-PCR followed by dot blot hybridization and nested PCR, and by virus isolation on cell cultures as well. The carrier state was virologically confirmed in 11 of 32 stallions (34%) during the first and in 9 of 20 (45%) during the second investigation. RT-PCR followed by confirmatory methods was more sensitive when compared to virus isolation on cell cultures. It is suggested to implement the national and EU directives by recommending RT-PCR as a routine diagnosis of EAV in semen samples.  相似文献   

10.
Sensitivity and specificity of an antigen-capture ELISA vs virus isolation in cell culture were evaluated for the detection of eastern equine encephalomyelitis (EEE) virus in the brain tissue of naturally infected equids. Brain specimens from 16 equids with neurologic disease were examined by ELISA and by inoculation onto baby hamster kidney cell cultures. Of 10 brain samples from which virus was isolated in the cell culture bioassay, all were correctly identified as containing EEE virus antigen by ELISA. None of the remaining 6 specimens, without detectable infectious EEE virus, contained detectable antigen. Sensitivity and specificity of the ELISA were 100% with no false-positive or false-negative results. The antigen-capture ELISA was a rapid, sensitive, specific, and simple alternative to a traditional bioassay for the detection of EEE virus.  相似文献   

11.
The occurrence of equine arteritis virus (EAV) induced equine abortions was studied with different laboratory methods during a 3-year period. Tissue samples from 96 aborted equine foetuses or newborn foals were collected from 57 farms located in different parts of Hungary. Virus isolation, polymerase chain reaction (PCR), immunohistochemistry and serology were used for the detection of EAV infection. The overall seroprevalence of EAV infection in mares was 65%. EAV induced abortion was diagnosed in eight (8.3%) cases from six (10.5%) herds. Abortion was sporadic in all herds except for one, where epidemic abortion happened. Fetal serology in six (75%) cases, the virus isolation in one (12.5%) case whereas PCR in all of the four investigated cases were positive. The virus could be observed with immunohistochemistry in seven (87.5%) cases mostly in the spleen followed by other organs and the allantochorion. In conclusion, PCR and immunohistochemistry seem to be the most sensitive and useful tests for the diagnosis of EAV induced equine abortion.  相似文献   

12.
Two 5-year-old grade male horses presented with epiphora, rhinorrhea, conjunctival and nasal mucosal hyperemia, and dorsal and thoracic macropapular rash. Skin biopsies were collected from the affected areas, and serial sections were evaluated following hematoxylin and eosin and immunoperoxidase histochemistry staining by using a murine monoclonal antibody of the immunoglobulin G2A isotype recognizing the 30-kDa membrane protein of equine arteritis virus (EAV). In both horses, lesions consisted of mild to moderate diffuse superficial dermal edema and vasculitis with mild perivascular lymphocytic infiltrates, occasional endothelial hypertrophy, and single-cell necrosis of tunica media myocytes. Immunohistochemically, a few endothelial cells, myocytes, and pericytes containing intracytoplasmic EAV antigen were identified. Immunoperoxidase histochemistry of skin biopsies can be used as an ancillary test for the clinical diagnosis of equine viral arteritis in horses, especially when a cutaneous macropapular rash is evident.  相似文献   

13.
Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.  相似文献   

14.
AIM: To review laboratory aspects of the equine viral arteritis (EVA) control scheme in New Zealand between 1989 and 2002. METHODS: The optimisation and performance of the virus neutralisation test (VNT) for equine arteritis virus (EAV) antibody, and the cell culture test to detect EAV in semen were analysed. Laboratory data and control scheme results were reviewed. RESULTS: Using optimised tests, it has been shown that antibody prevalence in Standardbred horses has steadily declined from 54% to <20%. Prevalences in Thoroughbred horses have remained at a low level of around 3%. The number of horses shedding EAV (all Standardbreds) has steadily declined from a maximum at any one time of 20 to the current figure of three. CONCLUSION: Eradication of EVA from the horse population in New Zealand is achievable in the near future.  相似文献   

15.
In an effort to further characterize the humoral immune response of horses to equine arteritis virus (EAV), direct and competitive enzyme-linked immunosorbent assays (c-ELISAs) were developed using monoclonal and polyclonal anti-sera to structural (G(L), N and M) and non-structural (nsp1) viral proteins. A nsp1-specific monoclonal antibody was produced to facilitate development of a c-ELISA to this protein. Data obtained using the various c-ELISAs confirm that the M protein is a major target of the antibody response of horses to EAV. However, none of the c-ELISAs that were developed were as sensitive in detecting EAV-specific antibodies in horse sera as the existing serum neutralization test.  相似文献   

16.
以灭活马流感病毒(EIV)A/Equine/Jilin/1/1989(H3N8)为免疫原,免疫Balb/c小鼠,经常规细胞融合后,用血凝抑制试验(H1)和间接ELISA方法筛选获得3株(3C2、5G10和5A10)能稳定分泌H3N8亚型马流感病毒单克隆抗体(mAb)的杂交瘤细胞株.其中3C2和5G10为IgG2α,5A...  相似文献   

17.
A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.  相似文献   

18.
Outbreaks of both equine viral arteritis and contagious equine metritis (CEM) in the United States have occurred in recent years. Shipped semen has been implicated in disease transmission and rapid spread. A survey was performed of state regulations regarding testing for equine arteritis virus (EAV) and CEM before interstate semen or embryo shipment. Results demonstrated lack of any requirements in 31 states. Four states had regulations regarding EAV; 17 required a Certificate of Veterinary Inspection (CVI) to accompany a semen or embryo shipment. Ten states required a negative equine infectious anemia test, primarily as a requirement of a CVI versus because of risk of dissemination of disease. No states required CEM testing. A comparison of state department of agriculture or veterinary medical association Web sites and direct communication with state veterinarians or their offices demonstrated contradictory information in six states. The lack of uniform regulations concerning CEM and EAV testing for shipped semen and embryos, and the threat they pose to the equine breeding industry and horse health, should be alarming to veterinarians and horse owners. Routine testing of animals before shipment of semen or embryos and veterinary involvement in collection and breeding activities are appropriate and necessary to help prevent future outbreaks and protect equine health.  相似文献   

19.
H3N8亚型马流感病毒间接ELISA抗体检测方法建立及应用   总被引：5，自引：0，他引：5  
为建立马流感血清学ELISA诊断方法,本研究以马流感病毒中国分离株A/马/新疆/07(H3N8)通过SPF鸡胚培养和增殖,收取含病毒尿囊液经蔗糖密度梯度离心纯化后作为ELISA包被抗原,首次在我国建立了检测H3N8亚型马流感抗体的间接ELISA诊断方法。试验的最佳反应条件为:最佳抗原稀释度7μg/mL,封闭液5%脱脂乳,血清稀释度1∶100,二抗稀释度1∶10000,稀释液PBS(pH7.4),血清反应时间1.5h,二抗反应时间1h。通过本方法对555份临床样品进行检测并与血凝抑制(HI)试验检测结果比较,证明本方法特异、敏感,具有良好的稳定性和可重复性,适于马流感的流行病学调查和监测工作。  相似文献   

20.
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