Indirect hemagglutination test in equine infectious anemia.

An indirect hemagglutination was developed for the diagnosis of equine infectious anemia using sheep red blood cells coated with group specific virus antigen which had been highly purified by affinity chromatography. The presence of indirect hemagglutination antibodies was demonstrated in horses with equine infectious anemia since the cells were specifically agglutinated by all the serum samples obtained from experimentally infected horses. Antibodies appeared within 35 days after inoculation, and development of which coincided well with that of precipitating and complement fixing antibodies. Titer of indirect hemagglutination antibodies were ten to 320 times greater than those of precipitating antibodies. Test results could be read more clearly by the indirect hemagglutination test especially in weakly positive cases. Ninety-six samples from suspected field cases collected from every region of Japan which were positive on the immunodiffusion test were also positive on indirect hemagglutination test. Serum samples from 420 horses in one race track were examined by both the indirect hemagglutination and immunodiffusion tests to determine the reliability of the indirect hemagglutination test for diagnosis of equine infectious anemia. The same result was obtained on both tests. Based on this evidence, the indirect hemagglutination test can be employed as a very sensitive serological test for the diagnosis of equine infectious anemia.     

Equine Infectious Anemia: Preliminary Investigation of the Complement-Fixation Test for the Demonstration of Antibodies and Antigen

Clinical field cases of equine infectious anemia were studied and the disease was reproduced experimentally in horses. Attempts were made to adapt the complement-fixation test to the detection of antibodies in the serum of infected animals and to the demonstration of antigens in tissue extracts.

A moderate complement-fixing antibody response was demonstrated in the serum of horses shortly after primary exposure to the infectious agent. However, this reactivity was of short duration and occurred with normal as well as with infected saline tissue extracts. It was therefore concluded that this reaction was not specific for equine infectious anemia. Possibly it is due to the appearance of auto-tissue antibodies. The value of this reaction in the diagnosis of the infection was limited because of its short duration and absence in chronic infection and following re-exposure to the infectious agent.

     

Structural proteins of equine infectious anemia virus and their antigenic activity

Using purified equine infectious anemia (EIA) virus labeled with 3H-glucosamine or 14C-protein hydrolysate, structural proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, 2 glycoproteins and 10 proteins with molecular weights (mol wt) ranging from 12,000 to 115,000 daltons were demonstrated. Of 12 structural proteins, 3 proteins, namely a glycoprotein with mol wt of 76,000 (gp76) and 2 proteins with mol wt of 25,000 (p25) and 12,000 (p12), respectively, had distinct antigenic activity from one another in immunodiffusion. Development of antibodies against gp76 and p25 was compared in infected horses. The antibody to gp76 appeared earlier and stronger than to p25 in horses infected with the homologous virus strain. The fraction with glycoproteins was found to have hemagglutinating activity which was inhibited by the serum sample from horses infected with equine infectious anemia virus.     

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5′-LTR/tat segment of PCR product revealed 83–93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/tat region represents an important target for the detection of non-clinical horses.     

Equine ehrlichiosis in northern California: 49 cases (1968-1981)

Case records of horses with equine ehrlichiosis (Ehrlichia equi) at the University of California Veterinary Medical Teaching Hospital and Ackerman Creek Large Animal Clinic were analyzed for evaluation of clinical signs, time of onset, hematologic values, response to treatment, and recovery. Equine ehrlichiosis was found to be seasonal in horses in the foothills of northern California, with higher incidence than reported previously. The horses developed fever, anorexia, depression, limb edema, icterus, and ataxia. Hematologic changes were leukopenia, thrombocytopenia, icterus, anemia, and inclusion bodies in the neutrophils and eosinophils. Diagnosis was made by observing the characteristic inclusion bodies, using a standard Wright's stain. Mortality was low, although complications of opportunistic secondary infection and injury due to ataxia did develop. Treatment with tetracycline resulted in prompt clinical improvement within 24 hours. Chronic cases were not detected. Equine ehrlichiosis should be differentiated from diseases with similar clinical signs including encephalitis, liver disease, purpura hemorrhagica, equine infectious anemia, and equine viral arteritis.     

马传染性贫血病毒弱毒疫苗S2基因在体内变异分析

为研究马传染性贫血病毒(EIAV)驴白细胞弱毒疫苗EIAVDLV121的S2基因在马体内的变异规律,本研究选用4匹成年马,其中2匹(#1、#2)接种EIAVDLV121,另外2匹作为对照.免疫后监测马体温、血小板含量以及病毒载量结果显示,免疫马未出现马传染性贫血体征.通过RT-PCR方法检测病毒S2基因在感染马体内不同时期的基因序列,结果显示,免疫马体内EIAVDLV121 S2蛋白的突变主要发生在氨基酸第17位、22位、39位、41位、51位和55位.另外,#1马免疫后70 d以及#2马免疫后第14 d和第28 d检测疫苗毒S2蛋白序列与EIAVDLV121亲缘关系较近,而#1马免疫后第42 d、第112 d和第140 d的疫苗毒S2蛋白序列与EIAVDLV121的致病性亲本强毒株EIAVDN40亲缘关系最近.本研究结果有助于对EIAV及EIAV疫苗株在马体内感染进程的研究.     

