An enzyme-linked immunosorbent assay (ELISA) test for the diagnosis of equine infectious anemia in Brazil

An enzyme-linked immunosorbent assay (ELISA) test was developed for the detection of specific antibodies against the unique infectious anemia (EIA) virus in equine sera. The ELISA test was faster and more sensitive when compared with the classic test of agar gel immunodiffusion (AGID). A total of 200 sera were tested: 100 from negative horses and 100 from positive horses by AGID. The ELISA test showed 92 horse sera negative and 100 horse sera positive by AGID with values of optical density (OD) less than 0.139 and higher than 0.139, respectively. Eight horse sera were negative by AGID and higher than 0.139 by ELISA. Six of these became AGID positive also when re-tested 30 days later, and two were of the horses that showed clinical signs of EIA and died before re-testing.     

Comparison of antibody detection assays for the diagnosis of equine herpesvirus 1 and 4 infections in horses

OBJECTIVE: To compare methods of detecting equine herpesvirus type 1 (EHV1)- and EHV4-specific antibodies in horse sera. SAMPLE POPULATION: 33 acute and convalescent serum samples from experimentally or naturally infected horses after confirmed EHV1 or EHV4 infection. PROCEDURE: For each sample, serum antibody titers against EHV1 and EHV4 were determined by use of virus neutralization (VN) and complement fixation (CF) assays. The ELISA absorbance values for each serum sample were determined against the EHV1 and EHV4 recombinant ELISA antigens. Values obtained for acute and convalescent sera in each assay were compared. RESULTS: Following experimental infection of foals, EHV1 or EHV4 antibodies that were specific for the inoculating virus were detected only by use of the ELISA. Results of VN and CF assays indicated that the foals seroconverted to EHV1 and EHV4 following infection with EHV4 only. After EHV1-induced abortion, myeloencephalitis, or respiratory tract disease, the VN and CF assay results revealed seroconversion to EHV1 and EHV4, whereas results of the ELISA revealed seroconversion to EHV1 only. Similarly, after confirmed EHV4-induced respiratory tract disease, increases in EHV4-specific antibodies were detected only by use of the ELISA with no indication of an increase in EHV1 antibodies. The CF and, to a lesser degree, VN assays revealed that seroconversion to EHV1 and EHV4 occurred between the time of obtaining acute and convalescent serum samples. CONCLUSIONS AND CLINICAL RELEVANCE: The EHV1/EHV4 type-specific antibody ELISA clearly identifies horses that have been infected with EHV1 or EHV4 by use of acute and convalescent sera. Results of VN and CF assays indicate that cross-reactive antibodies greatly limit their use.     

African horse sickness in naturally infected,immunised horses

To determine whether subclinical cases, together with clinical cases, of African horse sickness (AHS) occur in immunised horses in field conditions, whole blood samples were collected and rectal temperatures recorded weekly from 50 Nooitgedacht ponies resident in open camps at the Faculty of Veterinary Science, University of Pretoria, Onderstepoort, during 2008–2010. The samples were tested for the presence of African horse sickness virus (AHSV) RNA by a recently developed real‐time RT‐PCR. It was shown that 16% of immunised horses in an AHS endemic area were infected with AHSV over a 2 year period, with half of these (8%) being subclinically infected. The potential impact of such cases on the epidemiology of AHS warrants further investigation.     

An investigation of equine infectious anaemia infection in the central Anatolia region of Turkey

In this study, 162 horses, 80 donkeys and 51 mule serum samples were collected in Konya city. Additionally, 64 horse serum samples from Ankara and 49 samples from Kayseri city were included in the study. A total of 406 serum samples were examined by agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for antibody to equine infectious anaemia virus (EIAV) and no positive result was detected.     

