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1.  Evaluation of Five Enzyme-Linked Immunosorbent Assays and an Agar Gel Immunodiffusion Test for Detection of Antibodies to Small Ruminant Lentiviruses▿  
In the framework of the Dutch control program for small ruminant lentiviral (SRLV) infections, too many drawbacks were encountered with respect to serological testing. To improve the quality of testing, five enzyme-linked immunosorbent assays (ELISAs) and an agar gel immunodiffusion test (AGIDT) were evaluated. The focus was on the sensitivity, specificity, and variances of the commercially available tests. Clear differences were found among the tests in analytical and diagnostic sensitivity and overall diagnostic performance, whereas no significant differences in specificity were found. For serodiagnosis of sheep with clinical symptoms of maedi-visna virus (MVV) (histopathologically confirmed), one ELISA was significantly more sensitive than the other ELISAs and than the AGIDT, while for asymptomatic sheep originating from infected flocks, three ELISAs and the AGIDT demonstrated similar performance. The diagnostic performance appeared to be related to animal species and virus infection (MVV or caprine arthritis encephalitis virus [CAEV]) as well as the phase of infection/progression of disease. Receiver operating characteristic analysis, demonstrating the diagnostic potential of tests irrespective of defined cutoffs, again revealed clear differences between tests with respect to diagnostic performance for detection of antibodies against CAEV or MVV. An indirect ELISA, of which the solid phase is sensitized with a combination of the core protein p27 of MVV produced in Escherichia coli and a peptide derived from the transmembrane protein gp46, appeared to be the test of choice for serodiagnosis of SRLV infections in sheep and goats.
doi:10.1128/CVI.00282-06
PMCID: PMC2043322  PMID: 17609394
2.  Monoclonal Antibodies to Conformational Epitopes of the Surface Glycoprotein of Caprine Arthritis-Encephalitis Virus: Potential Application to Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detecting Antibodies in Goat Sera 
Four immunoglobulin G1 monoclonal antibodies (MAbs) to the gp135 surface envelope glycoprotein (SU) of the 79–63 isolate of caprine arthritis-encephalitis virus (CAEV), referred to as CAEV-63, were characterized and evaluated for their ability to compete with antibody from CAEV-infected goats. Three murine MAbs (MAbs GPB16A, 29A, and 74A) and one caprine MAb (MAb F7-299) were examined. All MAbs reacted in nitrocellulose dot blots with native CAEV-63 SU purified by MAb F7-299 affinity chromatography, whereas none reacted with denatured and reduced SU. All MAbs reacted in Western blots with purified CAEV-63 SU or the SU component of whole-virus lysate following denaturation in the absence of reducing agent, indicating that intramolecular disulfide bonding was essential for epitope integrity. Peptide-N-glycosidase F digestion of SU abolished the reactivities of MAbs 74A and F7-299, whereas treatment of SU with N-acetylneuraminate glycohydrolase (sialidase A) under nonreducing conditions enhanced the reactivities of all MAbs as well as polyclonal goat sera. MAbs 29A and F7-299 were cross-reactive with the SU of an independent strain of CAEV (CAEV-Co). By enzyme-linked immunosorbent assay (ELISA), the reactivities of horseradish peroxidase (HRP)-conjugated MAbs 16A and 29A with homologous CAEV-63 SU were <10% of that of HRP-conjugated MAb 74A. The reactivity of HRP-conjugated MAb 74A was blocked by sera from goats immunized with CAEV-63 SU or infected with CAEV-63. The reactivity of MAb 74A was also blocked by sera from goats infected with a CAEV-Co molecular clone, although MAb 74A did not react with CAEV-Co SU in Western blots. Thus, goats infected with either CAEV-63 or CAEV-Co make antibodies that inhibit binding of MAb 74A to CAEV-63 SU. A competitive-inhibition ELISA based on displacement of MAb 74A reactivity has potential applicability for the serologic diagnosis of CAEV infection.
