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.     

Development of a Polyclonal Competitive Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Ehrlichia ruminantium

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater.     

Validation of the Indirect MAP1-B Enzyme-Linked Immunosorbent Assay for Diagnosis of Experimental Cowdria ruminantium Infection in Small Ruminants

The major antigenic protein 1 fragment B (MAP1-B) enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Cowdria ruminantium infections was validated to determine cutoff values and evaluate its diagnostic performance with sheep and goat sera. Cowdria-infected populations consisted of 48 sheep and 44 goats, while the noninfected populations consisted of 64 sheep and 107 goats. Cutoff values were determined by two-graph receiver-operating characteristic (TG-ROC) curves. The cutoff value was set at 31 and 26.6% of the positive control reference samples for sheep and goat sera, respectively. The test’s diagnostic performance was evaluated with measurements of the area under the concentration-time curve (AUC) of the ROC curves and by the valid range proportion (VRP). The AUCs were 0.978 for sheep sera and 0.989 for goat sera. The VRP for both sheep and goat sera was approximately 1.0. The intermediate range (IR), which defines results that are neither positive nor negative, was 0 for goat sera and 2.81 for sheep sera. In an ideal test, the AUC and VRP would be 1.0 and the IR would be 0. In this study these parameters were close to those of an ideal test. It is concluded that the MAP1-B ELISA is a useful test for the diagnosis of C. ruminantium infection in small ruminants.     

Development of a polyclonal competitive enzyme-linked immunosorbent assay for detection of antibodies to Ehrlichia ruminantium

A polyclonal competitive enzyme-linked immunosorbent assay (PC-ELISA) is described for detection of antibodies to Ehrlichia (Cowdria) ruminantium by using a soluble extract of endothelial cell culture-derived E. ruminantium as the antigen and biotin-labeled polyclonal goat immunoglobulins as the competitor. For goats, the diagnostic sensitivity and specificity were both 100% with a cutoff of 80% inhibition (80 PI), with detection of antibodies for 550 days postinfection. For cattle, diagnostic sensitivity and specificity were 86 and 100%, respectively, with a cutoff of 50 PI and 79 and 100% with a cutoff of 70 PI. Cross-reactions with high-titer experimental or field antisera to other Ehrlichia and Anaplasma species were observed at up to 68 PI in cattle and up to 85 PI in sheep, and therefore to exclude these cross-reactions, cutoffs of 70 PI for bovine serology and 85 PI for small-ruminant serology were selected. Application of the PC-ELISA to bovine field sera from South Africa gave a higher proportion of positive results than application of the murine macrophage immunofluorescent antibody test or indirect ELISA, suggesting a better sensitivity for detection of recovered cattle, and results with bovine field sera from Malawi were consistent with the observed endemic state of heartwater and the level of tick control practiced at the sample sites. Reproducibility was high, with average standard deviations intraplate of 1.2 PI and interplate of 0.6 PI. The test format is simple, and the test is economical to perform and has a level of sensitivity for detection of low-titer positive bovine sera that may prove to be of value in epidemiological studies on heartwater.     

Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay.

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)     

Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater

Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C. ruminantium antigens are down regulated in cattle despite repeated exposure to C. ruminantium via ticks. Significantly, serological responses to the MAP 1B antigen may not be a reliable indicator of C. ruminantium exposure in cattle in areas of endemic heartwater infection.     

Development of Rapid Immunochromatographic Test with Recombinant NcSAG1 for Detection of Antibodies to Neospora caninum in Cattle

An immunochromatographic test (ICT) with recombinant surface antigen 1 of Neospora caninum (NcSAG1) was developed for the rapid detection of antibodies to N. caninum in cattle. The ICT was used to clearly discriminate between immunofluorescent-antibody test (IFAT)-positive bovine sera and IFAT-negative bovine sera. Serum samples collected from cattle in Yanbian, China, were examined by the ICT. Of the 96 serum samples, 23 (24.0%) were positive by the ICT, and 19 (19.8%) samples were positive by a previously developed enzyme-linked immunosorbent assay (ELISA). Eighteen of 19 ELISA-positive samples were positive according to the ICT. A good agreement was found between the results of the ICT and the ELISA. The results presented here suggest that the ICT with recombinant truncated NcSAG1 fused to glutathione S-transferase is a useful and reliable method for the detection of antibodies to N. caninum in cattle.     

