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1.  Serological Diagnosis of Ovine Enzootic Abortion by Enzyme-Linked Immunosorbent Assay with a Recombinant Protein Fragment of the Polymorphic Outer Membrane Protein POMP90 of Chlamydophila abortus 
Journal of Clinical Microbiology  2002;40(11):4235-4243.
Ovine enzootic abortion (OEA) resulting from infection of sheep and goats with Chlamydophila abortus is of major economic importance worldwide. Over the last 50 years the serological diagnosis of infection has been based mainly on the complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen of sheep, Chlamydophila pecorum. In the present study, a series of overlapping recombinant antigens representing the polymorphic outer membrane protein POMP90 of C. abortus was assessed by enzyme-linked immunosorbent assay (ELISA) with a panel of 143 serum samples from sheep experimentally infected with C. abortus, from sheep clinically free of OEA, and from specific-pathogen-free lambs experimentally infected with different subtypes of C. pecorum. The results were compared to those obtained by CFT and another recently described test, an indirect ELISA (iELISA) with the recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D. Longbottom, E. Psarrou, M. Livingstone, and E. Vretou, FEMS Microbiol. Lett. 195:157-161, 2001). The rOMP90-3 and rOMP90-4 ELISAs were identified as being more sensitive and specific than CFT. Assays with both fragments were evaluated further with a panel of 294 field serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which various C. pecorum subtypes had been isolated. ELISAs with both POMP90 fragments outperformed CFT with serum samples from C. pecorum-infected animals, producing no false-positive results. However, the ELISA with the rOMP90-4 fragment appeared to be more sensitive than the one with rOMP90-3, as it identified more of the OEA-positive samples. The ELISA with the rOMP90-4 fragment was also able to identify apparently healthy animals that were infected with an enteric strain of C. abortus in flocks that were probably infected with both enteric C. abortus and C. pecorum strains. The identification of animals infected with enteric C. abortus is extremely important in controlling the spread of OEA. Overall, the new rOMP90-4 ELISA was found to be a more sensitive and specific test than CFT for differentiating animals infected with C. abortus from those infected with C. pecorum.
doi:10.1128/JCM.40.11.4235-4243.2002
PMCID: PMC139646  PMID: 12409404
2.  Diagnostic sensitivity and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams. 
An enzyme-linked immunosorbent assay (ELISA) for antibody to Brucella ovis was compared with a standard complement fixation test. Sera of 176 rams from uninfected flocks gave 175 negative and one suspect ELISA reaction (diagnostic specificity 99.4%) whereas the complement fixation test yielded 167 negative, seven suspect and two anticomplementary reactions (diagnostic specificity of 96.0%). Diagnostic sensitivity was evaluated on sera of 79 rams from which B. ovis had been isolated. The ELISA showed 75 positive and four suspect reactions, while complement fixation test revealed 64 positive, 13 suspect and two negative results. Considering both positive and suspect reactions, the diagnostic sensitivity was 100% for ELISA and 97.5% for complement fixation test. The ELISA method was considered more specific, more sensitive and technically more advantageous than complement fixation test as a serodiagnostic test for B. ovis infection in rams.
PMCID: PMC1255281  PMID: 3567755
3.  Serological diagnosis of ovine enzootic abortion by comparative inclusion immunofluorescence assay, recombinant lipopolysaccharide enzyme-linked immunosorbent assay, and complement fixation test. 
Journal of Clinical Microbiology  1996;34(6):1512-1518.
Since the 1950s, serological diagnosis of ovine enzootic abortion (OEA), caused by strains of Chlamydia psittaci, has been based mainly on the complement fixation test (CFT), which is neither particularly sensitive nor specific since antibodies to other chlamydial and enterobacterial pathogens may be detected. In this study. a recombinant enzyme-linked immunosorbent assay (rELISA) (medac, Hamburg, Germany), based on a unique chlamydial genus-specific epitope of Chlamydia trachomatis L2 lipopolysaccharide, was evaluated for sensitivity and specificity as a primary screening assay for OEA by comparison with the CFT. A comparative inclusion immunofluorescence assay (IFA), in which antibody titers to C. psittaci and Chlamydia pecorum were examined, was used as the reference test for 573 serum samples from four flocks. Reactivity to C. pecorum was measured since inapparent intestinal infections by C. pecorum are believed to be common in British flocks. In detecting positive sera from an abortion-affected flock, in which a C. pecorum infection was also suggested by IFA, the rELISA outperformed the CFT with significant evidence for increased sensitivity (P = 0.003). In two flocks in which C. pecorum infections alone were suggested by IFA, the rELISA and CFT were prone to detect low levels of false-positive results, but the values were not significant. The rELISA provided results in one flock in which sera that were anticomplementary could not be resolved by the CFT. In another flock in which abortion had not occurred but infection by both chlamydial species was suspected, no significant difference was found between the sensitivities of the rELISA and CFT. The rELISA could not differentiate ovine C. psittaci and C. pecorum infections but was shown to be a more sensitive primary screening test for OEA than was the CFT, particularly where abortion had occurred and even when antibodies due to additional inapparent infection(s) by C. pecorum were present.