doi:10.1128/CDLI.8.1.44-51.2001
PMCID: PMC96009  PMID: 11139194
3.  Detection of Serum Antibodies to Ovine Progressive Pneumonia Virus in Sheep by Using a Caprine Arthritis-Encephalitis Virus Competitive-Inhibition Enzyme-Linked Immunosorbent Assay 
A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) for detection of antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) was recently reported (L. M. Herrmann, W. P. Cheevers, T. C. McGuire, D. Scott Adams, M. M. Hutton, W. G. Gavin, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 10:267-271, 2003). The cELISA utilizes CAEV-63 SU captured on microtiter plates using the monoclonal antibody (MAb) F7-299 and measures competitive displacement of binding of the anti-CAEV MAb GPB 74A by goat serum. The present study evaluated the CAEV cELISA for detection of antibodies to ovine progressive pneumonia virus (OPPV) in sheep. Three hundred thirty-two sera were randomly selected from 21,373 sheep sera collected throughout the United States to determine the sensitivity and specificity of cELISA and agar gel immunodiffusion (AGID) based on immunoprecipitation (IP) of [35S]methionine-labeled OPPV antigens as a standard of comparison. A positive cELISA test was defined as >20.9 percent inhibition (% I) of MAb 74A binding based on two standard deviations above the mean % I of 191 IP-negative sheep sera. At this cutoff, there were 2 of 141 false-negative sera (98.6% sensitivity) and 6 of 191 false-positive sera (96.9% specificity). Sensitivity and specificity values for IP-monitored AGID were comparable to those for cELISA for 314 of 332 sera with unambiguous AGID results. Concordant results by cELISA and IP resolved 16 of the 18 sera that were indeterminate by AGID. Additional studies evaluated cELISA by using 539 sera from a single OPPV-positive flock. Based on IP of 36 of these sera, there was one false-negative by cELISA among 21 IP-positive sera (95.5% sensitivity) and 0 of 15 false-positives (100% specificity). We conclude that the CAEV cELISA can be applied to detection of OPPV antibodies in sheep with high sensitivity and specificity.
doi:10.1128/CDLI.10.5.862-865.2003
PMCID: PMC193903  PMID: 12965917
4.  Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to Caprine Arthritis-Encephalitis Virus: Diagnostic Tool for Successful Eradication 
A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was evaluated for the detection of serum antibodies to the surface envelope (SU) of caprine arthritis-encephalitis virus (CAEV) in goats. This assay utilized 96-well microtiter plates containing CAEV-63 SU captured by monoclonal antibody (MAb) F7-299 and measured the competitive displacement of horseradish peroxidase-conjugated MAb GPB 74A binding by undiluted goat sera (F. Özyörük, W. P. Cheevers, G. A. Hullinger, T. C. McGuire, M. Hutton, and D. P. Knowles, Clin. Diagn. Lab. Immunol. 8:44-51, 2001). Two hundred serum samples from goats in the United States were used to determine the sensitivity and specificity of cELISA based on the immunoprecipitation (IP) of [35S]methionine-labeled viral antigens as a standard of comparison. A positive cELISA was defined as >33.2% inhibition of MAb 74A binding based on 2 standard deviations above the mean percent inhibition of 140 IP-negative serum samples. At this cutoff value, there were 0 of 60 false-negative sera (100% sensitivity) and 5 of 140 false-positive sera (96.4% specificity). Additional studies utilized IP-monitored cELISA to establish a CAEV-free herd of 1,640 dairy goats.
doi:10.1128/CDLI.10.2.267-271.2003
PMCID: PMC150544  PMID: 12626453
5.  Development and Field Testing of a Real-Time PCR Assay for Caprine Arthritis-Encephalitis-Virus (CAEV) 
Caprine arthritis/encephalitis (CAE) of goats and occasionally sheep are persistent virus infections caused by a lentivirus (CAEV). This viral infection results in arthritis in adult animals and encephalitis in kids. Prognosis for the encephalitic form is normally poor, with substantial economic loss for the farm. In this context an early/fast laboratory diagnosis for CAEV infection could be useful for effective prophylactic action. In this work we performed a quantitative real time PCR designed on the CAEV env gene to detect/quantify in goat/sheep samples, viral RNA or proviral DNA forms of CAEV. This procedure was validated in 15 sheep, experimentally infected with CAEV or with a highly correlated lentivirus (visna maedi, MVV); in addition, a total of 37 clinical goat specimens recruited in CAEV positive herds were analyzed and compared using serological analysis (Elisa and AGID). All samples infected with MVV resulted negative. In sheep experimentally infected with CAEV, proviral DNA was detectable 15 days post infection, whereas the serological methods revealed an indicative positivity after 40-60 days.This method showed a sensitivity of 102 env fragments/PCR) with a linear dynamic range of quantitation from 103 to 107 env fragments/PCR; the R2 correlation coefficient was 0.98. All subjects with a clinical diagnosis for Caprine Arthritis-Encephalitis (CAE) resulted CAEV DNA positive.
doi:10.2174/1874357901206010082
PMCID: PMC3414719  PMID: 22888382
Caprine arthritis-encephalitis; CAEV; lentivirus; real time PCR; agar gel immunodiffusion (AGID); enzyme-linked immunosorbent assay (ELISA).
6.  A New Sensitive Serological Assay for Detection of Lentivirus Infections in Small Ruminants 
Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99.8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.
PMCID: PMC95764  PMID: 10473527
7.  Development of an enzyme-linked immunosorbent assay for caprine arthritis-encephalitis virus. 
Journal of Clinical Microbiology  1988;26(5):971-975.