High Prevalence of Human Anti‐bovine IgG Antibodies as the Major Cause of False Positive Reactions in Two‐Site Immunoassays Based on Monoclonal Antibodies

Abstract

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n?=?54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities—a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti‐animal IgG (bovine, mouse, horse, and swine) antibodies and human anti‐bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti‐bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n?=?54) were positively correlated to the false positive signals in the PP14 ELISA (r?=?0.923; p?<?0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti‐bovine serum albumin antibodies in the donor sera (r?=?0.639; p?<?0.0001; n?=?103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two‐site immunoassays. The apparent presence of human anti‐mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Competitive Enzyme-Linked Immunosorbent Assay for Detection of Leptospira interrogans Serovar pomona Antibodies in Bovine Sera

A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to Leptospira interrogans serovar pomona. This assay was evaluated using field sera (n = 190) with serovar pomona microscopic agglutination test (MAT) titers of ≥100 as the positive population (group A); field sera (n = 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [n = 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.     

High prevalence of human anti-bovine IgG antibodies as the major cause of false positive reactions in two-site immunoassays based on monoclonal antibodies

A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p < 0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti-bovine serum albumin antibodies in the donor sera (r = 0.639; p < 0.0001; n = 103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two-site immunoassays. The apparent presence of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.     

Comparison between microscopic agglutination test (MAT) and enzyme-linked immunosorbent assay (ELISA) for detection of leptospiral antibodies in cattle

Leptospirosis is a worldwide zoonosis caused by Leptospira interrogans. It causes financial loss to the cattle industry with decreased milk production, abortion, stillbirth, infertility, and mortality. This study was conducted to evaluate two serological techniques, the microscopic agglutination test (MAT) and the enzyme-linked immunosorbent assay (ELISA), for detection of leptospiral antibodies in cattle. Three hundred and fifty five serum samples were collected from five commercial dairy farms in Tehran suburb from December 2004 to June 2005. The results of the MAT protocol showed that 55 serum samples (15.49%) had detectable antibodies against at least one serovar of L. interrogans at a dilution of ≥ 1:100. Icterohaemorrhagiae was the most (42.52%) and ballum the least (1.15%) prevalent serovars among 87 positive reactions against different serovars of L. interrogans. Leptospiral antibodies were also detected by the ELISA in 77 sera (21.69%) of 355 samples. The results of this study also showed that 2.25% of the sera that reacted positive in the MAT were negative by the ELISA, and 8.45% of the MAT negative sera were positive by the ELISA. Comparison between two techniques showed that there was a high correlation (0.61 < kappa?=?0.649 < 0.8) between the ELISA and MAT for detection of leptospiral antibodies in cattle.     

Occurrence and Potential Diagnostic Applications of Serological Cross-Reactivities between Brucella and Other Alpha-Proteobacteria

Agrobacterium, Sinorhizobium, and Ochrobactrum are genera closely related to Brucella but, in contrast to the latter, are not pathogenic for humans and animals. We studied by an indirect enzyme-linked immunosorbent assay (ELISA) the reactivities of brucellosis sera against cytosolic (CYT) and membrane (MA) antigens from these nonpathogenic bacteria, and we evaluated the potential usefulness of these cross-reactions for the diagnosis of brucellosis in humans, sheep, cows, and dogs. Canine infection by Brucella canis was detected with high specificity by CYT antigen-based ELISAs (96% for Agrobacterium, 96% for Sinorhizobium, and 91% for Ochrobactrum), while sensitivity was variable (58% for Agrobacterium, 88% for Sinorhizobium, and 84% for Ochrobactrum). In addition, it was possible to diagnose canine disease shortly after exposure to the pathogen (15 days). Similar results for canine brucellosis were obtained with MA antigens. In contrast, normal sera from humans, sheep, and cattle reacted strongly with all the antigens (CYT and MA antigens from the three bacteria), producing high cutoff values and, consequently, low sensitivities. While for some host species the reactivity patterns of normal sera by Western blotting were similar to those produced with sera from infected individuals, the reactivity pattern of bovine sera against Sinorhizobium meliloti antigens exhibited some differential bands for the two groups of sera. These results show that crude fractions from nonpathogenic alpha-proteobacteria can be used to diagnose canine brucellosis but may need to be further separated into simpler fractions to have diagnostic usefulness in ovine, bovine, or human infection. By reducing the biosafety requirements, the use of antigens derived from these nonpathogenic bacteria would simplify the production of diagnostic kits for brucellosis, especially in settings where biosafety level-3 facilities are scarce or absent.     