PMCID: PMC229052  PMID: 8735108
4.  Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. 
Journal of Clinical Microbiology  1994;32(8):1835-1840.
A total of 291 unvaccinated sheep from Brucella melitenesis-infected flocks were examined for delayed-type hypersensitivity (DTH) responses with Brucellergene commercial allergen and with cold saline extract and cytosol from rough B. melitensis 115, and their sera were tested in the rose bengal test (RBT), complement fixation test (CFT), and enzyme-linked immunosorbent assay (ELISA) with lipopolysaccharide. DTH reactions were maximal after 72 h, with no intensity differences among allergens, inoculation sites (eyelid and tail), and doses tested. There were no differences in the results recorded by visual inspection and palpation of inoculation sites, by measuring skin thickness with a caliper, or by microscopic examination of samples taken at necropsy. Six days after DTH testing, energy was observed in 100% of the animals, and 100% reactivity was recovered only after 24 days. All animals were necropsied, and thorough bacteriological searches were performed. The sensitivities found with the 140 animals from which B. melitensis was isolated were ELISA, 100%; DTH, 97.1%; RBT, 92.1%; and CFT, 88.6%. Those results put into question the value of RBT and CFT as screening and confirmatory tests for sheep brucellosis and at least indicate that their standardization should be modified. For 151 tested sheep from which B. melitensis was not isolated, the percentages of positive animals were ELISA, 100%; DTH, 94.0%; RBT, 57.6%; and CFT, 53.6%. All tests were negative for 100 tested sheep from Brucella-free flocks. The different results of bacteriological and immunological tests suggest the usefulness of developing indirect tests able to distinguish truly infected animals from those that have developed an immunological response.
PMCID: PMC263887  PMID: 7989528
5.  Evaluation of a Novel Commercial Enzyme-Linked Immunosorbent Assay Detecting Coxiella burnetii-Specific Immunoglobulin G for Q Fever Prevaccination Screening and Diagnosis 
Journal of Clinical Microbiology  2002;40(9):3526-3529.
A novel commercially available enzyme-linked immunosorbent assay (ELISA) for prevaccination screening and diagnosis of Q fever (PanBio Coxiella burnetii immunoglobulin G [IgG] ELISA) was compared to the complement fixation test (CFT), and the indirect fluorescent-antibody test (IFAT) was used to resolve discrepant results between the other two tests. A total of 214 serum samples was tested. The ELISA demonstrated a specificity of 96% (46 of 48 samples) and a sensitivity of 71% (95 of 134 samples). Of the six serum pairs showing CFT seroconversion, three pairs showed a corresponding ELISA seroconversion. No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma, brucella, and chlamydia infections. One of the 13 patients with leptospirosis demonstrated a positive result in the ELISA but not in the CFT or the IFAT, and Legionella pneumophila serogroup 4 antibody was found in one of the two sera that were false-positive by ELISA. The results presented in this study suggest that the PanBio Q fever IgG ELISA is a specific alternative method for prevaccination testing and an aid for the diagnosis of Q fever. This test is suitable for use as a screening assay, with CFT and/or IFAT used to confirm negative results.
doi:10.1128/JCM.40.9.3526-3529.2002
PMCID: PMC130813  PMID: 12202611
6.  Evaluation of Competitive ELISA for Detection of Antibodies to Brucella Infection in Domestic Animals 
Croatian Medical Journal  2010;51(4):314-319.
Aim
To evaluate competitive enzyme-linked immunosorbent assay (cELISA) for its suitability as an additional serological test for the diagnosis of animal brucellosis.
Methods
cELISA, which was developed at the Veterinary Laboratories Agency, has been evaluated for its accuracy and suitability as an additional serological test for the diagnosis of animal brucellosis. Samples from naturally and experimentally infected animals and those from Brucella-free flocks and herds were tested.
Results
Data obtained since 1991 were analyzed from routine surveillance, animals experimentally infected with Brucella, and stored sera to validate cELISA for the detection of antibodies to Brucella in cows, small ruminants, and pigs. The sensitivity of the test ranged from 92.31% to 100%, in comparison with 77.14% to 100% for the complement fixation test (CFT). Specificities for cELISA, indirect enzyme-linked immunosorbent assay, and CFT were greater than 90%.
Conclusion
cELISA can be used on a variety of animal species, and an added advantage is its suitability for use on poor-quality samples such as those affected by hemolysis.
doi:10.3325/cmj.2010.51.314
PMCID: PMC2931436  PMID: 20718084
7.  A serological and bacteriological survey of brucellosis in wild boar (Sus scrofa) in Belgium 
Background
Brucellosis is frequently reported among wild boar populations in Europe. The aim of the study was to assess the epidemiological situation in Belgium, regarding the steady increase of wild boar populations over the last decades. Several serological tests were used and compared with culture and IS711 polymerase chain reaction (PCR), to determine the most suitable combination of diagnostic tools for conducting a successful prevalence study in wildlife.