Because relatively few caprine arthritis-encephalitis virus (CAEV)-infected animals exhibit clinical signs of illness, efforts to control and eradicate this virus will depend heavily on a sensitive diagnostic test that can be easily carried out. The currently utilized tests are of limited usefulness because of relatively low sensitivity or because of incomplete cross-reactivity of goat sera with heterologous test antigens. An enzyme-linked immunosorbent assay (ELISA) with purified CAEV antigen and biotin-avidin amplification steps was therefore developed and compared with a radioimmunoassay (RIA) against CAEV p28. Of over 500 sera tested, there was 99% concordance between the two tests. On the other hand, 23 of 24 sera obtained from animals with clinical signs of disease that were negative by agar gel immunodiffusion test (with ovine progressive pneumonia virus antigen) were positive by ELISA and RIA. These results suggest that an ELISA with CAEV antigen is superior to the agar gel immunodiffusion test and is easier and faster than an RIA, and therefore may be the method of choice for diagnosing CAEV infection.
PMCID: PMC266498  PMID: 2838510
8.  Glycosylation of Immunodominant Linear Epitopes in the Carboxy-Terminal Region of the Caprine Arthritis-Encephalitis Virus Surface Envelope Enhances Vaccine-Induced Type-Specific and Cross-Reactive Neutralizing Antibody Responses 
Journal of Virology  2004;78(17):9190-9202.
This study evaluated type-specific and cross-reactive neutralizing antibodies induced by immunization with modified surface glycoproteins (SU) of the 63 isolate of caprine arthritis-encephalitis lentivirus (CAEV-63). Epitope mapping of sera from CAEV-infected goats localized immunodominant linear epitopes in the carboxy terminus of SU. Two modified SU (SU-M and SU-T) and wild-type CAEV-63 SU (SU-W) were produced in vaccinia virus and utilized to evaluate the effects of glycosylation or the deletion of immunodominant linear epitopes on neutralizing antibody responses induced by immunization. SU-M contained two N-linked glycosylation sites inserted into the target epitopes by R539S and E542N mutations. SU-T was truncated at 518A, upstream from the target epitopes, by introduction of termination codons at 519Y and 521Y. Six yearling Saanen goats were immunized subcutaneously with 30 μg of SU-W, SU-M, or SU-T in Quil A adjuvant and boosted at 3, 7, and 16 weeks. SU antibody titers determined by indirect enzyme-linked immunosorbent assay demonstrated anamnestic responses after each boost. Wild-type and modified SU-induced type-specific CAEV-63 neutralizing antibodies and cross-reactive neutralizing antibodies against CAEV-Co, a virus isolate closely related to CAEV-63, and CAEV-1g5, an isolate geographically distinct from CAEV-63, were determined. Immunization with SU-T resulted in altered recognition of SU linear epitopes and a 2.8- to 4.6-fold decrease in neutralizing antibody titers against CAEV-63, CAEV-Co, and CAEV-1g5 compared to titers of SU-W-immunized goats. In contrast, immunization with SU-M resulted in reduced recognition of glycosylated epitopes and a 2.4- to 2.7-fold increase in neutralizing antibody titers compared to titers of SU-W-immunized goats. Thus, the glycosylation of linear immunodominant nonneutralization epitopes, but not epitope deletion, is an effective strategy to enhance neutralizing antibody responses by immunization.
doi:10.1128/JVI.78.17.9190-9202.2004
PMCID: PMC506968  PMID: 15308714
9.  Immune responses of goats persistently infected with caprine arthritis-encephalitis virus. 
Infection and Immunity  1980;28(2):421-427.
Eight cesarean-derived goat kids were inoculated with caprine arthritis-encephalitis virus (CAEV), and proliferative responses of their peripheral blood mononuclear cells to mitogens and CAEV antigen were monitored for 9 months. Antibody specific for CAEV was measured by an enzyme-linked immunosorbent assay. Five cesarean-derived noninfected goats were tested simultaneously. Significant differences between the infected and control mononuclear cell proliferation reactions to CAEV began 14 days post-inoculation and continued in a fluctuant manner until 134 days post-inoculation. The magnitude of the proliferative reaction steadily increased in infected goats until the end of the experiment at 271 days post-inoculation. Responses to mitogens were not significantly different between infected and control goats. Virus-inoculated goats produced CAEV-specific antibody that reached a maximum level between 49 and 77 days post-inoculation and then declined to lower levels through 271 days post-inoculation. The virus-inoculated goats developed mild but characteristic clinical evidence of caprine arthritis-encephalitis, and CAEV was reisolated from four goats at 286 days post-inoculation. The five control goats developed neither an anti-CAEV immune response nor clinical disease, and CAEV could not be reisolated from them.
