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1.  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
2.  Characterization of Brucella ovis lipopolysaccharide and its use for diagnosis of ram epididymitis by enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1984;20(6):1159-1164.
Rough lipopolysaccharide, extracted by a mixture of phenol, chloroform, and petroleum ether from freeze-dried Brucella ovis cells with a yield of 0.71%, contained relatively small amounts of protein and nucleic acid contaminants as compared with lipopolysaccharides from other Brucellae. The crude lipopolysaccharide was suitable as a diagnostic antigen in an enzyme-linked immunosorbent assay for the sensitive and specific detection of ram epididymitis caused by B. ovis infection. In comparative serological tests, the enzyme-linked immunosorbent assay with B. ovis lipopolysaccharide gave better identification of infections and fewer false-negative results than the enzyme-linked immunosorbent assay with sonicated antigen or the complement fixation test.
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PMCID: PMC271537  PMID: 6394618
3.  Comparison of lipopolysaccharide and outer membrane protein-lipopolysaccharide extracts in an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection. 
Journal of Clinical Microbiology  1986;23(5):938-942.
Brucella ovis hot saline extracts and petroleum ether-chloroform-phenol lipopolysaccharide were compared in an enzyme-linked immunosorbent assay for the diagnosis of B. ovis ram epididymitis. Hot saline extracts detected greater numbers of infected rams. Chemical characterization of the antigens showed that, although both contained lipopolysaccharide, hot saline extracts also contained outer membrane proteins. These proteins were active as antigens in Western blot tests with sera of infected rams, and therefore they explained the better diagnostic results obtained with hot saline extracts. However, compared with lipopolysaccharide, hot saline extracts showed a higher degree of cross-reactivity with sera from smooth B. melitensis-infected animals. This observation might be explained by the presence of B. ovis outer membrane proteins in hot saline extracts which lack the specificity necessary for serological identification of the Brucella species present.
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PMCID: PMC268754  PMID: 3711279
4.  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
5.  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
6.  Lack of evidence of Brucella ovis infection in rams infection in rams in Quebec 
The Canadian Veterinary Journal  2004;45(4):312-314.
Abstract
A study was conducted to estimate the seroprevalence of Brucella ovis infection in rams in the Estrie and Bas-Saint-Laurent regions (Quebec). Rams sera (n = 258) were serologically evaluated from 224 rams in 30 commercial flocks and from 34 rams at 2 slaughterhouses by using an enzyme linked immunosorbent assay. Epididymides and testes were examined by palpation on farms and microscopically for culled rams. No ram was seropositive to Brucella ovis or had lesions suggestive of brucellosis from the farm or slaughterhouse surveys.
PMCID: PMC548616  PMID: 15144103
7.  Putative ATP-Binding Cassette Transporter Is Essential for Brucella ovis Pathogenesis in Mice▿  
Infection and Immunity  2011;79(4):1706-1717.
Brucella ovis is a major cause of reproductive failure in sheep, which is associated with epididymitis and infertility in rams. Importantly, B. ovis is one of the few Brucella species that is not zoonotic. Due to the scarcity of studies on B. ovis infection, a murine model of infection was developed. The roles of B. ovis genes encoding a putative hemagglutinin and an ABC transporter were investigated in the mouse model. The kinetics of B. ovis infection were similar in BALB/c and C57BL/6 mice, and both strains of mice developed multifocal microgranulomas in the liver and spleen, but only minimal colonization and histopathological changes were observed in the genital tract. Therefore, the mouse was considered a suitable infection model for B. ovis but not for B. ovis-induced genital disease. Two mutant strains were generated in this study (the ΔabcAB and Δhmg strains). The B. ovis ΔabcAB strain was attenuated in the spleens and livers of BALB/c mice compared to the wild-type (WT) strain (P < 0.001). Conversely, the Δhmg strain infected mice at the same level as WT B. ovis, suggesting that a putative hemagglutinin is not required for B. ovis pathogenesis. Additionally, the ΔabcAB strain did not survive in peritoneal macrophages, extracellularly in the peritoneal cavity, or in RAW 264.7 macrophages. Moreover, infection with the ΔabcAB strain was not lethal for male regulatory factor 1-knockout mice, whereas infection with the B. ovis WT strain was 100% lethal within 14 days postinfection. These results confirm that the predicted ABC transporter is required for the full virulence and survival of B. ovis in vivo.
doi:10.1128/IAI.01109-10
PMCID: PMC3067543  PMID: 21300772
8.  Serodiagnosis of ram epididymitis by counterimmunoelectrophoresis, using Brucella ovis surface R antigen. 
Journal of Clinical Microbiology  1979;10(4):451-453.
