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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.
doi:10.1128/JCM.41.9.4049-4053.2003
PMCID: PMC193860  PMID: 12958224
4.  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.
doi:10.1128/JCM.01442-08
PMCID: PMC2643663  PMID: 19020058
5.  Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1746-6148-8-109
PMCID: PMC3439703  PMID: 22776779
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.
doi:10.1128/JCM.39.8.2814-2822.2001
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.  A survey of Mycoplasma agalactiae in dairy sheep farms in Spain 
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1746-6148-8-171
PMCID: PMC3514350  PMID: 23006445
Mycoplasma agalactiae; Contagious agalactia; Real time PCR; Sheep; Dairy; Spain
10.  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
11.  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.
doi:10.1128/IAI.70.1.171-176.2002
PMCID: PMC127643  PMID: 11748179
12.  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.
doi:10.1128/JCM.42.5.2108-2114.2004
PMCID: PMC404611  PMID: 15131177
13.  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.
doi:10.1128/CDLI.10.5.917-925.2003
PMCID: PMC193896  PMID: 12965927
14.  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.
doi:10.1371/journal.pone.0088328
PMCID: PMC3919759  PMID: 24520369
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.
Images
PMCID: PMC264094  PMID: 7529242
17.  Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae 
The sip gene encoding for a conserved highly immunogenic surface protein of Streptococcus agalactiae was amplified using polymerase chain reaction (PCR) and subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in E. coli BL21 (DE3). An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified Sip protein as a coating antigen, which could identify S. agalactiae specific antibody in sera. The coating antigen at a concentration of 3.125 μg/ml, serum diluted to 1:160, and HRP-conjugated secondary antibody concentration at 1:4000 was found to be most effective in exhibiting positive result. The ELISA was found to be highly specific for S. agalactiae that may be used for the detection of the pathogen in mastitis cases, for epidemiological studies and for surveillance.
PMCID: PMC4782699  PMID: 27175190
Bovine mastitis; ELISA; Streptococcusagalactiae
18.  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
19.  Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro 
PLoS ONE  2016;11(9):e0163603.
Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae’s induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.
doi:10.1371/journal.pone.0163603
PMCID: PMC5035028  PMID: 27662492
20.  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.
doi:10.1371/journal.pone.0057775
PMCID: PMC3585158  PMID: 23469065
21.  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.
doi:10.1128/IAI.70.10.5612-5621.2002
PMCID: PMC128363  PMID: 12228289
22.  Evaluation of three 3ABC ELISAs for foot-and-mouth disease non-structural antibodies using latent class analysis 
Background
Foot-and-mouth disease (FMD) is a highly contagious viral disease of even-toed ungulates. Serological diagnosis/surveillance of FMD presents several problems as there are seven serotypes worldwide and in the event of vaccination it may be necessary to be able to identify FMD infected/exposed animals irrespective of their vaccination status. The recent development of non-structural 3ABC protein (NSP) ELISA tests has greatly advanced sero-diagnosis/surveillance as these tests detect exposure to live virus for any of the seven serotypes of FMD, even in vaccinated populations. This paper analyses the performance of three NSP tests using a Bayesian formulation of the Hui-Walter latent class model to estimate test sensitivity and specificity in the absence of a "gold-standard" test, using sera from a well described cattle population in Cameroon with endemic FMD.
Results
The analysis found a high sensitivity and specificity for both the Danish C-ELISA and the World Organisation for Animal Health (O.I.E.) recommended South American I-ELISA. However, the commercial CHEKIT kit, though having high specificity, has very low sensitivity. The results of the study suggests that for NSP ELISAs, latent class models are a useful alternative to the traditional approach of evaluating diagnostic tests against a known "gold-standard" test as imperfections in the "gold-standard" may give biased test characteristics.
Conclusion
This study demonstrates that when applied to naturally infected zebu cattle managed under extensive rangeland conditions, the FMD ELISAs may not give the same parameter estimates as those generated from experimental studies. The Bayesian approach allows for full posterior probabilities and capture of the uncertainty in the estimates. The implications of an imperfect specificity are important for the design and interpretation of sero-surveillance data and may result in excessive numbers of false positives in low prevalence situations unless a follow-up confirmatory test such as the enzyme linked immunoelectrotransfer blot (EITB) is used.
doi:10.1186/1746-6148-2-30
PMCID: PMC1629010  PMID: 17042948
23.  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
24.  In vitro and in vivo cell invasion and systemic spreading of Mycoplasma agalactiae in the sheep infection model 
Generally regarded as extracellular pathogens, molecular mechanisms of mycoplasma persistence, chronicity and disease spread are largely unknown. Mycoplasma agalactiae, an economically important pathogen of small ruminants, causes chronic infections that are difficult to eradicate. Animals continue to shed the agent for several months and even years after the initial infection, in spite of long antibiotic treatment. However, little is known about the strategies that M. agalactiae employs to survive and spread within an immunocompetent host to cause chronic disease. Here, we demonstrate for the first time its ability to invade cultured human (HeLa) and ruminant (BEND and BLF) host cells. Presence of intracellular mycoplasmas is clearly substantiated using differential immunofluorescence technique and quantitative gentamicin invasion assays. Internalized M. agalactiae could survive and exit the cells in a viable state to repopulate the extracellular environment after complete removal of extracellular bacteria with gentamicin. Furthermore, an experimental sheep intramammary infection was carried out to evaluate its systemic spread to organs and host niches distant from the site of initial infection. Positive results obtained via PCR, culture and immunohistochemistry, especially the latter depicting the presence of M. agalactiae in the cytoplasm of mammary duct epithelium and macrophages, clearly provide the first formal proof of M. agalactiae's capability to translocate across the mammary epithelium and systemically disseminate to distant inner organs. Altogether, the findings of these in vitro and in vivo studies indicate that M. agalactiae is capable of entering host cells and this might be the strategy that it employs at a population level to ward off the host immune response and antibiotic action, and to disseminate to new and safer niches to later egress and once again proliferate upon the return of favorable conditions to cause persistent chronic infections.
doi:10.1016/j.ijmm.2014.07.011
PMCID: PMC4282308  PMID: 25129554
Mycoplasma agalactiae; Cell invasion; Systemic spreading; Persistence; Immunohistochemistry; Intracellular
25.  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

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