Rapid and sensitive detection of African horse sickness virus by real-time PCR

A highly sensitive and specific TaqMan-MGB real-time RT-PCR assay has been developed and standardised for the detection of African horse sickness virus (AHSV). Primers and MGB probe specific for AHSV were selected within a highly conserved region of genome segment 7. The robustness and general application of the diagnostic method were verified by the detection of 12 AHSV isolates from all of the nine serotypes. The analytical sensitivity ranged from 0.001 to 0.15 TCID₅₀ per reaction, depending on the viral serotype. Real-time PCR performance was preliminarily assessed by analysing a panel of field equine samples. The same primer pair was used to standardise a conventional RT-PCR as an affordable, useful and simple alternative method in laboratories without access to real-time PCR instruments. The two techniques present novel tools to improve the molecular diagnosis of African horse sickness (AHS).     

A serological survey of African horse sickness in Botswana

A retrospective serological survey of African horse sickness (AHS) in Botswana covering a 10-year period (1995-2004) is reported. The survey involved horses showing clinical symptoms of the disease; the horses had not been vaccinated against AHS. Over the period surveyed, serological evidence suggestive of infection with AHS virus (AHSV) was found in 99 clinical cases out of which 41.4% (41/99) cases were found during the 1st half (1995-1999) and 58.6 % (58/99) cases were found in the 2nd half of the survey period (2000-2004). These serological findings are discussed in relation to AHSV serotypes isolated from diseased horses in Botswana before and during the period of this serological survey.     

非洲马瘟病毒VP7蛋白研究进展

非洲马瘟(African horse sickness, AHS)是由非洲马瘟病毒(African horse sickness virus, AHSV)引起的一种通过库蜢等昆虫传播的、主要感染马科动物的传染病。我国是世界动物卫生组织认可的非洲马瘟无疫国,随着AHS疫情在东南亚的传播,增大了疫情传入我国的风险。AHSV编码了7种结构蛋白(VP1～VP7),其中VP7是病毒内衣壳蛋白的主要组成部分,在AHSV 9个血清型中高度保守,常作检测的靶标。此外,VP7蛋白的自组装特点对于AHSV亚单位疫苗和病毒样颗粒疫苗(VLP)的研究有基础性作用。对当前AHSV VP7蛋白相关研究进展进行了综述,以期为AHSV检测方法及疫苗等研究提供参考。     

Design and validation of an ELISA for equine infectious anemia (EIA) diagnosis using synthetic peptides

Three peptides derived from the equine infectious anemia virus (EIAV) surface proteins were synthesized to design and validate an ELISA for EIA diagnosis. Peptides identified as gp90-I and gp90-II correspond to the N- and C-terminal part of the surface glycoprotein gp90. Peptide gp45-1 overlaps the immunodominant epitope CIERTHVFC of the transmembrane glycoprotein gp45, and includes a hydrophilic chain close to the N-terminal end of this nonapeptide loop. Serum samples from 140 naturally infected horses with EIAV and a panel of 167 non-immune equine sera obtained from non-infected animals were used. Differences in reactivity between positive and negative serum samples were clearly distinguished. Samples considered weak positive to the agar gel immunodiffusion (AGID) test were "true" positive in the ELISA. These results are consistent with the improved sensitivity of the ELISA in comparison with the AGID test. The cyclic peptide that mimics the immunodominant sequence of gp45 showed excellent reactivity, thus suggesting that its functional activity depends significantly on its conformation, since very low reactivity was observed in the linear form of the peptide. The detectability indices of positive and negative sera reached 98% when gp90-II and gp45-I synthetic peptides were used in the same assay, illustrating the high specificity and sensitivity of the assay. Our study represents a first approach for the design of a diagnostic kit, which would allow the rapid analysis of a large numbers of serum samples from horses, and could be applied in endemic areas with different prevalence of infection.     

Laboratory confirmation of African horsesickness in the western Cape: application of a F(ab')2-based indirect ELISA

Recently a suspected outbreak of African horsesickness in the Western Cape Province resulted in the deaths of four foals and one adult horse. Spleen samples from these animals were subjected to analysis by an enzyme-linked immunosorbent assay (ELISA) which uses F(ab')2 fragments of immunoglobulins to detect African horse sickness virus (AHSV) antigens. The results of the immunoassay were compared with those obtained by isolation followed by serotyping as is currently applied by the Reference Centre at the Veterinary Research Institute, Onderstepoort. Samples of spleen tissue from the four foals contained sufficient antigen to be readily detectable by ELISA. A marginally positive signal was obtained with the tissue from the adult horse. This sample was inoculated onto VERO cells and four days were allowed for viral multiplication. Subsequently, when the cell culture was assayed by F(ab')2-ELISA, a much higher absorbance value than that obtained with the original spleen sample resulted, thus confirming the presence of AHSV in the initial specimen. The F(ab')2-ELISA has potential to be used as an initial diagnostic test to screen for AHSV.     