The counterimmunoelectrophoresis (CIE) technique was developed as a diagnostic procedure for ram epididymitis caused by Brucella ovis. CIE test results with sera from naturally and experimentally infected male sheep compared favorably with those obtained by gel diffusion and complement fixation employing the same B. ovis surface R antigen. The main advantage of CIE over gel diffusion consists in a significant reduction of the time required to detect precipitin formation, whereas both methods obviate several of the difficulties encountered with complement fixation tests for B. ovis.
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PMCID: PMC273195  PMID: 118983
9.  Profiles of serological reactivity against cytosoluble antigens of Brucella ovis in experimentally infected rams. 
Journal of Clinical Microbiology  1990;28(12):2647-2652.
Sera from rams infected with and excreting Brucella ovis in the semen (shedders), as well as from animals which had recovered from previous experimental challenge with B. ovis, were analyzed for their serological reactivities against cytosolic antigens of the bacterium. Membrane vesicles, including outer and inner membrane components, were precluded from the analyses by subjecting French-pressed bacteria to ultracentrifugation. The resulting cytosolic supernatant was fractionated into four major antigenic fractions, fractions A, B, C, and D, by high-pressure liquid chromatography. Temporal enzyme-linked immunosorbent assays with the A antigen revealed that all shedder rams displayed a rise-and-surge response, while rams which recovered from experimental challenge showed a rise-and-fall profile. The B antigen was less discriminatory in detecting a difference between the two ram groups, while C and D antigens were serologically unreactive in the enzyme-linked immunosorbent assay. In contrast to the reactivity patterns shown by native high-pressure liquid chromatography-fractionated cytosolic supernatant antigens, immunoblotting of C and D polypeptides generated by boiling in the presence of sodium dodecyl sulfate and mercaptoethanol was particularly useful in distinguishing between sera collected at the mid-surge phase of infected rams from sera obtained at the mid-fall stage of recovered animals. It is likely that native or denatured antigens of different cytosolic fractions may provide useful serological reagents for differentiating between infected rams and those which have recovered from exposure to B. ovis.
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PMCID: PMC268250  PMID: 2279996
10.  Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection 
Veterinary Research  2014;45(1):61.
Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG1, IgG2a and IgG2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.
doi:10.1186/1297-9716-45-61
PMCID: PMC4057616  PMID: 24898325
11.  Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams 
Background
Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections.
Results
The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples.
Conclusions
The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.
doi:10.1186/1746-6148-9-51
PMCID: PMC3614447  PMID: 23514236
Actinobacillus seminis; Brucella ovis; Histophilus somni; Epididymitis; Multiplex polymerase chain reaction
12.  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
13.  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
14.  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
15.  Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp. 
Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals.
PMCID: PMC170519  PMID: 9144364
16.  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
17.  Epididymitis Caused by Brucella ovis in a Southern Ontario Sheep Flock 
The Canadian Veterinary Journal  1985;26(10):293-296.
Epididymitis was diagnosed in three rams in a commercial sheep flock in southern Ontario. The affected rams had palpably enlarged epididymides and two rams had semen which contained inflammatory cells and was of poor quality. Serum compliment fixation titers for Brucella ovis were 1:20, 1:80 and 1:90. Five other rams in the flock were clinically normal and without titers. Two of the affected rams had lesions similar to those produced by experimental infection with B. ovis. The infection in the rams had no apparent affect on ewe performance. The source of the infection remains unknown, but the rams were purchased from a flock which had imported ewes from the western U.S.A.
