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1.  Use of recombinant human parvovirus B19 antigens in serological assays. 
Journal of Clinical Pathology  1993;46(9):840-845.
AIMS--To compare the sensitivity, specificity, and practicality of recombinant proteins in serological tests for the detection of human parvovirus B19 IgG and IgM. METHODS--Indirect enzyme linked immunosorbent assays using B19 structural proteins expressed in Escherichia coli were developed for the detection of B19 specific IgG and IgM (rELISA-G and rELISA-M). Cells infected with baculovirus expressing B19 structural proteins were also used in an indirect immunofluorescence assay for IgG and IgM antibodies (IFA-G and IFA-M). Antibody capture radioimmunoassays for IgG and IgM (GACRIA and MACRIA) were used as comparative assays. RESULTS--Twenty nine pools of intravenous immunoglobulin were clearly positive for B19 IgG by rELISA-G and contained low IgG titres by GACRIA. From 113 samples tested by all methods, sensitivities of 92% (77/84) and 97% (68/70) were obtained for ELISA and immunofluorescence, respectively, when compared with GACRIA. One hundred and sixteen samples from patients presenting with rash or arthralgia were compared by MACRIA, rELISA-M, and IFA-M. Sensitivities of both recombinant tests were more than 95%. Despite pretreatment to remove IgG or rheumatoid factor, false positive results were a problem in the rELISA-M but were not seen with the IFA-M. CONCLUSIONS--The limited supply of native antigen has severely restricted the wide application of serology for parvovirus B19. The use of recombinant antigens permitted the introduction of local screening tests which had many advantages, including quicker results and relief of the burden on the Reference Laboratory. The use of rELISA-M for sensitivity and IFA-M for specificity and confirmation proved a useful and practical combination for diagnosis of recent infection with B19, and rELISA-G allowed the immune response to be determined in selected populations.
PMCID: PMC501521  PMID: 8227436
2.  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
3.  Importance of Methodology in Determination of Chlamydia pneumoniae Seropositivity in Healthy Subjects and in Patients with Coronary Atherosclerosis 
Journal of Clinical Microbiology  2003;41(9):4049-4053.
Enzyme immunoassays (EIAs) for the detection of Chlamydia pneumoniae antibodies were compared to the microimmunofluorescence (MIF) test, the reference method. Furthermore, we assessed the hypothesis that a possible relationship between Chlamydia pneumoniae immunoglobulin G (IgG) antibodies and coronary artery disease is dependent on the type of EIA. Sera from 112 healthy men (mean age, 50.1 years) were tested for antibodies against Chlamydia pneumoniae by five commercial test kits: Focus Chlamydia MIF IgG test, Labsystems Chlamydia pneumoniae IgG EIA (LS EIA), R-Biopharm Elegance Chlamydia pneumoniae IgG EIA (RB EIA), Medac Chlamydia pneumoniae IgG sandwich enzyme-linked immunosorbent assay ELISA (MCp sELISA) and Medac Chlamydia IgG recombinant enzyme-linked immunosorbent assay ELISA (MC rELISA). Sera from 106 consecutive male patients (mean age, 63.6 years) undergoing diagnostic coronary angiography were also examined using the Focus MIF, LS EIA, MCp sELISA, and MC rELISA techniques. The agreement between LS EIA (65 to 83% [controls-patients]) or MC rELISA (49 to 61%) and Focus MIF (78 to 83%) was average to fair (κ = 0.597 and 0.234, respectively). MCp sELISA and RB EIA showed good agreement with MIF (κ = 0.686 and 0.665, respectively), with 80 to 89 and 79% of individuals reacting positively. A significant difference in seroprevalence between patients and healthy subjects was observed with the LS EIA, while seropositivities in the two study groups appeared equal when the Focus MIF assay was applied. The MC rELISA and MCp sELISA gave statistically significant differences in antibody seroprevalence in patients with two-vessel disease or when the patient group combined individuals with a two- or a three-vessel disease, respectively. The concordance between MIF and other commonly used serological assays for C. pneumoniae IgG antibody detection is good to fair. The choice of serological assay has important implications for C. pneumoniae antibody seroprevalence, as well as for the relationship between C. pneumoniae seropositivity and coronary artery disease.
PMCID: PMC193860  PMID: 12958224
4.  Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae 
Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated.
Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating:
i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae;
ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections.
A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test.
The average diagnostic sensitivity was 56% [51.8–59.8] for the fusion protein ELISA and 84% [81.3–87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9–100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4–99.9] and 95.7% [93.8–97.2] respectively.
Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples.
Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor.
These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.
PMCID: PMC3439703  PMID: 22776779
5.  Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples▿ † 
Journal of Clinical Microbiology  2008;47(2):445-450.
We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R2 > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.
PMCID: PMC2643663  PMID: 19020058
6.  Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of Mycoplasma agalactiae 
Journal of Clinical Microbiology  2001;39(8):2814-2822.
The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two −10 boxes and a possible −35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both −10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.
PMCID: PMC88244  PMID: 11473997
7.  Neutralization receptor-based immunoassay for detection of neutralizing antibodies to Escherichia coli verocytotoxin 1. 
Journal of Clinical Microbiology  1990;28(12):2830-2833.
The NeutRELISA, a modification of the receptor enzyme-linked immunosorbent assay developed for the detection of verocytotoxin 1 (VT1) which permits the rapid detection of neutralizing antibodies (NAb) against this toxin, has been performed. A standard concentration of VT1 was preincubated with VT-immune or nonimmune rabbit serum. The serum-toxin mixtures were then added to microtiter plates coated with deacylated globotriosyl ceramide (lyso-Gb3). The reduction of VT1 binding to lyso-Gb3 in the immune serum-toxin mixtures compared with the VT1-Gb3 binding in the nonimmune serum-toxin mixtures was detected by using mouse monoclonal antibody to VT1. After standardization of the NeutRELISA with rabbit sera, 57 human control serum samples were tested to establish a cutoff value below which NeutRELISA results would be considered positive. Thirty-three single serum samples known to demonstrate NAb to VT1 by biological assay reproducibly demonstrated VT1 NAb when tested by the NeutRELISA. There was a close correlation between the biological VT1 neutralization assay and the NeutRELISA. This assay offers a practical, rapid, and reliable approach for the detection of NAb to VT1 and other verocytotoxins.
PMCID: PMC268288  PMID: 2280020
8.  Evaluation of Chlamydia immunoglobulin M (IgM), IgG, and IgA rELISAs Medac for diagnosis of Chlamydia pneumoniae infection. 
Chlamydia pneumoniae is an important pathogen responsible for a variety of respiratory diseases in humans. Cell culture remains the most specific method for C. pneumoniae diagnosis, but it is labor-intensive and time-consuming. Thus, serology, particularly microimmunofluorescence (MIF) testing, is frequently utilized. However, the MIF test has a significant subjective component. We evaluated a new serological test: Chlamydia Immunoglobulin M (IgG, IgA, and IgM rELISAs Medac, based on a recombinant Chlamydia-specific lipopolysaccharide (LPS) fragment, for the diagnosis of C. pneumoniae infection. The results of this study demonstrated that the use of rELISAs Medac with single sera does not appear to be sensitive or specific for diagnosis of C. pneumoniae infection compared to culture. In children, sensitivities of the rELISAs compared to culture did not exceed 34.2%, and the specificities ranged from 68.4% (IgG) to 91.2% (IgA). In adults, the sensitivities of the rELISAs were slightly higher, up to 77.8% (IgA or IgG), but the specificities ranged from a very low 20.8% for IgA or IgG to 81.1% for IgM. When multiple sera were tested, the results of the rELISAs Medac correlated with culture results in five of eight (62.5%) patients. However, this offers only a retrospective diagnosis, which makes it difficult to manage these patients prospectively.