Blocking ELISA for distinguishing infectious bovine rhinotracheitis virus (IBRV)-infected animals from those vaccinated with a gene-deleted marker vaccine.

A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of > 0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N < 0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values > 0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a borderline (+/-) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115/116 sera with VN titers of 1:2 to 1:256 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48.     

Comparison of Two Recombinant Major Outer Membrane Proteins of the Human Granulocytic Ehrlichiosis Agent for Use in an Enzyme-Linked Immunosorbent Assay

Enzyme-linked immunosorbent assay (ELISA) for human granulocytic ehrlichiosis (HGE) using two different recombinant P44 proteins (rP44 and rP44-2hv) of the HGE agent as antigens was evaluated. Sera from a total of 72 healthy humans both from regions where HGE is nonendemic and regions where HGE is endemic were used as negative controls to determine the cutoff value for ELISA. Sera from a total of 14 patients (nine from whom the HGE agent was isolated and five who were HGE-PCR positive) were used as positive controls. One hundred nine sera from 72 patients in an area where HGE is endemic who were suspected of having HGE were examined by ELISA and indirect immunofluorescence assay (IFA). All IFA-negative sera were negative by both ELISAs. Of 39 sera that were IFA positive, 35 and 27 were positive by ELISA using rP44 and rP44-2hv, respectively, indicating that the use of rP44 is more sensitive. Western blot analysis of the four rP44-ELISA-negative IFA-positive sera using whole HGE agent as antigen suggests that these four sera were false IFA positive. There was no difference in results with or without the preabsorption of sera with Escherichia coli or with or without the cleavage of the fused protein derived from the vector. There was a significant positive correlation between IFA titers and optical densities of ELISAs. Four Ehrlichia chaffeensis-positive and 10 Borrelia burgdorferi-positive sera were negative by ELISA. However, two Babesia microti-positive sera showed strong cross-reactivity to the fused vector protein, which was eliminated after cleavage of the protein. Thus, ELISA using rP44 nonfusion protein would provide a simple, specific, and objective HGE serologic test which can be easily automated.     

Development of an Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Ringworm Infection in Cattle

The aim of this study was to develop an in-house enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of ringworm infection in cattle. We used available recombinant forms of Trichophyton rubrum dipeptidyl peptidase V (TruDppV) and T. rubrum leucin aminopeptidase 2 (TruLap2), which are 98% identical to Trichophyton verrucosum orthologues. Field serum samples from 135 cattle with ringworm infection, as confirmed by direct microscopy, fluorescence microscopy, and PCR, and from 55 cattle without any apparent skin lesions or history of ringworm infection that served as negative controls were used. Sensitivities, specificities, and positive and negative predictive values were determined to evaluate the diagnostic value of our ELISA. Overall, the ELISAs based on recombinant TruDppV and TruLap2 discriminated well between infected animals and healthy controls. Highly significant differences (P < 0.0001, Mann-Whitney U test) were noted between optical density values obtained when sera from infected versus control cattle were tested. The ELISA developed for the detection of specific antibodies against DppV gave 89.6% sensitivity, 92.7% specificity, a 96.8% positive predictive value, and a 78.4% negative predictive value. The recombinant TruLap2-based ELISA displayed 88.1% sensitivity, 90.9% specificity, a 95.9% positive predictive value, and a 75.7% negative predictive value. To the best of our knowledge, this is the first ELISA based on recombinant antigens for assessing immune responses to ringworm infection in cattle; it is particularly suitable for epidemiological studies and also for the evaluation of vaccines and/or vaccination procedures.     