Results
An indirect enzyme-linked immunosorbent assay (iELISA) was used on 1168 sera from hunter-killed wild boar sampled between 2003 and 2007 in 4 natural regions of southern Belgium. Results gave an apparent prevalence of 54.88% (95% CI 52.03-57.73). Prevalence was significantly affected by age and by the year of study, but not by sex nor by the region of sampling. The relative sensitivities of the complement fixation test (CFT), the Rose Bengal test (RBT), and the slow agglutination test (SAT) versus the iELISA differed widely between tests, reaching 62.67%, 46.68%, and 34.77%, respectively. The relative specificities of the CFT, RBT and SAT versus the iELISA were respectively 99.01%, 92.49%, and 99.1%. From seropositive animals (iELISA), 9% were positive by culture and 24% by PCR when testing spleen and/or tonsils. Sensitivity of the PCR was higher on tonsils than on spleen. All bacterial isolates were identified as Brucella suis biovar 2.
Conclusions
Brucellosis is widespread among wild boar in southern Belgium, with seroprevalences having increased over ten years, and constitutes a growing risk of spillback to outdoor-farmed pig herds. The iELISA showed a better sensitivity than the CFT, RBT and SAT. Serological tests must be associated with direct diagnosis and PCR proved more sensitive than culture under wildlife sampling conditions. Spleen and tonsils are lymphoid tissues usually sampled in multi-disease monitoring programs. They remain top-grade organs for direct diagnosis of brucellosis, with a preference for tonsils.
doi:10.1186/1746-6148-8-80
PMCID: PMC3503824  PMID: 22709889
8.  Serodiagnosis of Human Cysticercosis by Using Antigens from Vesicular Fluid of Taenia crassiceps Cysticerci 
Neurocysticercosis (NC), caused by the presence of Taenia solium metacestodes in tissues, is a severe parasitic infection of the central nervous system with universal distribution. To determine the efficiency of enzyme-linked immunosorbent assay (ELISA) and immunoblot with antigens of T. crassiceps vesicular fluid (Tcra) compared to standard techniques (indirect immunofluorescence test [IFT] and complement fixation test [CFT]) using T. solium cysticerci (Tso) for the serodiagnosis of NC, we studied serum samples from 24 patients with NC, 30 supposedly healthy individuals, 76 blood bank donors, 45 individuals with other non-NC parasitoses, and 97 samples from individuals screened for cysticercosis serology (SC). The sensitivity observed was 100% for ELISA-Tso and ELISA-Tcra, 91.7% for the IFT, and 87.5% for the CFT. The specificity was 90% for ELISA-Tso, 96.7% for ELISA-Tcra, 50% for IFT, and 63.3% for CFT. The efficiency was highest for ELISA-Tcra, followed by ELISA-Tso, IFT, and CFT. Of the 23 samples from SC group, which were reactive to ELISA-Tso and/or ELISA-Tcra, only 3 were positive to immunblot-Tcra (specific peptides of 14- and 18-kDa) and to glycoprotein peptides purified from Tcra antigen (gp-Tcra), showing the low predictive value of ELISA for screening. None of the samples from the remaining groups showed specific reactivity in immunoblot-Tcra. These results demonstrate that ELISA-Tcra can be used as a screening method for the serodiagnosis of NC and support the need for specific tests for confirmation of the results. The immunoblot can be used as a confirmatory test both with Tcra and gp-Tcra, with the latter having an advantage in terms of visualization of the results.
doi:10.1128/CDLI.8.6.1140-1144.2001
PMCID: PMC96240  PMID: 11687454
9.  Minor Nucleotide Substitutions in the omp31 Gene of Brucella ovis Result in Antigenic Differences in the Major Outer Membrane Protein That It Encodes Compared to Those of the Other Brucella Species 
Infection and Immunity  2001;69(11):7020-7028.
The gene coding for the major outer membrane protein Omp31 was sequenced in five Brucella species and their biovars. Although the omp31 genes appeared to be highly conserved in the genus Brucella, nine nucleotide substitutions were detected in the gene of Brucella ovis compared to that of Brucella melitensis. As shown by differential binding properties of monoclonal antibodies (MAbs) to the two Brucella species, these nucleotide substitutions result in different antigenic properties of Omp31. The antigenic differences were also evidenced when sera from B. ovis-infected rams were tested by Western blotting with the recombinant B. melitensis or B. ovis Omp31 proteins. Twelve available sera reacted with recombinant B. ovis Omp31, but only four of them reacted with recombinant B. melitensis Omp31. These results validate previous evidence for the potential of Omp31 as a diagnostic antigen for B. ovis infection in rams and demonstrate that B. ovis Omp31, instead of B. melitensis Omp31, should be used to evaluate this point. The antigenic differences between the B. melitensis and B. ovis Omp31 proteins should also be taken into account when Omp31 is evaluated as a candidate for the development of subcellular vaccines against B. ovis infection. No reactivity against recombinant B. melitensis Omp31 was detected, by Western blotting, with sera from B. melitensis-infected sheep. Accordingly, Omp31 does not seem to be a good diagnostic antigen for B. melitensis infections in sheep. Two immunodominant regions were identified on the B. ovis Omp31 protein by using recombinant DNA techniques and specific MAbs. Sera from B. ovis-infected rams that reacted with the recombinant protein were tested by Western blotting against one of these immunodominant regions shown to be exposed at the bacterial surface. Only 4 of the 12 sera reacted, but with strong intensity.
doi:10.1128/IAI.69.11.7020-7028.2001
PMCID: PMC100082  PMID: 11598077
10.  Ovine Brucellosis in Alberta 
The Canadian Veterinary Journal  1986;27(6):245-249.