Prevalence of antibodies to equine viruses in the Netherlands

Summary The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested. The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi-1, M. Influenzae A / equi-2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested. The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.     

马传染性贫血病毒自然感染马LTR序列分析

我国从20世纪70年代开始使用EIAV弱毒疫苗后,在全国范围内基本控制了马传贫的发生,我们从现地少数EIAV琼扩阳性马外周血中扩增并克隆了二株EIAV前病毒5'LTR序列,并与国内外毒株5'LTR序列进行了比较分析,发现中国EIAV LTR特有的特异性细胞转录因子结合基序,同时发现PEA2位点不是强毒特有的基序,在弱毒中亦发现该基序.     

四川省马传染性贫血病防控的回顾与思考

马传染性贫血病（简称马传贫）是严重危害马属动物的一种传染病。四川省于1989年在凉山州越西、昭觉两县发现了阳性马匹,经确诊后,即采取了普查、检疫、免疫注射、扑杀等综合性防控措施,1998年全省达到稳定控制马传贫标准,2003年达到消灭马传贫标准。从2003年至今,各项防控措施不停,监测表明均未发现阳性病例,成效显著。     

Prevalence of antibodies to equine viruses in the Netherlands

Summary

The prevalence of antibodies to various viruses was investigated in a series of serum samples collected from horses in the Netherlands between 1963 and 1966 and from 1972 onwards. Neutralizing antibodies to equine rhinopneumonitis virus, equine arteritis virus and to equine rhinovirus types 1 and 2 were detected in respectively 76%, 14%, 66% and 59% of the equine serum samples tested.

The observed incidence of serum samples positive to equine adenovirus in the complement fixation test was 39%. Precipitating antibodies to equine infectious anaemia virus were detected only in serum samples from two horses imported from abroad. Haemagglutination inhibiting antibodies to Myxovirus influenzae A / equi‐1, M. Influenzae A / equi‐2, and Reovirus types 1, 2, and 3 were present in respectively 82%, 50%, 10%, 33% and 3.6% of the serum samples tested.

The most frequently observed incidence of antibodies to the various equine respiratory viruses occurred in the groups of horses having repeatedly contact with other horses.     

A propagating epizootic of equine infectious anemia on a horse farm

An epizootic of equine infectious anemia (EIA) involved 35 horses on a farm in south Georgia. During a 126-day period, 21 of these horses became seropositive for EIA. After the initial diagnosis in July, the horses were tested every 7 to 10 days. At least one additional horse was found to be seropositive on each testing day. As soon as they were determined to be seropositive, the horses were removed from the herd and sent to slaughter. The removal of the seropositive horses, however, did not stop the epizootic. We believe the initial infection was from a 7-year-old stallion that recently had been purchased or from 1 of 2 mares that were seropositive for EIA on the first test. None of the horses had been tested for EIA at the time of purchase or within 60 days before the epizootic.     

马传贫驴白细胞弱毒疫苗株基质蛋白基因的克隆与表达

从感染驴白细胞的马传贫驴白细胞弱毒疫苗株前病毒DNA中克隆了编码基质蛋白（p15)的基因，并在大肠杆菌中进行了表达，所表达的蛋白是一种可溶性的融合蛋白，其氨基端带有6个组氨酸的标签，因此可以用固定化金属离子亲和层析法在非变性条件下进行纯化，在间接ELISA和免疫印迹试验中，重组的基质蛋白可与马传贫阳性血清样品发生反应，而与健康马血清无任何反应，这表明该重组蛋白具有良好的抗原性和特异性，可用于马传贫弱毒疫苗株在体内外复制及在接种马体人免疫应答的研究中。     

Immune-mediated hemolytic anemia induced by penicillin in horses

Three horses developed severe, immune-mediated hemolytic anemia after treatment with penicillin. The horses had positive direct antiglobulin (Coombs) tests and high titers of IgG antibody that agglutinated penicillin-coated equine red cells. Two of the horses were tested for antibodies to autologous red cell antigens; autoantibodies were not present. Titers of antipenicillin antibody decreased after penicillin was discontinued but IgG antibody was detectable months after recovery. One of the horses was challenged with penicillin; antibody titer increased slightly, but anemia did not develop. Antipenicillin antibody of the IgM class was present in low titer in 23 (77%) of 30 non-anemic horses tested. Apparently, the horse is similar to man in that penicillin-induced anemia is rare but the percentage of individuals with antipenicillin antibody is high.     

Effects of equine infectious anemia virus on hematopoietic progenitors in vitro.