PMCID: PMC170504  PMID: 9067658
9.  The indirect hemagglutination test for the detection of antibodies in cattle naturally infected mycoplasmas. 
Stable mycoplasma antigens for the indirect hemagglutination test (IHA) were prepared employing glutaraldehyde treated sheep erythrocytes sensitized with Mycoplasma agalactiae subsp. bovis and Mycoplasma bovigenitalium antigens. Employing these antigens mycoplasma antibodies were detected in sera from cattle which had mastitic symptoms due to natural infection with either M. agalactiae subsp. bovis or M. bovigenitalium. A total of 200 cows from four herds were examined at varying intervals for the presence of M. agalactiae subsp. bovis and for the detection of antibody using growth inhibition and IHA tests. Mycoplasmas were isolated from 37 animals. Growth inhibiting antibody was detected from 56 of the 200 animals. In the IHA tests, antibody titer greater than or equal to 1:80 were detected in 148 animals, 76 of these having antibody titers greater than or equal to 1:160, while sera of 116 normal control animals had no growth inhibiting antibody and none had IHA antibody titers greater than 1:40. M. bovigenitalium was isolated from the milk of three of 26 animals in a fifth herd during an outbreak of mastitis. Growth inhibiting antibodies were demonstrated in the sera of ten of the 26 animals. However, the IHA test detected antibody titers of greater than or equal to 1:160 in 13 animals and of 1:80 in one of the 26 animals. To determine the specificity of the IHA tests, M. agalactiae subsp. bovis and M. bovigenitalium antigens were reacted with rabbit hyperimmune typing sera produced against 12 species of bovine mycoplasmatales. Homologous antisera showed IHA antibody titers of 1:1280 and 1:2560 against M. agalactiae subsp. bovis and M. bovigenitalium respectively, whereas heterologous antisera showed IHA antibody titers of less than or equal to 1:20. Also eight type-specific bovine antisera were reacted with M agalactiae subsp. bovis and M. bovigenitalium antigens in homologous and heterologous tests. Homoogous reactions showed IHA antibody titers greater than or equal to 1:320, whereas heterologous reactions showed IHA titers of less than or equal to 1:20. This IHA test promises to be useful for the detection of bovine mycoplasma antibodies in sera from cattle infected with M. agalactiae subsp. bovis or M. bovigenitalium. Thes test is sensitive, reproducible and specific and the technique is relatively simple and rapid. The antigens were stable for at least seven months.
PMCID: PMC1277514  PMID: 1000374
10.  Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides  
Infection and Immunity  2002;70(1):171-176.
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.
PMCID: PMC127643  PMID: 11748179
11.  Identification of Novel Immunogenic Proteins from Mycoplasma bovis and Establishment of an Indirect ELISA Based on Recombinant E1 Beta Subunit of the Pyruvate Dehydrogenase Complex 
PLoS ONE  2014;9(2):e88328.
The pathogen Mycoplasma bovis (M. bovis) is a major cause of respiratory disease, mastitis, and arthritis in cattle. Screening the key immunogenic proteins and updating rapid diagnostic techniques are necessary to the prevention and control of M. bovis infection. In this study, 19 highly immunogenic proteins from M. bovis strain PD were identified using 2-dimensional gel electrophoresis, immunoblotting and MALDI-TOF/TOF MS. Of these 19 proteins, pyruvate dehydrogenase E1 component beta subunit (PDHB) showed excellent immune reactivity and repeatability. PDHB was found to be conserved in different M. bovis isolates, as indicated by Western blot analysis. On the basis of these results, a rPDHB-based indirect ELISA (iELISA) was established for the detection of serum antibodies using prokaryotically expressed recombinant PDHB protein as the coating antigen. The specificity analysis result showed that rPDHB-based iELISA did not react with other pathogens assessed in our study except M. agalactiae (which infects sheep and goats). Moreover, 358 serum samples from several disease-affected cattle feedlots were tested using this iELISA system and a commercial kit, which gave positive rates of 50.8% and 39.9%, respectively. The estimated Kappa agreement coefficient between the two methods was 0.783. Notably, 39 positive serum samples that had been missed by the commercial kit were all found to be positive by Western blot analysis. The detection rate of rPDHB-based iELISA was significantly higher than that of the commercial kit at a serum dilution ratio of 1∶5120 to 1∶10,240 (P<0.05). Taken together, these results provide important information regarding the novel immunogenic proteins of M. bovis. The established rPDHB-based iELISA may be suitable for use as a new method of antibody detection in M. bovis.
PMCID: PMC3919759  PMID: 24520369
12.  A survey of Mycoplasma agalactiae in dairy sheep farms in Spain 
Contagious Agalactia (CA) is one of the major animal health problems in small ruminants because of its economic significance. Currently, four Mycoplasma spp. have been associated with this syndrome: M. agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. Their presence has been evaluated in several studies conducted in CA-endemic countries. However, previous Spanish studies have been focused on caprine CA, and there is a knowledge gap regarding which Mycoplasma species are present in sheep flocks from Spain, which has the second highest number of sheep amongst the 27 European Union member states. Consequently, we investigated the presence and geographic distribution of the four CA-causing mycoplasmas in Spanish dairy sheep farms. This is the first time such an investigation has been performed.