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PMCID: PMC1680171  PMID: 17422577
Sheep; epididymitis; ram; brucellosis; Brucella ovis
18.  Use of Recombinant BP26 Protein in Serological Diagnosis of Brucella melitensis Infection in Sheep 
Previously a Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. In the present study we evaluated antibody responses of infected and B. melitensis Rev.1-vaccinated sheep to the BP26 protein using purified recombinant BP26 protein produced in Escherichia coli in an indirect enzyme-linked immunosorbent assay (I-ELISA). The specificity of the I-ELISA determined with sera from healthy sheep (n = 106) was 93%. The sensitivity of the I-ELISA assessed with sera from naturally infected and suspected sheep found positive in the current conventional diagnostic tests was as follows: 100% for bacteriologically and serologically positive sheep (n = 50), 88% for bacteriologically negative but serologically and delayed-type hypersensitivity-positive sheep (n = 50), and 84% for bacteriologically and serologically negative but delayed-type hypersensitivity-positive sheep (n = 19). However, the absorbance values observed did not reach those observed in an I-ELISA using purified O-polysaccharide (O-PS) as an antigen. In sheep experimentally infected with B. melitensis H38 the antibody response to BP26 was delayed and much weaker than that to O-PS. Nevertheless, the BP26 protein appears to be a good diagnostic antigen to be used in confirmatory tests and for serological differentiation between infected and B. melitensis Rev.1-vaccinated sheep. Weak antibody responses to BP26 in some of the latter sheep suggest that a B. melitensis Rev.1 bp26 gene deletion mutant should be constructed to ensure this differentiation.
doi:10.1128/CDLI.8.4.772-775.2001
PMCID: PMC96141  PMID: 11427425
19.  Serological Diagnosis of Brucella Infections in Odontocetes▿  
Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a “gold” standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (Ri/g2 = 0.65 and κi/g = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (Ri/c2 = 0.46, Rg/c2 = 0.37 and κi/c = 0.62, κg/c = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.
doi:10.1128/CVI.00413-08
PMCID: PMC2691062  PMID: 19386800
20.  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
21.  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
22.  Detection of Anaplasma ovis infection in goats by major surface protein 5 competitive inhibition enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1995;33(3):675-679.
A competitive inhibition enzyme-linked immunosorbent assay (ELISA) based on a major surface protein 5 (MSP5) B-cell epitope conserved among Anaplasma species was used to detect goats infected with Anaplasma ovis. We examined strains of A. ovis isolated from goats in Kenya and demonstrated that MSP5 and the target B-cell epitope, bound by monoclonal antibody ANAF16C1, were conserved. Sera from 149 goats in four regions of Kenya and from 302 goats in six U.S. states were tested for the presence of epitope-specific antibodies with the MSP5 competitive inhibition ELISA. Evidence that the assay can be used to detect A. ovis-infected goats includes the following: (i) 53 goats raised in confinement with arthropod control were all seronegative; (ii) six goats experimentally infected with A. ovis seroconverted at the same time that they developed detectable rickettsemia; (iii) seroconverted goats remained seropositive, consistent with the persistence of A. ovis in goats and the presence of anti-MSP5 antibody in cattle persistently infected with Anaplasma marginale; and (iv) 119 of 127 known A. ovis-infected goats in Kenya were seropositive. A. ovis infection, as determined serologically and by demonstration of infected erythrocytes, in goats from the four regions in Kenya was highly prevalent. In contrast, despite the presence of A. ovis and competent arthropod vectors in the United States, the prevalence of infection appeared to be very low. The high prevalence in Kenya and the occurrence of anemia in persistently infected goats may be impediments to current efforts to increase milk yields on small farms.
PMCID: PMC228012  PMID: 7538510
23.  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
24.  Field Evaluation of the Gel Diffusion Test for the Diagnosis of Ram Epididymitis Caused by Brucella ovis 
Applied Microbiology  1973;26(6):855-857.
A comparison was made with the microslide gel diffusion technique, the complement fixation test, cultural isolation, and clinical examination in detecting ram epididymitis caused by Brucella ovis. The results of the gel diffusion method are shown to be similar to those obtained by the complement fixation test and isolation of B. ovis from cultures of semen. The technique offers a reliable diagnostic method adaptable to field use in controlling ovine brucellosis and is more practical.
PMCID: PMC379921  PMID: 4203332
25.  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

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