Direct effects of equine infectious anemia virus (EIAV) on hematopoiesis in vitro were studied. Bone marrow mononuclear cells from clinically normal horses were incubated with 100 TCID50 of EIAV/10(7) cells. These cells were cultured to assay for colonies derived from erythroid progenitors, granulocyte/monocyte progenitors, and fibroblastic progenitors. The EIAV had a selective suppressive effect on the erythroid progenitors. Colony-forming units-erythroid were suppressed to 80% of that for medium controls (P = 0.011). Burst-forming units-erythroid were suppressed to 70% of that for medium controls (P = 0.003). Significant effect was not apparent on colony-forming units-granulocyte/macrophage or on colony-forming units-fibroblastic.     

Association between the presence of serum antibodies against Neospora spp. and fetal loss in equines

A study of the association between the presence of serum antibodies against Neospora spp. and fetal loss was performed using serum samples of horses submitted to the laboratory for the detection of antibodies to Equine Herpesvirus-1 and Equine Infectious Anemia Virus. The sera submitted for equine infectious anemia testing were from horses declared healthy and those submitted for the detection of antibodies to Equine Herpesvirus-1 were from mares with late clinical signs of reproductive disorders or males living in close contact with diseased mares. For the detection of Neospora spp. infection, the immunofluorescent antibody test was employed, using a cut-off titer of 50 as significant for the presence of antibodies. Among the 483 mares in the diseased group, 15.1% (73/483) was reactant, while 5.8% (19/325) was seropositive in the healthy group. The results show that late clinical signs of reproductive disorders in mares are positively associated (p<0.001) to infection with protozoa belonging to the genus Neospora and point to the fact that the participation of this group of coccidia in the genesis of reproductive disorders in equine must be investigated.     

Seroprevalence of antibodies against Neospora caninum in diagnostic equine serum samples and their possible association with fetal loss

A case-control study of the association between the presence of serum antibodies against Neospora spp. and fetal loss was performed on serum samples submitted to a veterinary diagnostic laboratory in northwestern United States. Control sera were randomly selected from those submitted from healthy horses for routine equine infectious anemia testing required for regulatory health certification. Case sera were randomly selected from those submitted from aborting mares for diagnostic workup. Based on a 1:50 or greater titer on the indirect fluorescent antibody test, 8% of the 160 control sera and 13% of the 140 case sera were titer positive. The association odds ratio of 1.67 fell short of statistical significance (p=0.124).     

Leukoencephalitis associated with selective viral replication in the brain of a pony with experimental chronic equine infectious anemia virus infection

Neurologic disease occurs sporadically in horses infected with the equine infectious anemia virus (EIAV). This report describes a case of clinically severe neurologic disease in a pony experimentally infected with EIAV. This pony did not have fever or anemia, which are the characteristic clinical signs of disease. The histopathologic changes were characterized as lymphohistiocytic periventricular leukoencephalitis. Polymerase chain reaction and in situ hybridization data showed that the brain lesions were directly associated with viral replication and that high-level viral replication occurred selectively within the lesion and not in other tissues. These findings suggest that EIAV-associated neurologic disease is the direct result of viral replication.     

Cross-neutralizing and subclass characteristics of antibody from horses with equine infectious anemia virus

Antibody responses in horses with equine infectious anemia virus (EIAV) were examined to determine their cross-neutralizing capacity. Antibodies induced by infection with any of six biologically cloned variants of EIAV cross-neutralized multiple variants from the group. Anti-EIAV antibody was found in both the IgG and IgG(T) subclasses in plasmas with virus-neutralizing activity and the majority of antiviral antibody was of the IgG(T) subclass. Depletion of IgG(T) did not increase the neutralization indexes of either neutralizing or non-neutralizing plasma samples.     

Cloning and large-scale expansion of epitope-specific equine cytotoxic T lymphocytes using an anti-equine CD3 monoclonal antibody and human recombinant IL-2

Cytotoxic T lymphocytes are involved in controlling intracellular pathogens in many species, including horses. Particularly, CTL are critical for the control of equine infectious anemia virus (EIAV), a lentivirus that infects horses world-wide. In humans and animal models, CTL clones are valuable for evaluating the fine specificity of epitope recognition, and for adoptive immunotherapy against infectious and neoplastic diseases. Cloned CTL would be equally useful for similar studies in the horse. Here we present the first analysis of a method to generate equine CTL clones. Peripheral blood mononuclear cells were obtained from an EIAV-infected horse and stimulated with the EIAV Rev-QW11 peptide. Sorted CD8+ T cells were cloned by limiting dilution, and expanded without further antigen addition using irradiated PBMC, anti-equine CD3, and human recombinant IL-2. Clones could be frozen and thawed without detrimental effects, and could be subsequently expanded to numbers exceeding 2 x 10(9)cells. Flow cytometry of expanded clones confirmed the CD3+/CD8+ phenotype, and chromium release assays confirmed CTL activity. Finally, sequencing TCR beta chain genes confirmed clonality. Our results provide a reliable means to generate large numbers of epitope-specific equine CTL clones that are suitable for use in downstream applications, including functional assays and adoptive transfer studies.