Three hundred thirty nine out of 922 sheep flocks were positive for M. agalactiae by real time PCR (36.8%) and 85 by microbiological identification (9.2%). Interestingly, all 597 milk samples assessed for the presence of M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens tested negative. To evaluate the intermittent excretion of the pathogen in milk, we sampled 391 additional farms from 2 to 5 times, resulting that in 26.3% of the cases a previously positive farm tested negative in a later sampling.
M. agalactiae was the only Mycoplasma species detected in the study area showing a high frequency of presence and wide distribution. Therefore, the establishment of a permanent surveillance network is advantageous, as well as the implementation of control and prevention measures to hinder the dissemination of M. agalactiae and to prevent the entrance of other Mycoplasma species.
PMCID: PMC3514350  PMID: 23006445
Mycoplasma agalactiae; Contagious agalactia; Real time PCR; Sheep; Dairy; Spain
13.  Comparison of Two 3ABC Enzyme-Linked Immunosorbent Assays for Diagnosis of Multiple-Serotype Foot-and-Mouth Disease in a Cattle Population in an Area of Endemicity 
Journal of Clinical Microbiology  2004;42(5):2108-2114.
The development of a serological test for foot-and-mouth disease virus (FMDV) which is quick and easy to use, which can identify all seven serotypes, and which can differentiate vaccinated from convalescing or potential virus carriers would be a major advance in the epidemiological toolkit for FMDV. The nonstructural polyprotein 3ABC has recently been proposed as such an antigen, and a number of diagnostic tests are being developed. This paper evaluates the performance of two FMDV tests for antibodies to nonstructural proteins in an unvaccinated cattle population from a region of Cameroon with endemic multiple-serotype FMD. The CHEKIT-FMD-3ABC bo-ov (CHEKIT) enzyme-linked immunosorbent assay (ELISA) (Bommeli Diagnostics/Intervet) is a commercially available test that was compared with a competitive 3ABC ELISA (C-ELISA) developed in Denmark. The tests were compared with the virus neutralization test as the “gold standard.” Diagnostic sensitivity and specificity were examined over a range of test cutoffs by using receiver operating characteristic curves, which allowed comparison of the overall performance of each test. The results indicated that the CHEKIT ELISA kit was 23% sensitive and 98% specific and the Danish C-ELISA was 71% sensitive and 90% specific at the recommended cutoff. These results have important implications if the tests are to be used to screen herds or individual cattle in surveillance programs, at border crossings for import-export clearance, or following emergency vaccination in an outbreak situation.
PMCID: PMC404611  PMID: 15131177
14.  Detection by Two Enzyme-Linked Immunosorbent Assays of Antibodies to Ehrlichia ruminantium in Field Sera Collected from Sheep and Cattle in Ghana 
Two serological tests for detection of antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, were compared by using field sera collected from sheep and cattle as part of serosurveys in Ghana. Sera selected as either negative or positive by a new polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) were tested by the indirect MAP1-B ELISA. Cutoff values of 14 percent positivity (14 PP) for both ruminant species were obtained for the MAP1-B ELISA by using preseroconversion Ghanaian sera and were compared with previously recommended cutoff values of 29 PP for sheep and 38 PP for cattle. With the 14-PP cutoff, of 151 sheep sera which tested negative by PC-ELISA, 89% were also negative by MAP1-B ELISA, while of 419 sheep sera positive by PC-ELISA, 98% were also positive by MAP1-B ELISA. Of 261 bovine sera negative by PC-ELISA, 82% were also negative by MAP1-B ELISA. Of 511 bovine sera positive by PC-ELISA, only 47% were positive by MAP1-B ELISA; these included 168 sera collected from cattle following first seroconversion as detected by both tests, with 125 of these sera positive by PC-ELISA but only 59 and 5 positive by MAP1-B ELISA with the 14- and 38-PP cutoff levels, respectively. These results indicate that both assays are highly sensitive and specific for detection of E. ruminantium exposure in sheep but that the MAP1-B ELISA lacks sensitivity for postseroconversion bovine sera in comparison to the PC-ELISA. Both tests confirm E. ruminantium seroprevalence of at least 70% in Ghanaian sheep; levels of exposure among Amblyomma variegatum-infested Ghanaian cattle are likely to be higher than the seroprevalence value of 66% obtained with the PC-ELISA.