Two parallel surveys of rams from Alberta sheep flocks were conducted to determine the presence of infection with Brucella ovis. In a retrospective study over a period of 24 months, using complement fixation test, 12 flocks out of 142 tested were considered infected. In another 17-month survey of slaughter rams by serology and culture methods 11 flocks out of 124 were found to be infected. The overall prevalence of ovine brucellosis was 8.6% of the flocks tested which represented 12.5% of the estimated sheep flocks in Alberta. Up to 67% of rams in infected flocks reacted to complement fixation test.
The complement fixation test was evaluated for its efficiency in the diagnosis of ovine brucellosis and compared with a limited number of an enzyme-linked immunosorbent assay (ELISA) results and clinical criteria. The complement fixation test as well as ELISA identified all culture positive rams. Both serological tests appeared satisfactory for the diagnosis of B. ovis epididymitis when the results could be interpreted in the light of flock history and clinical findings.
PMCID: PMC1680265  PMID: 17422669
Brucella ovis; ram epididymitis; sheep diseases
11.  Comparison of diagnostic tests for the detection of Brucella spp. in camel sera 
BMC Research Notes  2011;4:525.
Background
Brucellosis in livestock causes enormous losses for economies of developing countries and poses a severe health risk to consumers of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. To date, little information on application of real-time PCR for detection of Brucella in camel serum is available. Therefore, this study was performed to compare the diagnostic efficiency of different serological tests and real-time PCR in order to identify the most sensitive, rapid and simple combination of tests for detecting Brucella infection in camels.
Findings
A total of 895 serum samples collected from apparently healthy Sudanese camels was investigated. Sudan is a well documented endemic region for brucellosis with cases in humans, ruminants, and camels. Rose Bengal Test (RBT), Complement Fixation Test (CFT), Slow Agglutination Test (SAT), Competitive Enzyme Linked Immunosorbant Assay (cELISA) and Fluorescence Polarization Assay (FPA) as well as real-time PCR were used. Our findings revealed that bcsp31 kDa real-time PCR detected Brucella DNA in 84.8% (759/895) of the examined samples, of which 15.5% (118/759) were serologically negative. Our results show no relevant difference in sensitivity between the different serological tests. FPA detected the highest number of positive cases (79.3%) followed by CFT (71.4%), RBT (70.7%), SAT (70.6%) and cELISA (68.8%). A combination of real-time PCR with one of the used serological tests identified brucellosis in more than 99% of the infected animals. 59.7% of the examined samples were positive in all serological tests and real-time PCR. A subpopulation of 6.8% of animals was positive in all serological tests but negative in real-time PCR assays. The high percentage of positive cases in this study does not necessarily reflect the seroprevalence of the disease in the country but might be caused by the fact that the camels were imported from brucellosis infected herds of Sudan, accidentally. Seroprevalence of brucellosis in camels should be examined in confirmatory studies to evaluate the importance of brucellosis in this animal species.
Conclusion
We suggest combining bcsp31 real-time PCR with either FPA, CFT, RBT or SAT to screen camels for brucellosis.
doi:10.1186/1756-0500-4-525
PMCID: PMC3284514  PMID: 22145943
12.  Comparison of a Commercial Enzyme-Linked Immunosorbent Assay with Immunofluorescence and Complement Fixation Tests for Detection of Coxiella burnetii (Q Fever) Immunoglobulin M 
Journal of Clinical Microbiology  2000;38(4):1645-1647.
A commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Q fever (PanBio Coxiella burnetii immunoglobulin M [IgM] ELISA, QFM-200) was compared to the indirect fluorescent antibody test (IFAT) for C. burnetii IgM and the complement fixation test (CFT). The ELISA demonstrated 92% agreement with the reference method (IFAT), and gave a sensitivity of 99% (69 of 70 samples) and a specificity of 88% (106 of 121). Specificity can be increased with confirmation by IFAT. CFT was found to have a specificity of 90% (107 of 119), although it was lacking in sensitivity (73%; 51 of 70). No cross-reactivity was observed in the ELISA with serum samples from patients with mycoplasma (n = 6), chlamydia (n = 5), or legionella (n = 4) infections, although 2 of 5 patients with leptospirosis and 1 of 4 samples containing rheumatoid factor (RF) demonstrated positive results in the ELISA. Results indicate that the performance of the PanBio C. burnetii (Q fever) IgM ELISA (F = 187) is superior to that of CFT (F = 163), and consequently the ELISA should be a useful aid in the diagnosis of acute Q fever.
PMCID: PMC86512  PMID: 10747159
13.  Detection of Equine Antibodies to Babesia caballi by Recombinant B. caballi Rhoptry-Associated Protein 1 in a Competitive-Inhibition Enzyme-Linked Immunosorbent Assay 
Journal of Clinical Microbiology  1999;37(7):2285-2290.
A competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was developed for detection of equine antibodies specific for Babesia caballi. The assay used recombinant B. caballi rhoptry-associated protein 1 (RAP-1) and monoclonal antibody (MAb) 79/17.18.5, which is reactive with a peptide epitope of a native 60-kDa B. caballi antigen. The gene encoding the recombinant antigen was sequenced, and database analysis revealed that the gene product is a rhoptry-associated protein. Cloning and expression of a truncated copy of the gene demonstrated that MAb 79/17.18.5 reacts with the C-terminal repeat region of the protein. The cELISA was used to evaluate 302 equine serum samples previously tested for antibodies to B. caballi by a standardized complement fixation test (CFT). The results of cELISA and CFT were 73% concordant. Seventy-two of the 77 serum samples with discordant results were CFT negative and cELISA positive. Further evaluation of the serum samples with discordant results by indirect immunofluorescence assay (IFA) demonstrated that at a serum dilution of 1:200, 48 of the CFT-negative and cELISA-positive serum samples contained antibodies reactive with B. caballi RAP-1. Four of five CFT-positive and cELISA-negative serum samples contained antibodies reactive with B. caballi when they were tested by IFA. These data indicate that following infection with B. caballi, horses consistently produce antibody to the RAP-1 epitope defined by MAb 79/17.18.5, and when used in the cELISA format, recombinant RAP-1 is a useful antigen for the serologic detection of anti-B. caballi antibodies.
PMCID: PMC85139  PMID: 10364599
14.  Use of synthetic antigens improves detection by enzyme-linked immunosorbent assay of antibodies against abortigenic Chlamydia psittaci in ruminants. 
Journal of Clinical Microbiology  1997;35(9):2293-2298.
Synthetic peptide antigens were prepared for use in enzyme-linked immunosorbent assays (ELISAs) to detect serum antibodies against abortigenic strains of Chlamydia psittaci in livestock. Peptide antigens were identified with C. psittaci B577-immune sera by solid-phase scanning of overlapping octapeptides of variable domains (VDs) of the major outer membrane protein of C. psittaci serovar 1 (omp1 type C. psittaci B577). Two VD 4 regions and one VD 2 region were strongly reactive with all C. psittaci B577 antisera. Peptides encompassing these regions were synthesized with biotin and a serine-glycine-serine-glycine spacer at the N terminus and were attached to streptavidin-coated microtiter plates. In direct ELISAs with these plates, the synthetic peptides reacted with C. psittaci B577 antisera, but not with sera from specific-pathogen-free animals. Serum specimens from 40 sheep and 40 cattle, obtained from herds with abortion problems, were screened for antibodies by these C. psittaci B577 peptide ELISAs and an ELISA with recombinant, genus-specific Chlamydia lipopolysaccharide (LPS) antigen. Results from these newly developed ELISAs were compared to those from the reference C. psittaci B577 elementary body (EB) ELISA and the Chlamydia complement fixation test (CFT). The C. psittaci B577 peptide ELISAs, the LPS ELISA, and the EB ELISA correctly identified the presence or absence of antibodies against chlamydiae in all sheep and bovine sera. The Chlamydia CFT, which is the most widely accepted serodiagnostic method for chlamydial infections in animals, correctly identified the presence or absence of antibodies against chlamydiae in only 78 and 4.9% of sheep and bovine sera, respectively. These results suggest that the C. psittaci B577-peptide and Chlamydia LPS ELISAs are superior for the serodiagnosis of ruminant infections with abortigenic chlamydiae, since they are more sensitive than the CFT, they are easy to standardize, and they use readily available synthetic antigens instead of organism-derived CFT antigen.
PMCID: PMC229957  PMID: 9276405
15.  Prevalence-dependent use of serological tests for diagnosing glanders in horses 
Background
The internationally mandatory complement fixation test (CFT) for testing of equine sera for the absence of glanders has repeatedly led to discrepant results. Not only do "false positive" sera pose a problem for the diagnostician and the animal health authorities but they can also result in significant financial losses for the animal owners.
Due to the very low prevalence of glanders in the horse population it is of major importance to use tests with a high specificity to overcome unreliable predictive values. We have compared formalin-fixed B. mallei whole cell antigen and a well characterised mouse monoclonal antibody with regard to their specificity and sensitivity for glanders serodiagnosis using CFT, an indirect (i) and a competitive (c) ELISA platform.
Results
Our results show that the CFT is still a very reliable technique in horse populations with very low glanders prevalence. The cELISA has a high sensitivity and specificity comparable to that of the CFT. The cELISA offers the possibility for automatisation, can be applied to non-complement fixing sera and used for various host species.