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PMCID: PMC550952  PMID: 7399671
10.  Evaluation of agar gel immunodiffusion serology using caprine and ovine lentiviral antigens for detection of antibody to caprine arthritis-encephalitis virus. 
Journal of Clinical Microbiology  1994;32(1):243-245.
The sensitivity of the agar gel immunodiffusion (AGID) test for the detection of antibody to caprine arthritis-encephalitis virus (CAEV) was investigated with CAEV or ovine progressive pneumonia virus (OPPV) as the source of antigen. A total of 218 goat serum specimens were tested for anti-CAEV antibody by AGID and immunoprecipitation of [35S]methionine-labeled CAEV. In comparison with that of immunoprecipitation, the sensitivity of the CAEV AGID test was 0.91, and that of the OPPV AGID test was 0.56. The AGID test with either antigen was 100% specific. The lower sensitivity of the OPPV AGID test in detecting caprine antibody to CAEV indicates that OPPV antigen is of limited value for use in CAEV diagnosis and control programs.
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PMCID: PMC263008  PMID: 8126191
11.  Epitope analysis of capsid and matrix proteins of North American ovine lentivirus field isolates. 
Journal of Clinical Microbiology  1991;29(7):1472-1479.
Monoclonal antibodies (MAbs) directed against two phenotypically distinct ovine lentivirus (OvLV) strains were generated by fusion of BALB/c SP2/0-Ag 14 myeloma cells with spleen cells from mice immunized with purified OvLV. Hybridomas were selected by indirect enzyme-linked immunosorbent assay (ELISA) and analysis of reactivity on immunoblots. The majority (17 of 21) of the MAbs recognized the gag-encoded capsid protein, CA p27, of both strains. Four other MAbs recognized a smaller structural protein, presumably a matrix protein, MA p17. Three distinct epitopes on CA p27 and one on MA p17 were distinguished by the MAbs with competition ELISA. MAbs from each epitope group were able to recognize 17 North American field isolates of OvLV and the closely related caprine arthritis-encephalitis virus (CAEV). Analysis of the data indicated that these epitopes were highly conserved among naturally occurring isolates. A representative MAb from each epitope group of anti-CA p27 MAbs reacted with four field strains of OvLV and CAEV on immunoblots. An anti-MA p17 MAb recognized the same OvLV strains on immunoblots but failed to recognize CAEV. MAbs which recognize conserved epitopes of gag-encoded lentivirus proteins (CA p27 and MA p17) are valuable tools. These MAbs can be used to develop sensitive diagnostic assays and to study the pathogenesis of lentivirus infections in sheep and goats.
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PMCID: PMC270137  PMID: 1715884
12.  Antibody reactivity to the immunodominant epitopes of the caprine arthritis-encephalitis virus gp38 transmembrane protein associates with the development of arthritis. 
Journal of Virology  1994;68(11):7139-7147.
High titers of antibodies to caprine arthritis-encephalitis virus (CAEV) envelope (Env) glycoproteins are found in infected goats developing a progressive arthritis. In order to identify linear B epitopes of the CAEV Env, which may be involved in the immunopathology of arthritis, we constructed a lambda gt11 Env expression library. By combining library screening with sera from naturally infected Swiss goats with an enzyme immunoassay with overlapping peptides (pepscan), four group-specific epitopes could be precisely defined in the transmembrane envelope proteins: TM1 to TM4, including a conserved structure (TM3) that corresponds to the immunodominant epitope of human immunodeficiency virus type 1 and other lentiviruses. A panel of 190 CAEV naturally infected goat serum samples, obtained from animals with defined clinical status, was tested for reactivity to synthetic peptides corresponding to the TM epitopes in an enzyme-linked immunosorbent assay. Antibody reactivity to two epitopes was highly associated (TM3, P = 0.002, and TM4, P < 0.001) with the presence of clinically detectable arthritis. Such an association is absent for anti-Gag antibody. Antibodies to the immunodominant structures of the TM glycoprotein could thus have an important role in the immunopathogenic process leading to disease.
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PMCID: PMC237153  PMID: 7933096
13.  Development of TaqMan-based qPCR method for detection of caprine arthritis–encephalitis virus (CAEV) infection 
Archives of Virology  2013;158(10):2135-2141.