PMCID: PMC193896  PMID: 12965927
15.  Cross-reactivity of Haemophilus somnus antibody in agglutination and complement fixation tests and in the enzyme-linked immunosorbent assay. 
Journal of Clinical Microbiology  1983;17(3):500-506.
The specificity and sensitivity of agglutination, complement fixation, and enzyme-linked immunosorbent assay (ELISA) procedures in the detection of antibodies to Haemophilus somnus was investigated. H. somnus rabbit immune sera were found to agglutinate Pasteurella multocida, Staphylococcus aureus, and Haemophilus agni and, in some instances, also Pasteurella haemolytica, Salmonella dublin, Streptococcus agalactiae, and Corynebacterium pyogenes. In complement fixation tests with saline extracts as antigens, only H. agni reacted with H. somnus antisera to any significant degree. In ELISA tests with sonicated or heat-extracted antigens, cross-reactions were seen with the two Pasteurella spp. and with H. agni. When whole cells and saline extracts were used as antigens in ELISAs, only H. agni showed any cross-reactivity. The greatest specificity in distinguishing homologous from heterologous reactions was achieved by ELISA with saline extracts as antigens. Escherichia coli and Brucella abortus antigens failed to react with H. somnus antibody in any of the tests. A rabbit serum containing antibody to bovine type isolates of P. multocida, P. haemolytica, S. aureus, S. agalactiae, S. dublin, C. pyogenes, and E. coli gave no positive reaction in ELISA tests with saline extract of H. somnus as antigen. It is concluded that such saline extract, which appears to consist largely of H. somnus common antigen, has the potential of being a useful diagnostic reagent in the study by ELISA of antibody response to H. somnus.
PMCID: PMC272674  PMID: 6841584
16.  Brucella melitensis cell envelope protein and lipopolysaccharide epitopes involved in humoral immune responses of naturally and experimentally infected sheep. 
Journal of Clinical Microbiology  1994;32(10):2514-2522.
Cell envelope fraction (CEF) of Brucella melitensis B115 was used to investigate antibody responses of B. melitensis naturally and strain H38 experimentally infected sheep by immunoblotting, indirect enzyme-linked immunosorbent assay (ELISA) (I-ELISA), and competitive ELISA (C-ELISA) with monoclonal antibodies (MAbs). MAbs used were directed to outer membrane proteins with molecular masses of 10, 16.5, 19, 25 to 27, 31 to 34, 36 to 38, and 89 kDa; to the heat shock protein DnaK, to O-polysaccharide (O-PS) common (C) and M epitopes; and to rough lipopolysaccharide (R-LPS) epitopes. In immunoblotting, all infected sheep sera tested recognized a large number of protein bands, including the above-cited proteins and other proteins for which MAbs have not been defined. The antibody response pattern was different from one animal to another, even within the experimentally infected sheep which were infected under the same experimental conditions. A number of protein bands were recognized by the sheep sera prior to experimental infection and by other uninfected sheep sera. The antibody reactivity to these antigens and others might explain the nonspecific antibody reactivity of sera in I-ELISA with CEF. C-ELISA confirmed also the individual variability of the antibody responses of infected sheep to protein antigens. Antibody responses to O-PS C and M epitopes were detected in all experimentally infected sheep and in half of the naturally infected sheep, but these responses were also heterogeneous in relation to their intensities. Antibody responses to R-LPS epitopes detected by use of C-ELISA with the anti-R-LPS MAbs were low or negative in most of the infected animals. Despite antibody response heterogeneity for CEF antigens, immunoblot and C-ELISA results indicated that, among the CEF antigens, the O-PS epitopes (C and M epitopes) and epitopes of the major 25- to 27- and 31- to 34-kDa outer membrane proteins seem to be the most promising for detecting B. melitensis infection in sheep.
PMCID: PMC264094  PMID: 7529242
17.  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
18.  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.
PMCID: PMC193903  PMID: 12965917
19.  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.
