Assessment of a fluorescent antibody test for the detection of antibodies against epizootic bovine abortion

Abstract

The current study was directed at developing and validating an indirect fluorescent antibody test (IFAT) capable of detecting antibodies specific for the agent of epizootic bovine abortion (aoEBA). Sensitivity and specificity was determined by comparing antibody titers from 114 fetuses infected with aoEBA with 68 fetuses diagnosed with alternate infectious etiologies. Data established specificity at 100% and sensitivity at 94.7% when cutoff criteria for a positive test were assigned at a titer of ≥1,000. Potential cross-reactivity was noted in samples from 3 fetuses with antibody titers of 10 or100; all were infected with Gram-positive organisms. The remaining 65 fetuses infected with microbes other than aoEBA, and an additional 12 negative reference sera, did not have detectable titers. The IFAT-based serology assay is rapid, reproducible, and unaffected by fluid color or opacity. Total fetal immunoglobulin (Ig)G was also evaluated as an aid for diagnosing EBA. Significantly higher concentrations of IgG were identified in fetuses infected with aoEBA as compared to those with alternate infectious etiologies. The presence of IgG is a sensitive indicator of EBA and increases the specificity of FAT-based serologic diagnosis when titers are 10 or 100. Taken together, serology and IgG analyses suggest that the incidence of EBA may be underestimated.

Protection of Cattle against Epizootic Bovine Abortion (EBA) Using a Live Pajaroellobacter abortibovis Vaccine

“…The P. abortibovis-specific IFAT assay described in these studies was reported to be >98% sensitive and >95% specific when diagnostically applied to bovine fetal fluids [29]; a small percentage of fetal fluid samples were believed to contain low-titer cross-reactive antibodies. Sensitivity and specificity of the IFAT assay have yet to be defined for mature animals.…”

“…Samples were also collected from heifers in Trial #4 at 0 DPV (n = 58) and on day of challenge at either 146 or 181 DPV (n = 34). An indirect fluorescent antibody test (IFAT) was conducted as previously described [29]. Briefly, single-cell suspensions of P. abortibovis-infected SCID mice splenocytes, adjusted to a final concentration of 3.3 × 10 7 cells/mL with PBS, were dispersed evenly over each well surface of 12-well Teflon-coated slides, fixed in acetone, air-dried and stored at −20 • C. Fluid samples were serially diluted with PBS, added to antigen-coated wells and incubated at room temperature for 20 min.…”

“…Fetal fluids were tested for antibodies directed against bovine herpesvirus-1 (infectious bovine rhinotracheitis; IBR), bovine viral diarrhea virus (BVDv), Neospora caninum and Leptospira spp. Assays to support EBA diagnoses included macro-and microscopic lesions [7,27] and elevated serum IgG [29,30] as well as P. abortibovis-specific immunohistochemistry (IHC; [19]), duplex PCR [26] and IFAT [29].…”

Genome Report: Identification and Validation of Antigenic Proteins from Pajaroellobacter abortibovis Using De Novo Genome Sequence Assembly and Reverse Vaccinology

“…Sera b, c, and d are referred to as immune sera and e, f, and g as EBA-negative controls. The presence of P. abortibovis antibody was confirmed in serum from P. abortibovis -exposed animals and absence in those with no known exposure by indirect fluorescent antibody test (IFAT) as previously described ( Blanchard et al 2014 ). Antibody binding was detected with alkaline phosphatase-conjugated anti-mouse IgG (H&L) (Vector Laboratories, Burlingame, CA) or anti-bovine IgG (H&L) (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as appropriate, followed by NBT/BCIP substrate (Roche Diagnostics, Indianapolis, IN).…”

“…Identification of this Deltaproteobacterium, Pajaroellobacter abortibovis , allowed for the development of new diagnostic methodologies ( Blanchard et al 2010 , 2014 ; Brooks et al 2011 ). The visualization of P. abortibovis through immunohistochemistry and DNA-based diagnostic techniques using PCR have increased diagnostic sensitivity ( Anderson et al 2006 ; Brooks et al 2016 ).…”