A specific and sensitive two-step TaqMan real-time PCR has been developed for rapid diagnosis of caprine arthritis-encephalitis virus (CAEV) infection by using a set of specific primers and a TaqMan probe targeting a highly conserved region within the gene encoding the viral capsid protein (CA). The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and isolated PBMCs, with a lower detection limit of 102 copies and a linear dynamic range of 105 to 1010 copies/ml. There was no cross-reaction with other animal viruses (e.g., goat pox virus, bovine leukemia virus, bovine mucosal disease virus, swine influenza virus and Nipah virus). When applied in parallel with serological AGID and conventional PCR for detection of CAEV in field samples, this assay exhibited a higher sensitivity than these traditional methods, and 7.8 % of the 308 specimens collected in the Shanxi and Tianjin regions of China from 1993 to 2011 were found to be positive. Thus, the TaqMan qPCR assay provides a fast, specific and sensitive means for detecting CAEV proviral DNA in goat specimens and should be useful for large-scale detection in eradication programs and epidemiological studies.
doi:10.1007/s00705-013-1728-1
PMCID: PMC3785178  PMID: 23670072
14.  Detection of caprine arthritis-encephalitis virus by polymerase chain reaction. 
Journal of Clinical Microbiology  1993;31(11):3042-3043.
Detection of caprine arthritis-encephalitis virus (CAEV) infection in goats is currently limited to serologic testing or cell culture. We developed a polymerase chain reaction (PCR) assay to detect CAEV sequences in peripheral blood mononuclear cells (PBMC), synovial fluid cells (SFC), and milk cells (MC) obtained from infected goats. Results were positive for 18 of 20 PBMC, 8 of 8 MC, and 5 of 5 SFC samples from seropositive goats, whereas 3 of 33 PBMC samples and none of 8 MC or 5 SFC samples from seronegative goats were positive. Two of the PCR-positive and seronegative goats seroconverted upon follow-up testing 2 months later. This PCR assay provides a useful method for detecting CAEV infection in goats.
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PMCID: PMC266206  PMID: 8263195
15.  Priming with tat-Deleted Caprine Arthritis Encephalitis Virus (CAEV) Proviral DNA or Live Virus Protects Goats from Challenge with Pathogenic CAEV 
Journal of Virology  1998;72(8):6796-6804.
We previously reported that infection of goats with caprine arthritis encephalitis virus (CAEV) tat− proviral DNA or virus results in persistent infection, since the animals seroconverted and direct virus isolation from cultures of blood-derived macrophages was positive. In this study we wanted to determine whether goats injected with CAEV tat− proviral DNA or virus were protected against challenge with the pathogenic homologous virus and to investigate whether CAEV tat− was still pathogenic. All animals injected with CAEV tat− became infected as indicated by seroconversion and virus isolation. Challenge at 8 or 9 months postinfection demonstrated protection in four of four animals injected with CAEV tat− but did not in three of three mock-inoculated challenged goats. Challenge virus was undetectable in the blood macrophages of protected animals during a period of 6 or 10 months postchallenge. In two of four protected animals, however, we were able to detect the challenge wild-type virus by reverse transcriptase PCR on RNA directly extracted from synovial membrane cells surrounding the inoculation site. This result suggests that protection was achieved without complete sterilizing immunity. Animals injected with CAEV tat− and mock challenged developed inflammatory lesions in the joints, although these lesions were not as severe as those in CAEV wild-type-injected goats. These results confirm the dispensable role of Tat in CAEV replication in vivo for the establishment of infection and pathogenesis and demonstrate in another lentivirus infection model the efficacy of live attenuated viruses to induce resistance to superinfection.
PMCID: PMC109888  PMID: 9658128
16.  Isolation of caprine arthritis encephalitis virus from goats in Mexico. 
A lentivirus was isolated from 2 goats in Mexico that were seropositive to caprine arthritis encephalitis virus (CAEV) by the agar gel immunodiffusion (AGID) test. The lentivirus was identified as CAEV by the observation of giant multinucleated cells (syncytia) in goat synovial membrane (GSM) monolayers co-cultivated with blood mononuclear (BMN) cells from the seropositive goats, and by amplifying a DNA segment of the CAEV gag gene using the polymerase chain reaction (PCR) technique. Subsequently, cell supernatants from the GSM cells co-cultivated with BMN cells were used to infect 2 CAEV-seronegative goats. These goats seroconverted to CAEV as determined by the AGID test, and CAEV was re-isolated from these goats. One of the goats developed polyarthritis 8 mo after inoculation. Previous serological surveys indicate that infection with CAEV is prevalent among goats in Mexico. To our knowledge this is the first report of CAEV isolation in Mexico. Because of globalization of markets and increased trading among nations, the rapid identification and reporting of diseases such as CAEV are important to prevent the dissemination of these diseases.