Conclusion
The CFT is still the method of choice for testing horses for the absence of glanders.
doi:10.1186/1746-6148-5-32
PMCID: PMC2745380  PMID: 19723336
16.  Enzyme Immunoassays for Serological Diagnosis of Bovine Brucellosis: A Trial in Latin America 
The results of a field trial conducted in Latin America with two indirect enzyme-linked immunosorbent assays (ELISAs) and two competitive ELISAs (CELISAs) for the detection of bovine antibody to Brucella abortus are reported. One of the CELISA formats performed most accurately. The percentage of positive reactions in the CELISA relative to the selected positive rose bengal agglutination test (RBT) and complement fixation test (CFT) results was 97.47%, the percentage of negatives relative to the selected negative RBT and CFT results for unexposed cattle was 98.32%, and the percentage of negatives in cattle vaccinated with B. abortus 19 was 96.51%. The same assay format under Canadian conditions had an actual sensitivity of 100%, a specificity of 99.90% in nonvaccinates, and a specificity of 97.7% in a strain 19-vaccinated population. Overall, the CELISA performed as expected and the results were not dissimilar from the results obtained in the Canadian study. This provided further evidence that this CELISA can in many instances differentiate infected cattle from those that are vaccinated or infected with a cross-reacting organism while still giving very few false-positive or false-negative results.
PMCID: PMC95636  PMID: 9729532
17.  Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. 
Journal of Clinical Microbiology  1994;32(5):1159-1165.
Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests.
PMCID: PMC263630  PMID: 8051240
18.  Use of the Brucella melitensis Native Hapten To Diagnose Brucellosis in Goats by a Rapid, Simple, and Specific Fluorescence Polarization Assay▿  
The performance of the fluorescence polarization assay (FPA) using the recently described Brucella melitensis native hapten and the Brucella abortus O-polysaccharide tracer was evaluated and compared with those of The World Organization for Animal Health tests related to indirect and competitive enzyme-linked immunosorbent assays as classification variables for goat sera obtained from a high-prevalence area where vaccination was performed; test series were also evaluated to increase the final specificity of the tests. Our results showed that the respective relative sensitivity and specificity were 99.7% and 32.5% for the rose Bengal test with a 3% cell concentration (RBT3), 92.8% and 68.8% for the rose Bengal test with 8% cell concentration (RBT8), 98.4% and 84.9% for the Canadian complement fixation test (CFT), 83.7% and 65.5% for the Mexican CFT, 98.4% and 81.0% for the buffered plate agglutination test (BPAT), and 78.1% and 89.3% for the fluorescence polarization assay (FPA). The use of the FPA as the secondary test significantly increased the final specificities of test combinations; the screening tests BPAT, RBT3, and RBT8 plus FPA resulted in 90%, 91.2%, and 91.3% final specificities, respectively, whereas for the combinations RBT3 plus Mexican CFT, RBT8 plus Mexican CFT, and BPAT plus Canadian CFT, the specificities were 65.5%, 63.2%, and 91.7%, respectively. The results suggested that the FPA may be routinely applied as an adaptable screening test for diagnosis of goat brucellosis, since its cutoff can be adjusted to improve its sensitivity or specificity, it is a rapid and simple test, it can be the test of choice when specificity is relevant or when an alternative confirmatory test is not available, and it is not affected by vaccination, thus reducing the number of goats wrongly slaughtered due to misdiagnosis.
doi:10.1128/CVI.00046-08
PMCID: PMC2446613  PMID: 18385457
19.  Serological testing of cattle experimentally infected with Mycoplasma mycoides subsp. mycoides Small Colony using four different tests reveals a variety of seroconversion patterns 
Background
To study the specific antibody response to infection with Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), the agent of Contagious Bovine Pleuropneumonia (CBPP), we examined three panels of sera collected during three experimental infection trials in African cattle. The methods used included an in-house complement fixation test (CFT), a commercially available CFT, a competitive antibody ELISA (cELISA) and the immunoblotting test (IBT). In addition, lung tissue samples were examined by culture.
Results
A total of 89% (51/59) of all experimentally infected animals tested positive on at least one of the serological tests throughout the trial. The specific antibody titres to the MmmSC infection became positive first by CFT (6 to 9 days post infection [dpi]), followed by IBT (9 to 13 dpi) and cELISA (13 to 16 dpi). Individual animals were found to display remarkably distinct seroconversion patterns, which allowed their classification into i) early high responders, ii) late high responders, and iii) low responders. In accordance with other studies, none of the present serological tests was capable of detecting all CBPP infected animals.
Conclusion
Comparison of the assays' performance in terms of sensitivity and specificity raises serious questions as to their reliability for identification of infected individuals in the field. In view of these limitations, a combination of CFT and cELISA can markedly improve CBPP diagnosis at single-animal level.
doi:10.1186/1746-6148-7-72
PMCID: PMC3377920  PMID: 22098816
20.  Comparison of enzyme-linked immunosorbent assay and complement fixation test for the detection of specific antibody in cattle vaccinated and challenged with Brucella abortus. 