Bovine respiratory syncytial virus antibodies in non-bovine species

Summary To study the role of non-bovine species in the epidemiology of bovine respiratory syncytial virus (RSV) infections, sera obtained from 9 non-bovine animal species and from humans were examined for bovine RSV specific antibodies. Sera were mainly from animals and humans which had been in contact with cattle. Forty sera of each species were tested in an RSV specific whole virus ELISA as well as in a peptide based ELISA, that was developed to measure antibodies specific for bovine RSV. Antibodies directed against RSV were detected in over 50% of sera obtained from sheep, goat, cattle and human beings, and anti-RSV activity was also found in some roe and dogs and one horse. Antibodies to bovine RSV were found in sera of all tested cattle, 11 (27.5%) goats and in some other individual animals: 3 horses, 2 roe, 1 cat and 1 dog. These results indicate that of the investigated species, besides cattle only goats might play a role in the epidemiology of bovine RSV.     

Cloning of the Bovine Immunodeficiency Virus gag Gene and Development of a Recombinant-Protein-Based Enzyme-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) was established for the rapid detection of specific bovine immunodeficiency virus (BIV) antibodies in cattle, using recombinant Gag protein as an antigen. The gag coding region from BIV was cloned into an expression vector, pQE32, which expressed high levels of recombinant protein from Escherichia coli. The ELISA was standardized by a checkerboard titration against known BIV-positive and -negative sera from cattle and a monoclonal antibody to the Gag protein. A total of 139 cattle serum samples, from the diagnostic laboratory at Kansas State University, Manhattan, and from the Dairy Station, Louisiana State University, Baton Rouge, were compared by ELISA and immunoblot assays for the detection of BIV-specific antibodies. Of 26 cattle sera samples which tested positive using the immunoblot assay, 23 were positive by ELISA, thus establishing a strong correlation between the two tests. The sensitivity and specificity of ELISA relative to immunoblotting were 0.88 and 0.93, respectively. ELISA proved to be as specific as immunoblotting but was much less time-consuming and easier to perform.     

Potential Value of Major Antigenic Protein 2 for Serological Diagnosis of Heartwater and Related Ehrlichial Infections

Cowdria ruminantium is the etiologic agent of heartwater, a disease causing major economic loss in ruminants in sub-Saharan Africa and the Caribbean. Development of a serodiagnostic test is essential for determining the carrier status of animals from regions where heartwater is endemic, but most available tests give false-positive reactions with sera against related Erhlichia species. Current approaches rely on molecular methods to define proteins and epitopes that may allow specific diagnosis. Two major antigenic proteins (MAPs), MAP1 and MAP2, have been examined for their use as antigens in the serodiagnosis of heartwater. The objectives of this study were (i) to determine if MAP2 is conserved among five geographically divergent strains of C. ruminantium and (ii) to determine if MAP2 homologs are present in Ehrlichia canis, the causative agent of canine ehrlichiosis, and Ehrlichia chaffeensis, the organism responsible for human monocytic ehrlichiosis. These two agents are closely related to C. ruminantium. The map2 gene from four strains of C. ruminantium was cloned, sequenced, and compared with the previously reported map2 gene from the Crystal Springs strain. Only 10 nucleic acid differences between the strains were identified, and they translate to only 3 amino acid changes, indicating that MAP2 is highly conserved. Genes encoding MAP2 homologs from E. canis and E. chaffeensis also were cloned and sequenced. Amino acid analysis of MAP2 homologs of E. chaffeensis and E. canis with MAP2 of C. ruminantium revealed 83.4 and 84.4% identities, respectively. Further analysis of MAP2 and its homologs revealed that the whole protein lacks specificity for heartwater diagnosis. The development of epitope-specific assays using this sequence information may produce diagnostic tests suitable for C. ruminantium and also other related rickettsiae.     

Diagnostic accuracy of a Mycobacterium phlei-absorbed serum enzyme-linked immunosorbent assay for diagnosis of bovine paratuberculosis in dairy cows.