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PMCID: PMC1189550  PMID: 10480464
17.  Simple and Rapid Method for Production of Whole-Virus Antigen for Serodiagnosis of Caprine Arthritis-Encephalitis Virus by Enzyme-Linked Immunosorbent Assay 
Polyethylene glycol (PEG) was used to produce whole-virus antigen derived from tissue culture cells infected with a Canadian strain of caprine arthritis-encephalitis virus. PEG antigen batches were obtained after precipitation and concentration of infected tissue culture material with PEG 8000 and final treatment with sodium dodecyl sulfate. The optimum time of harvest of tissue culture extracted material to produce the maximum amount of viral proteins was determined in roller bottles, after cocultivation of infected and noninfected fetal lamb corneal cells. Samples from day 9 to day 25 postculture were collected and processed. By Western blotting, the optimum time of harvest was found to be day 25 following the coculture. Two large batches of PEG antigen were prepared at the optimum time of harvest. Both batches gave similar results when tested by Western blotting and enzyme-linked immunosorbent assay (ELISA), using reference control sera from infected and noninfected goats. For further testing in ELISA, cutoff values and ratios were determined for PEG batch 1, using 200 known serum samples from goats free of the disease. The PEG antigen batch was compared with an in-house ELISA antigen in a kinetic mode, using 498 serum samples from field goats. The in-house ELISA antigen was produced following two rounds of ultracentrifugation and treatment with sodium dodecyl sulfate (R. A. Heckert, W. B. McNab, S. M. Richardson, and M. R. Briscoe, Can. J. Vet. Res. 56:237–241, 1992). The PEG antigen batch was found suitable for ELISA, with a relative specificity of 100% and a relative sensitivity of 99.4% compared to the in-house ELISA antigen. This method of antigen production for ELISA was found to be rapid, inexpensive, and reliable for the diagnosis of caprine-arthritis encephalitis, without requiring the use of sophisticated laboratory equipment.
doi:10.1128/CDLI.8.2.352-356.2001
PMCID: PMC96062  PMID: 11238221
18.  Evaluation of a Western Blot Method for the Detection of Yersinia Antibodies: Evidence of Serological Cross-Reactivity between Yersinia Outer Membrane Proteins and Borrelia burgdorferi 
Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.
doi:10.1128/CDLI.12.11.1269-1274.2005
PMCID: PMC1287758  PMID: 16275939
19.  Structure and genetic variability of envelope glycoproteins of two antigenic variants of caprine arthritis-encephalitis lentivirus. 
Journal of Virology  1991;65(11):5744-5750.
To define the structure of the caprine arthritis-encephalitis virus (CAEV) env gene and characterize genetic changes which occur during antigenic variation, we sequenced the env genes of CAEV-63 and CAEV-Co, two antigenic variants of CAEV defined by serum neutralization. The deduced primary translation product of the CAEV env gene consists of a 60- to 80-amino-acid signal peptide followed by an amino-terminal surface protein (SU) and a carboxy-terminal transmembrane protein (TM) separated by an Arg-Lys-Lys-Arg cleavage site. The signal peptide cleavage site was verified by amino-terminal amino acid sequencing of native CAEV-63 SU. In addition, immunoprecipitation of [35S]methionine-labeled CAEV-63 proteins by sera from goats immunized with recombinant vaccinia virus expressing the CAEV-63 env gene confirmed that antibodies induced by env-encoded recombinant proteins react specifically with native virion SU and TM. The env genes of CAEV-63 and CAEV-Co encode 28 conserved cysteines and 25 conserved potential N-linked glycosylation sites. Nucleotide sequence variability results in 62 amino acid changes and one deletion within the SU and 34 amino acid changes within the TM.
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PMCID: PMC250234  PMID: 1656067
20.  Caprine arthritis encephalitis virus dysregulates the expression of cytokines in macrophages. 
Journal of Virology  1997;71(10):7488-7497.
Caprine arthritis encephalitis virus (CAEV) is a lentivirus of goats that leads to chronic mononuclear infiltration of various tissues, in particular, the radiocarpal joints. Cells of the monocyte/macrophage lineage are the major host cells of CAEV in vivo. We have shown that infection of cultured goat macrophages with CAEV results in an alteration of cytokine expression in vitro. Constitutive expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) was increased in infected macrophages, whereas transforming growth factor beta1 (TGF-beta1) mRNA was down-regulated. When macrophages were infected with a CAEV clone lacking the trans-acting nuclear regulatory gene tat, IL-8 and MCP-1 were also increased. No significant differences from cells infected with the wild-type clone were observed, suggesting that Tat is not required for the increased expression of IL-8 and MCP-1 in infected macrophages. Furthermore, infection with CAEV led to an altered pattern of cytokine expression in response to lipopolysaccharide (LPS), heat-killed Listeria monocytogenes plus gamma interferon, or fixed cells of Staphylococcus aureus Cowan I. In infected macrophages, tumor necrosis factor alpha, IL-1beta, IL-6, and IL-12 p40 mRNA expression was reduced in response to all stimuli tested whereas changes in expression of granulocyte-macrophage colony-stimulating factor depended on the stimulating agent. Electrophoretic mobility shift assays demonstrated that, in contrast to effects of human immunodeficiency virus infection of macrophages, CAEV infection had no effect on the level of constitutive nuclear factor-kappaB (NF-kappaB) activity or on the level of LPS-stimulated NF-kappaB activity, suggesting that NF-kappaB is not involved in altered regulation of cytokine expression in CAEV-infected cells. In contrast, activator protein 1 (AP-1) binding activity was decreased in infected macrophages. These data show that CAEV infection may result in a dysregulation of expression of cytokines in macrophages. This finding suggests that CAEV may modulate the accessory functions of infected macrophages and the antiviral immune response in vivo.