Comparison of diagnostic tests for the detection of equine infectious anemia antibody

Two diagnostic tests are approved for detecting antibody to equine infectious anemia virus: the agar-gel immunodiffusion (AGID) test and the competitive enzyme-linked immunosorbent assay (ELISA). A total of 420 sera from National Veterinary Services Laboratories check sets were tested with the AGID and competitive ELISA. A 100% correlation was obtained. The AGID and competitive ELISA were further used to test difficult samples with low levels of equine infectious anemia antibody (weak positives). A third test (Western blot) was also used with these weak positive samples to resolve any discordant results.     

非洲马瘟病毒、西尼罗病毒和马冠状病毒的基因芯片检测技术研究

为探索建立马病病毒基因芯片检测方法,采用人工拼接的方式拼接了非洲马瘟病毒(ASHV)核酸序列,通过分子克隆技术获得西尼罗病毒(WNV)和马冠状病毒(ECV)的特异基因片段。用芯片点样仪逐点分配到处理过的玻片上,制备成检测芯片。以拼接、克隆的核酸序列为模板通过多重不对称RT-PCR进行特异性扩增和荧光标记后滴加到芯片上进行杂交,对杂交结果进行扫描检测和计算机软件分析。结果显示,制备的基因芯片可同时检测和鉴别上述3种病毒,ECV质粒样品、WNV质粒样品检测灵敏度为10²拷贝;AHSV质粒样品检测灵敏度为10⁴拷贝。其他病毒材料未出现荧光信号,验证了本方法的特异性。证明基因芯片技术可快速、准确和灵敏地同时进行多种病毒的检测。     

Serodiagnosis of experimental and natural Babesia equi and B. caballi infections

The sensitivity and specificity of the complement fixation (CF) test for the diagnosis of Babesia infections in equines was assessed, using the indirect fluorescent antibody (IFA) test as a reference. Antibodies were first detected between 11 and 20 days post infection (dpi) in the CF test and between 7 and 14 dpi in the IFA test in ponies infected experimentally with B. equi (USDA strain). The CF test became negative in four of five ponies 63-174 dpi although B. equi was demonstrated microscopically in two of these four ponies up to 364 and 455 dpi. The IFA test remained positive up to 476 dpi (end of the examination period). Ponies infected experimentally with B. caballi (USDA strain) showed positive reactions in the CF test at first between 13 and 15 dpi and in the IFA test 10 or 11 dpi. The CF test became negative in two of three ponies 80 and 140 dpi, whereas the IFA test remained positive up to 190 dpi (end of the examination period). Cross-reactions of sera with heterologous antigens occurred at dilutions of 1/5 in the CF test and up to 1/20 in the IFA test. A total of 3944 CF tests was performed on 3765 horses from various European countries during 1980-1984. Sera that gave positive or trace CF reactions were retested in the IFA test. All 123 CF-positive sera were also IFA-positive and 26 of 31 sera (B. equi) and 11 of 32 sera (B. caballi) showing CF trace reactions were positive in the IFA test. Sera of two CF-negative horses were positive in the IFA test (B. equi); one of these horses was also positive upon microscopic examination. In seven of 21 horses repeatedly examined over longer periods the IFA titers (B. equi) persisted for up to 454 days longer than the CF titers. Sera of horses from highly endemic areas gave the following reactions: Sudan, 62 of 91 sera CF- and 86 of 91 IFA-positive; Zaire, 58 of 75 sera CF- and 72 of 75 IFA-positive; Columbia, 51 of 56 sera CF- and 56 of 56 IFA-positive; Brazil, 17 of 25 sera CF- and 21 of 25 IFA-positive. Only B. equi infections were demonstrated in Zaire. The combined use of the CF and IFA tests is recommended for safe identification of equine Babesia infections.     