PMCID: PMC139646  PMID: 12409404
20.  Development of a Highly Sensitive and Specific Enzyme-Linked Immunosorbent Assay Based on Recombinant Matrix Protein for Detection of Avian Pneumovirus Antibodies 
Journal of Clinical Microbiology  2000;38(11):4010-4014.
The matrix (M) protein of avian pneumovirus (APV) was evaluated for its antigenicity and reliability in an enzyme-linked immunosorbent assay (ELISA) for diagnosis of APV infection, a newly emergent disease of turkeys in United States. Sera from APV-infected turkeys consistently contained antibodies to a 30-kDa protein (M protein). An ELISA based on recombinant M protein generated in Escherichia coli was compared with the routine APV ELISA that utilizes inactivated virus as antigen. Of 34 experimentally infected turkeys, 33 (97.1%) were positive by M protein ELISA whereas only 18 (52.9%) were positive by routine APV ELISA 28 days after infection. None of the serum samples from 41 uninfected experimental turkeys were positive by M protein ELISA. Of 184 field sera from turkey flocks suspected of having APV infection, 133 (72.3%) were positive by M protein ELISA whereas only 99 (53.8%) were positive by routine APV ELISA. Twelve serum samples, which were negative by M protein ELISA but positive by routine APV ELISA, were not reactive with either recombinant M protein or denatured purified APV proteins by Western analysis. This indicates that the samples had given false-positive results by routine APV ELISA. The M protein ELISA was over six times more sensitive than virus isolation (11.5%) in detecting infections from samples obtained from birds showing clinical signs of APV infection. Taken together, these results show that ELISA based on recombinant M protein is a highly sensitive and specific test for detecting antibodies to APV.
PMCID: PMC87534  PMID: 11060061
21.  A Virus-Like Particle Enzyme-Linked Immunosorbent Assay Detects Serum Antibodies in a Majority of Women Infected With Human Papillomavirus Type 16 
Previous studies have demonstrated that genital infection with high-risk types of human papillomavirus (HPV), most often HPV16, is the most significant risk factor for the development of cervical cancer. However, serologic assays that have been developed to identify high-risk HPV infection have either failed to associate serum reactivity with other indicators of HPV infection or have identified only a minority of HPV-infected individuals.
Our purpose was to determine whether a specifically developed enzyme-linked immunosorbent assay (ELISA) could detect IgG anti-HPV16 virion antibodies in the sera of women who had tested positive for genital HPV16 infection by DNA-based methods.
An ELISA was developed using newly developed HPV16 virus-like particles as antigens to detect anti-HPV16 virion IgG antibodies. These particles are comprised of HPV16 structural proteins that are self-assembled in insect cells after expression by recombinant baculo-viruses. The sera of 122 women, whose HPV status had been previously evaluated by nucleic acid-based methods, were tested by this ELISA.
The sera of 59% of women (32 of 54) positive for genital HPV16 DNA by polymerase chain reaction (PCR) were positive in the ELISA assay compared with sera from women who had tested negative for HPV DNA (P<.0005). In contrast, 6% of HPV DNA-negative women (two of 31) and 9% of women positive for low-risk HPV6/11 DNA (one of 11) were ELISA positive by this criterion. The sera of women who were DNA positive for two additional high-risk HPV types were evaluated; the sera of 31% of HPV18-positive (four of 13) and 38% of HPV31-positive women (five of 13) were positive in the HPV16 particle ELISA. The sera of 75% of HPV16 DNA-positive women with severe dysplasias (12 of 16) gave positive ELISA results. The sera of 67% of women (28 of 42) who tested positive for HPV16 DNA by both PCR and the less sensitive ViraType assay tested positive in the ELISA compared with 33% of women (four of 12) who were positive by PCR but negative by ViraType (P<.05).
The majority of women with cervical HPV16 infection generate an IgG antibody response to conformationally dependent epitopes of HPV16 L1 that can be detected by ELISA.
This particular ELISA, or a similar one incorporating virus-like particles of additional HPV types, may be useful in determining the natural history of high-risk HPV infection and perhaps help to identify women at risk for developing cervical cancer.
PMCID: PMC3935441  PMID: 8133532
22.  Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection 
PLoS ONE  2013;8(2):e57775.
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.
PMCID: PMC3585158  PMID: 23469065
23.  Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay. 
Journal of Clinical Microbiology  1995;33(9):2405-2410.