PMCID: PMC192095  PMID: 9311828
21.  Comparison of Two Rapid Diagnostic Assays for Detection of Immunoglobulin M Antibodies to Dengue Virus 
Two easy-to-use commercial diagnostic assays, a dipstick enzyme-linked immunosorbent assay (ELISA) (Integrated Diagnostics, Baltimore, Md.) and an immunochromatographic card assay (PanBio, Brisbane, Australia) were evaluated for detection of immunoglobulin M (IgM) antibody to dengue virus with an in-house IgM antibody capture microplate ELISA as a reference assay. The dipstick ELISA was based on the indirect-ELISA format using dengue 2 virus as the only antigen and enzyme-labeled goat anti-human IgM antibody as the detector. The total assay time was 75 min. The immunochromatographic card assay was based on the antibody capture format and separately measured both anti-dengue virus IgM and IgG in the same test. Colloidal-gold-labeled anti-dengue virus monoclonal antibody bound with dengue virus 1 to 4 antigen cocktail was the detector, and anti-human IgM and IgG were the capture antibodies. The total assay time was <10 min. Sera from 164 individuals classified as either anti-dengue virus IgM positive (94) or anti-dengue virus IgM negative (70) in the reference microplate ELISA with a dengue virus 1 to 4 antigen cocktail were tested in the two commercial assays. The dipstick ELISA missed 7 of 94 positive samples, for a sensitivity of 92.6%, while the immunochromatographic card assay missed two positive samples, for a sensitivity of 97.9%. Of the 70 negative samples, four were false positive by the dipstick ELISA and two were false positive in the immunochromatographic card assay, resulting in specificities of 94.3 and 97.1%, respectively. Both commercial assays provide sensitive and specific detection of anti-dengue virus IgM antibody and could prove useful in settings where the microplate ELISA is impractical.
PMCID: PMC95832  PMID: 10618287
22.  Antigenic variation of neutralization-sensitive epitopes of caprine arthritis-encephalitis lentivirus during persistent arthritis. 
Journal of Virology  1988;62(9):3488-3492.
Caprine arthritis-encephalitis virus (CAEV), a naturally occurring lentivirus of goats, causes disease characterized by virus persistence and recurrent arthritis. These studies demonstrate in vitro neutralization of CAEV infectivity by serum from goats infected with CAEV. Serum neutralizing activity was not detectable until 10 to 36 months postinfection, and titers were relatively low (less than or equal to 1:8). Serum neutralization was caused by antibody and was virus specific. Antigenic variants of CAEV were isolated from cell-free joint fluid of arthritic goats 9 to 18 months postinfection. The delayed appearance of neutralizing antibody and the subsequent development of antigenic variants may promote CAEV persistence in vivo and provide a stimulus for recurrent arthritis.
PMCID: PMC253474  PMID: 2457116
23.  Detection of human immunoglobulins G and M antibodies to Rift Valley fever virus by enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1984;19(2):225-229.
Rift Valley fever virus (RVFV) is an important human and animal pathogen in Africa and has been responsible for infections in travelers. Because of the aerosol infectivity and risk of dissemination of the virus, a need exists for simple, safe, serological tests for diagnosis. An enzyme-linked immunosorbent assay (ELISA) was developed to detect RVFV-specific immunoglobulins (immunoglobulin G [IgG] and IgM). In the test, a betapropiolactone-inactivated, sucrose-acetone-extracted, suckling mouse liver RVFV antigen was captured by mouse RVFV antibodies adsorbed to polystyrene plates. The test sample (human serum) was then added, and the binding of specific antibodies was indicated by alkaline phosphatase-conjugated swine anti-human IgG or IgM. A mu-capture IgM ELISA was also developed by using polystyrene plates coated with goat anti-human IgM incubated successively with serum sample, RVFV antigen, and indicator antibodies. The ELISA for RVFV-specific IgG proved to be more sensitive than hemagglutination inhibition or complement fixation tests and almost as sensitive as the plaque reduction neutralization test in detecting specific antibodies in human sera after vaccination. The two ELISA IgM tests could detect specific IgM antibodies during the first 6 weeks after RVFV vaccination. Three injections of inactivated vaccine were given on days 0, 6 to 8, and 32 to 34. ELISA IgM values for sera obtained on days 6 to 8 were negative or in the lower range of significance, on days 32 to 34 they were strongly positive, and on days 42 to 52 they were waning. Later sera were negative. The plague reduction neutralization test was negative on days 6 to 8 but rose progressively in later samples. These findings suggest that the three doses of RVFV vaccine induce a prolonged primary antibody response. The ELISA IgM could become an important tool for early diagnosis in acute human infection. A number of African sera, some of which were positive for RVFV by plaque reduction neutralization test, were also tested by ELISA IgG. There was good agreement between both tests.