非洲马瘟间接ELISA方法的建立及初步应用

为建立检测非洲马瘟间接酶联免疫吸附试验方法,利用重组杆状病毒感染昆虫细胞表达出具有良好抗原性的VP7蛋白,将感染VP7重组杆状病毒的昆虫细胞裂解液作为包被抗原,通过优化包被抗原浓度、二抗稀释度等,建立了检测非洲马瘟的间接ELISA方法,并进行了初步运用。结果表明,直接利用真核细胞表达VP7蛋白的昆虫细胞裂解液作为包被液可成功建立检测非洲马瘟的间接ELISA方法。     

An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses

A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(?)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from <10 to 40) 8 weeks after vaccination leading us to hypothesize that there could be a participation of cell mediated immunity (CMI) in protection against AHSV4. The present study aimed at characterizing the CMI induced by the experimental ALVAC(?)-AHSV4 vaccine. Six horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(?)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(?)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.     

Detection of equine infectious anemia virus in horse leukocyte cultures derived from horses in various stages of equine infectious anemia viral infection

The enzyme-linked immunosorbent assay (ELISA) antigen-positive and agar-gel immunodiffusion test (AGID)-negative horses do not have infective equine infectious anemia (EIA) virus. The ELISA testing of horse leukocyte culture (HLC) supernatants did detect EIA virus in a HLC that was infected with the Wyoming strain of EIA virus and in HLC derived from horses in febrile, acute, or subacute stages of EIA infection. In supernatants of HLC derived from chronic and inapparent carrier horses, EIA virus was not detected with ELISA. Direct fluorescent antibody tests detected EIA virus in HLC infected with 10(6)TCID50 of the Wyoming strain of EIA virus and in 50% of the HLC from febrile acute or subacute horses. The direct fluorescent antibody testing of HLC derived from chronic and inapparent carrier horses did not detect cell-associated EIA virus. The pony inoculation test proved to be the most reliable and accurate method for detecting infective EIA virus in horses in various stages of EIA infection and accurately correlated with the AGID test.     

重组马传染性贫血病病毒核心蛋白抗原在 A GID 和ELISA中应用的评价

用昆虫杆状病毒表达系统获得的马传染性贫血病病毒（ Ｅ Ｉ Ａ Ｖ） 核心蛋白（ Ｇａｇ） 和 Ｐ２６ 蛋白, 作为免疫琼脂双扩散（ Ａ Ｇ Ｉ Ｄ） 和酶联免疫吸附试验（ Ｅ Ｌ Ｉ Ｓ Ａ） 抗原。对７６ 份已知马传贫非特异性血清进行检查, 同时与市售 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒作比较。证明, 用表达蛋白作抗原的 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 检测结果均为阴性反应, 而用市售 Ａ Ｇ Ｉ Ｄ 试剂盒检查有５４ 份马血清出现非特异性反应, 市售 Ｅ Ｌ Ｉ Ｓ Ａ 试剂盒检查也出现了非特异性反应, Ｏ Ｄ 值比表达抗原 Ｅ Ｌ Ｉ Ｓ Ａ 高３５ 倍。初步证明在 Ａ Ｇ Ｉ Ｄ 和 Ｅ Ｌ Ｉ Ｓ Ａ 法中, 表达抗原优于常规马传贫病毒抗原。     

Evaluation of a polyvalent enzyme-linked immunosorbent assay incorporating a recombinant p44 antigen for diagnosis of granulocytic ehrlichiosis in dogs and horses

OBJECTIVE: To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. ANIMALS: 32 dogs and 43 horses. PROCEDURE: Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. RESULTS: For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, western immunoblotting, and the ELISA were in complete agreement. Results of the ELISA for 3 canine serum samples known to contain antibodies to Ehrlichia canis and 12 equine serum samples known to contain antibodies to E risticii were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results of the present study suggest that a polyvalent ELISA incorporating a recombinant p44 antigen is suitable for detecting antibodies to E equi in dogs and horses.