An enzyme-linked immunosorbent assay (ELISA) and a complement fixation test (CFT) were applied to sera collected regularly from 60 cattle challenged with Brucella abortus strain 544. Of the 60 cattle, 48 were vaccinated with either B. abortus strain 19 or B. abortus strain 45/20 as calves or adults. The remaining 12 cattle were not vaccinated. Of the 27 sera from cattle found to be infected at slaughter, 9 showed aberrant reactions to the CFT in that a positive titer after challenge was delayed or transient. The performance of the ELISA in these nine cattle with aberrant reactions and the other cattle in the trial was as follows. After vaccination with strain 19 or strain 45/20, the number of weeks at which the ELISA was positive was significantly greater (P less than 0.001) than that for the CFT. The strain 19 vaccine also induced positive responses to the CFT and the ELISA for a significantly longer period (P less than 0.001) than did the strain 45/20 vaccine. For cattle with aberrant reactions, the number of weeks after challenge when the ELISA was positive was significantly greater (P less than 0.001) than that for the CFT. For noninfected cattle, the average number of weeks after challenge when the CFT was negative (35 of 40) was higher than that for the ELISA (18 of 40).
PMCID: PMC268318  PMID: 3926817
21.  Coxsackievirus B1-based antibody-capture enzyme-linked immunosorbent assay for detection of immunoglobulin G (IgG), IgM, and IgA with broad specificity for enteroviruses. 
Journal of Clinical Microbiology  1993;31(12):3240-3246.
An antibody-capture enzyme-linked immunosorbent assay (ELISA) with coxsackievirus B1 as the antigen was evaluated for detection of immunoglobulin G (IgG), IgM, and IgA antibodies and showed broad specificity for enteroviruses. In total, 116 serum or cerebrospinal fluid samples from 62 patients were tested by ELISA and the complement fixation test (CFT). Additionally, 15 serum samples that contained poliovirus-specific IgM antibody were tested. Serum samples from 200 healthy blood donors were used for standardization of the assays. The sensitivity of the ELISA varied with time of serum sampling, with a relatively low sensitivity when serum was collected within 3 days after the onset of symptoms (23%; 5 of 22) but good sensitivity when serum was collected later (83%; 20 of 24). The sensitivity was better than that of the CFT. The ELISAs were broadly reactive as concluded from typing of virus isolates that were simultaneously obtained. The assay did, furthermore, detect antibody against poliovirus type 3. Sera that contained rheumatoid factor, antinuclear antibody, or cardiolipin antibody (by the Venereal Disease Research Laboratory test) did not react in this ELISA. Nonspecific reactivity did occur, however, in cases of infectious mononucleosis and in Mycoplasma pneumoniae infection. The enterovirus-specific ELISA is found to be simple to perform, more sensitive than the CFT, and far less laborious than the neutralization test.
PMCID: PMC266387  PMID: 8308117
22.  Competitive Enzyme Immunoassay for Diagnosis of Human Brucellosis 
Journal of Clinical Microbiology  1999;37(10):3245-3248.
The methods commonly used for human brucellosis serological testing are agglutination tests and the complement fixation test (CFT). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The competitive enzyme immunoassay (CELISA) for the detection of serum antibody to Brucella is a multispecies assay which appears to be capable of differentiating vaccinal and cross-reacting antibodies from antibodies elicited by field infection in cattle. The competing monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). In this study, we compared the CELISA to the classical tests for the diagnosis of human brucellosis. The CELISA cutoff value was determined to calculate its diagnostic specificity and sensitivity. A survey was performed with 911 sera. Of the sera, 341 were from an asymptomatic population that tested negative with conventional serological tests (screening and confirmatory). Based on these samples, the CELISA specificities were determined to be 99.7 and 100% with cutoff values of 28 and 30% inhibition (%I), respectively. In a further study with 393 additional sera from an asymptomatic population found negative by the conventional screening tests, the CELISA specificities were calculated to be 96.5 and 98.8% with cutoff values of 28 and 30%I. The CELISA sensitivities were determined to be 98.3 and 94.8% with cutoff values of 28 and 30%I, respectively, for sera from 116 individuals found positive by the classical tests. For the 51 culture-positive patients, CELISA was positive for 100%, the CFT was positive for 92%, and the standard tube agglutination test (TAT) was positive for 100%. The CELISA specificity was 100% for 31 sera from patients found negative by conventional serological tests but with brucellosis-like symptoms. The CELISA is fairly rapid to perform, somewhat faster than TAT, and cross-reacts less with other antigens (or antibodies) than the conventional tests. Further, the CELISA is simpler to perform that the CFT and may readily be standardized by the use of purified S-LPS antigen and monoclonal antibody for competition.
PMCID: PMC85539  PMID: 10488186
23.  Evaluation of an indigenous ELISA for diagnosis of Johne’s disease and its comparison with commercial kits 
Indian Journal of Microbiology  2007;47(3):251-258.