The purpose of this study was to describe the responses of sera from five groups of cattle to an enzyme-linked immunosorbent assay (ELISA) for paratuberculosis by using serum absorbed with Mycobacterium phlei at a single working dilution. The infection status of the cattle was determined by fecal culture. Cattle with different levels of exposure (high versus low prevalence and test negative) and disease manifestation (clinically suspect infection versus subclinical infection) were examined, as follows: (i) two paratuberculosis-negative herds; (ii) a fecal culture-confirmed, clinically suspect cases of paratuberculosis; (iii) cows from a paratuberculosis-infected herd with a high infection rate, as determined by fecal culture, but with no clinical cases at the time of sampling; (iv) cows from three paratuberculosis-infected herds known to have paratuberculosis diagnosed on the farm (low infection rate determined by fecal culture); and (v) one fecal culture-negative herd with known serologically positive cattle. Results generally showed a decreased ELISA response when absorbed rather than nonabsorbed serum from each animal was used. The results of the fecal culture confirmed clinically suspect cases, which were analyzed in relation to the amount of colonies isolated from the animals on fecal culture (0, +, ++,+++ , ++++, and above). There was a significant increase in the ELISA response for animals with heavy Mycobacterium paratuberculosis shedding ( ++++ or above), when both unabsorbed and absorbed sera were used, compared with the response in animals that were fecal culture negative or that shed M. paratuberculosis at lower levels (less than +) (P less than 0.05). The effects on sensitivity and specificity by using different cutoff points for the five groups of cattle with different levels of exposure is described, since sera were not discretely segregated into distinct groups of positive and negative samples. The specificity of the ELISA in the two fecal culture-negative herds was 100% at an ELISA cutoff of an optical density (OD) of 0.1 and above for absorbed serum. For unabsorbed serum the specificity was 62.9% at a similar cutoff value. Similarly, the specificity of the fecal culture-negative, serologically positive herd increased from 37.5 to 72.2 at an ELISA cutoff value of 0.1 to 0.2 (OD) by using absorbed versus unabsorbed serum from 75.0 to 94.4 at an ELISA cutoff value of 0.2 to 0.3 (OD).     

Crohn’s disease-specific pancreatic autoantibodies are specifically present in ruminants with paratuberculosis: Implications for the pathogenesis of the human disease

Abstract

Mycobacterium avium subspecies paratuberculosis (MAP) induces paratuberculosis (ptb) in ruminants and has clinical and histological features resebling Crohn’s disease (CD). Pancreatic autoantibodies (PAB) targeting glycoprotein 2 (GP2) are specifically found in CD, but it is currently unknown whether these autoantibodies can be found in ruminants with ptb. IgG anti-MAP and anti-GP2 antibodies were tested by ELISA in 286 ruminants (212 sheep and 74 cattle). PAB testing was performed by indirect immunofluorescence (IIF) using anti-sheep or anti-cattle specific antisera. PCR analysis confirmed the presence of MAP in anti-MAP positive samples. Anti-GP2 antibodies were more prevalent in anti-MAP antibody positive (26.9%) than in anti-MAP negative ruminants (8.7%, p?<?0.001). Anti-GP2 antibodies were found in 16/70 (22.9%) anti-MAP positive sheep compared to 10/142 (7%, p?=?0.001) anti-MAP antibody negative and in anti-MAP positive cattle than in negative counterparts (5/8 versus 8/66, p?=?0.003). Absorbance values for anti-GP2 antibodies were higher in cattle than in sheep (mean 21 AU/mL?±?25.4SD versus 12.2 AU/mL?±?23 SD, p?<?0.001). There was no correlation between anti-GP2 and anti-MAP antibody concentrations. Anti-GP2 antibodies persisted up to 1/1000 and showed the characteristic IIF pancreatic pattern seen by anti-GP2 antibody positive CD samples. This is the first study to demonstrate the presence of CD-specific GP2-reactive pancreatic autoantibodies in MAP-infected ruminants. Our data suggest that CD and ptb are characterised by an antigen-driven loss of immunological tolerance to GP2, implying commonalities in the immunopathogenesis of the human and ruminant inflammatory bowel disorder.