PMCID: PMC271026  PMID: 6538206
24.  Surveys on Coxiella burnetii infections in Swedish cattle, sheep, goats and moose 
Background
Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Prevalence data in ruminant species are important to support risk assessments regarding public and animal health. The aim was to investigate the presence of or exposure to C. burnetii in cattle, sheep, goats and moose, and to compare two enzyme-linked immunosorbent assays (ELISAs). National surveys of antibodies against C. burnetii were performed for dairy cattle (n=1537), dairy goats (n=58) and sheep (n=518). Bovine samples consisted of bulk milk, caprine of pooled milk, and ovine of pooled serum. Antibodies were investigated in moose samples (n=99) from three regions. A one-year regional cattle bulk milk survey was performed on the Isle of Gotland (n=119, four occasions). Cattle, sheep and goat samples were analysed with indirect ELISA and moose samples with complement fixation test. For the sheep, goat, and parts of the cattle survey, samples were run in parallel by ELISAs based on antigens from infected ruminants and ticks. Bulk milk samples from the regional cattle survey and vaginal swabs from a subset of the sheep herds (n=80) were analysed for the agent by polymerase chain reaction. Spatial clustering was investigated in the national cattle survey.
Results
The prevalence of antibodies in dairy herds was 8.2% with large regional differences. High risk clusters were identified in the southern regions. The prevalence among dairy herds on the Isle of Gotland varied from 55.9% to 64.6% and 46.4% to 58.9.0% for antibodies and agent, respectively, overall agreement between agent and antibodies was 85.2%. The prevalence of antibodies in sheep was 0.6%, the agent was not detected the vaginal swabs. Antibodies were not detected in goats or moose, although parts of the moose samples were collected in an area with high prevalence in cattle. The overall agreement between the two ELISAs was 90.4%.
Conclusions
The prevalence of antibodies against C. burnetii in dairy cattle in Sweden shows large regional differences. The results suggest that C. burnetii is a rare pathogen among Swedish moose, dairy goat and sheep. ELISAs based on ruminant and tick antigen performed in a similar manner under Swedish conditions.
doi:10.1186/1751-0147-56-39
PMCID: PMC4112654  PMID: 25007979
Coxiella burnetii; Surveillance; Epidemiology; Cattle; Goat; Sheep; Moose; Cervids; Antibodies; Test evaluation
25.  Comparison of Three Immunoassays for Detection of Antibodies to Strongyloides stercoralis 
Due to the limited sensitivities of stool-based microscopy and/or culture techniques for Strongyloides stercoralis, the detection of antibodies to this intestinal nematode is relied upon as a surrogate for determining exposure status or making a diagnosis of S. stercoralis infection. Here, we evaluated three immunoassays, including the recently released InBios Strongy Detect IgG enzyme-linked immunosorbent assay (ELISA) (InBios International, Inc., Seattle, WA), the SciMedx Strongyloides serology microwell ELISA (SciMedx Corporation, Denville, NJ), and the luciferase immunoprecipitation system (LIPS) assay performed at the National Institutes of Health (NIH), for their detection of IgG antibodies to S. stercoralis. A total of 101 retrospective serum samples, previously submitted for routine S. stercoralis antibody detection using the SciMedx assay, were also evaluated by the InBios and LIPS assays. The qualitative results from each assay were compared using a Venn diagram analysis, to the consensus result among the three assays, and each ELISA was also evaluated using the LIPS assay as the reference standard. By Venn diagram analysis, 65% (66/101) of the samples demonstrated perfect agreement by all three assays. Also, the numbers of samples considered positive or negative by a single method were similar. Compared to the consensus result, the overall percent agreement of the InBios, SciMedx, and LIPS assays were comparable at 87.1%, 84.2%, and 89.1%, respectively. Finally, the two ELISAs performed analogously but demonstrated only moderate agreement (kappa coefficient for the two assays, 0.53) with the LIPS assay. Collectively, while the two commercially available ELISAs perform equivalently, neither should be used independently of clinical evaluation to diagnose strongyloidiasis.
doi:10.1128/CVI.00041-14
PMCID: PMC4018881  PMID: 24648484

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