Country lacks sensitive and indigenous diagnostic kits for the screening of goats and sheep against Johne’s disease. Therefore an indigenous ELISA kit was developed using protoplasmic antigen from native Mycobacterium avium subspecies paratuberculosis ‘Bison Type’ strain of goat origin (Kit 1). In the present study, kit 1 and two commercial kits (kit 2 and 3) were evaluated with respect to ‘Gold Standard’ fecal culture in 71 animals (55 goats and 16 sheep). Kit 1 using indigenous antigen (protoplasmic antigen) was sensitive at very low concentration (0.1 μgm / well) as compared to purified commercial protoplasmic antigen (4 μgm / well) used in kit 2, in the Type 1 reactors (strong positive as positive). Screening of 71 animals by fecal culture detected 38.0% animals (goats-40.0%, sheep-31.2%) as positive (clinical shedders of bacilli) from these farm animals. Of the farm animals located at Central Institute for Research on Goats, herds of goat were endemic whereas, sheep flocks were comparatively resistant to Johne’s disease. The 29.5 and 61.9, 15.4 and 57.7 and 4.2 and 14.0% animals (goats and sheep) were in the category of sero-reactors type 1 and 2 of the ELISA kits 1, 2 and 3, respectively. In the type 1 sero-reactors, sensitivity and specificity of kit 1, 2 and 3 was 53.7 and 86.0, 17.8 and 86.0 and 3.5 and 94.7%, respectively. Indigenous ELISA test (kit 1) was significantly superior for the screening of goatherds and sheep flocks against JD as compared to commercial ELISA kits (Kit 2 and 3). In comparison to kit 2 and 3, kit 1 had highest sensitivity, comparable specificity and substantial to nearly perfect proportional agreement (Kappa Scores) with respect to ‘Gold standard’ fecal culture in goats and sheep. Disease being endemic in herds and flocks screened using ELISA kits, Type I sero-rectors had better correlation with fecal culture in comparison to Type II sero-reactors therefore, used for estimation of sero-prevalence. Newly developed Indigenous ELISA kit was simple, inexpensive, sensitive and reliable for screening of goats and sheep population against Johne’s disease. The study reports high prevalence of Johne’s disease in farm goatherds and sheep flocks, using sensitive tests (fecal culture and ELISA kit). Results of Type 1 reaction in kit 1 were optimally correlated with culture and were good for estimating the sero-prevalence. For controlling Johne’s disease in endemic herds initial removal of the animals in strong positive category (Tyep 1 reactors), may help to remove heavy shedders.
doi:10.1007/s12088-007-0046-2
PMCID: PMC3450340  PMID: 23100673
Johne’s disease; Mycobacterium avium subsp. paratuberculosis; Protoplasmic antigen; Diagnosis; Fecal culture; ELISA kit
24.  Use of a Recombinant Burkholderia Intracellular Motility A Protein for Immunodiagnosis of Glanders ▿ 
Glanders, caused by the Gram-negative, nonmotile bacterium Burkholderia mallei, is a contagious and highly fatal disease of equines. During the last decade, the number of glanders outbreaks has increased steadily. The disease also has high zoonotic significance and B. mallei is listed biological warfare agent. The complement fixation test (CFT) is a routinely used and internationally recognized test to screen equine sera for the glanders. However, discrepant results have been observed using the CFT. The low sensitivity and specificity of the CFT and enzyme-linked immunosorbent assay (ELISA) have been linked to the use of crude test antigens. We expressed a novel recombinant Burkholderia intracellular motility A (rBimA) protein in Escherichia coli for the diagnosis of equine glanders. Purified rBimA was used in an indirect ELISA format. All of the 21 true-positive serum samples used in the study tested positive, whereas only 17 of the 1,524 potentially negative sera tested positive by indirect ELISA, thus exhibiting 100% sensitivity and 98.88% specificity. Also, rBimA protein did not react with melioidosis patient and normal healthy human serum samples, showing its high specificity. The developed assay can be used as a simple and rapid tool for diagnosis of glanders in equine serum samples. An Indian patent (1328/DEL/2010) has been filed for the reagent.
doi:10.1128/CVI.05185-11
PMCID: PMC3165212  PMID: 21752949
25.  Serological, Bacteriological, and Molecular Diagnosis of Brucellosis in Domestic Animals in Croatia 
Croatian Medical Journal  2010;51(4):320-326.
Aim
To present the surveillance data on Brucella melitensis, B. suis, and B. ovis infection in cattle, sheep, goats, and swine in Croatia obtained in 2008 by serological, bacteriological, and molecular methods for diagnostics of brucellosis in domestic animals.
Methods
We serologically tested 42 785 cattle serums, 22 686 sheep and goat serums, and 28 520 swine serums using the Rose Bengal test, complement fixation test, and various immunosorbent assays. We also tested 10 173 ram blood samples for B. ovis infection using the complement fixation test. Bacteriological examination was conducted on 214 samples collected from 34 serologically positive animals. Different molecular methods were employed in the identification and typing of 20 isolates from the samples.
Results
B. melitensis biovar (bv.) 3 was confirmed with different identification methods in 2 flocks in 2 Croatian counties and B. suis bv. 2 in 3 herds in 3 counties. B. melitensis in cows was confirmed for the first time in Croatia. Infection with B. ovis was serologically confirmed in 202 rams in 12 counties.
Conclusions
In 2008, the size of the brucellosis-affected area in Croatia and the efficiency of detection and prevention of brucellosis in sheep, goats, and swine were satisfactory. Infection with B. melitensis in cattle was confirmed for the first time and possible links for infection in humans were detected. More efficient measures for suppression and control of ovine epididymitis are required and a new strategy may be necessary for complete eradication of this disease.
doi:10.3325/cmj.2010.51.320
PMCID: PMC2931437  PMID: 20718085

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