Currently available serological tests for cowdriosis (Cowdria ruminantium infection) in domestic ruminants are hampered by their low specificities because of cross-reactivity with Ehrlichia spp. The use of recombinant major antigenic protein (MAP1) of C. ruminantium for serodiagnosis was investigated. Overlapping fragments of the MAP1 protein were expressed in Escherichia coli and were reacted with sera from sheep infected with either C. ruminantium or Ehrlichia ovina. Two immunogenic regions on the MAP1 protein, designated MAP1-A and MAP1-B, were identified. MAP1-A was reactive with C. ruminantium antisera, E. ovina antisera, and three MAP1-specific monoclonal antibodies, whereas MAP1-B reacted only with C. ruminantium antisera. An indirect enzyme-linked immunosorbent assay (ELISA) based on MAP1-B was further developed and validated with sera from animals experimentally infected with C. ruminantium or several Ehrlichia spp. Antibodies raised in sheep, cattle, and goats against nine isolates of C. ruminantium reacted with MAP1-B. Cross-reactivity with MAP1-B was limited to Ehrlichia canis and Ehrlichia chaffeensis, two rickettsias which do not infect ruminants. Antibodies to Ehrlichia spp. which do infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not react with MAP1-B. Antibody titers to C. ruminantium in sera from experimentally infected cattle, goats, and sheep were detectable for 50 to 200 days postinfection. Further validation of the recombinant MAP1-B-based ELISA was done with sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. A total of 159 of 169 samples which were considered to be false positive by immunoblotting or indirect ELISA did not react with MAP1-B.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC228424  PMID: 7494037
24.  Characterization of P40, a Cytadhesin of Mycoplasma agalactiae  
Infection and Immunity  2002;70(10):5612-5621.
An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.
PMCID: PMC128363  PMID: 12228289
25.  Development of a competitive enzyme-linked immunosorbent assay for detection of bovine, ovine, porcine, and equine antibodies to vesicular stomatitis virus. 
Journal of Clinical Microbiology  1993;31(7):1860-1865.
Two competitive (C) enzyme-linked immunosorbent assays (ELISAs) were developed for the detection of antibodies to vesicular stomatitis virus (VSV) in animal sera. The assays are based upon the availability of polyclonal antibodies (PAbs) from mouse ascitic fluids prepared against the New Jersey (NJ) and the Indiana (IN) VSV serotypes. The assays were performed by the immobilization of VSV-NJ and VSV-IN antigens on a solid phase (microtiter plate). Appropriately diluted test serum mixed with an equal volume of serotype-specific PAb was allowed to incubate in the presence of the relevant VSV antigen and finally with an enzyme-conjugated anti-mouse immunoglobulin. In the absence of anti-VSV antibodies in the test serum, the VSV antigen sites are reactive with the relevant PAb (NJ or IN) as indicated by color development after enzyme degradation of substrate. If the test serum contains the homologous VSV-NJ or VSV-IN antibodies, they compete with the relevant PAb for immobilized antigen sites and quantitatively block and inhibit the PAb reaction and subsequent color development. The performance of C-ELISAs in detecting anti-VSV antibodies in serum samples from four calves, two horses, four sheep, and seven pigs experimentally infected with VSV-NJ and VSV-IN was evaluated. The sensitivity and specificity of the C-ELISAs were compared with those of the standard microtiter serum neutralization (MTSN) tests. Homologous antibodies were demonstrable by C-ELISAs as early as 5 days postinfection (DPI) in one horse and one sheep infected with VSV-IN serotype. Seroconversion was demonstrable by C-ELISAs and MTSN tests in all animals by 9 DPI except in one sheep that received VSV-NJ and one horse inoculated with VSV-IN serotype which, on the basis of the MTSN test results, did not seroconvert until 14 and 11 DPI, respectively. The dynamics of homologous antibody response in all animals as revealed by the corresponding type-specific C-ELISAs paralleled the results of the MTSN tests. The type-specific antibodies to VSV serotypes increased exponentially during the first 2 to 4 weeks postinfection and remained relatively stable for about 6 months in some animals. The results suggest that the C-ELISAs offer many advantages over the MTSN tests and have potential applications as rapid and inexpensive tests in serodiagnosis of VSV infections in animals.
PMCID: PMC265646  PMID: 8394377

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