DOI: 10.5061/dryad.9zw3r22t9
title: Development and validation of enzyme-linked immunosorbent assays for the serodiagnosis of canine Bartonelloses

description: Bartonella species, emerging vector-borne pathogens of dogs, are
 increasingly associated with severe, chronic sequelae, as well as
 potentially life-threatening diseases such as endocarditis and
 myocarditis. Diagnosis of Bartonelloses is mainly based on PCR, culture,
 and serological assays. Despite molecular and biotechnological advances,
 serological assays employing Immunofluorescence antibody (IFA), Western
 Blotting (WB), and enzyme-linked-immunosorbent-assay (ELISA) technologies
 have encountered diagnostic limitations, primarily due to poor
 sensitivity. Using sera from Bartonella-infected and naïve dogs, we
 applied an immunoproteomic approach to develop a reliable ELISA assay for
 the diagnosis of Bartonelloses in dogs. Five recombinant Bartonella
 henselae immunodominant proteins (rATP-β, rGroEL, rLemA, rSucB,
 and rVirB5) were tested in an ELISA format. Dogs comprised Group I: 36
 Bartonella spp. naturally infected dogs (all B. henselae IFA seroreactive)
 and Group II: 34 Bartonella spp. PCR negative and IFA negative dogs. Based
 upon the ELISA seroreactivity results, rATP-β and rGroEL represented the
 most sensitive and specific candidate peptide targets for utilization in a
 canine diagnostic ELISA assay. rGroEL resulted in the sensitivity of 83%
 and specificity of 94% at an OD cutoff value of 0.439 and AUC score of
 0.93 (95% CI 0.87-0.99), while the sensitivity and specificity of rATP-β
 was 69% and 94%, respectively, at a cutoff value of 0.565. The combination
 of rATP-β with rGroEL resulted in an improved sensitivity of 88% and
 specificity of 92% at an OD cutoff value of 0.505. A ROC curve analysis
 for the rATP-β plus rGroEL yielded an AUC score of 0.899 (95% CI
 0.809-0.989). Combining rATP-β with rGroEL could potentially further
 improve both the diagnostic sensitivity and specificity of an ELISA assay
 for diagnosis of canine Bartonelloses. 
description: # Development and validation of enzyme-linked immunosorbent assays for the
 serodiagnosis of canine Bartonelloses We have submitted our raw data
 (**Dogs_ELISA_OD_data.csv**), rATP-β analysis (**rATP-β_analysis.csv**),
 rATP-β plus rGroEL analysis (**rATP-β_plus_rGroEL_analysis.csv**), rGroEL
 analysis (**rGroEL_analysis.csv**), rLemA analysis (**rLemA
 analysis.csv**), rSucB analysis (**rSucB_analysis.csv**), and rVirB5
 analysis (**rVirB5_analysis.csv**). [Access this dataset on
 Dryad](https://doi.org/10.5061/dryad.9zw3r22t9) In this study, we
 evaluated the diagnostic utility of B. henselae recombinant proteins by
 ELISA using sera from 70 dogs (36 Bartonella spp. IFA-positive (naturally
 infected) and 34 Bartonella spp. IFA-negative and PCR-negative (control
 dogs). ELISA optical density (OD) values were measured at 450 nm. The data
 and analysis files are standalone and can be retrieved or used without any
 additional dependencies. ### Dogs_ELISA_OD_data.csv This file contains the
 immunofluorescence antibody assay (IFA), Bartonella species PCR,
 Bartonella alpha Proteobacteria growth medium (BAPGM) enrichment blood
 culture, and enzyme-linked immunosorbent assay (ELISA) results for all
 dogs in Group I (Bartonella henselae IFA-positive naturally infected dogs)
 and Group II (Bartonella species PCR-negative and IFA-negative control
 dogs). Sera were initially screened at 1:16 to 1:64 dilutions. Two-fold
 serial dilutions of reactive sera ranging from 1:16 to 1:8192 were then
 tested and IFA titer was determined as the highest dilution of the
 dogs' serum that shows detectable fluorescence.A cutoff titer of
 ≥1:64 was used to define an IFA seropositive titer. All 34 Group II
 control dogs were previously tested IFA negative (titers, ≤1:16 screening
 dilution) for three Bartonella spp. (B. henselae, B. vinsonii subsp.
 berkhoffii, and B. koehlerae). Therefore, these dogs were presumably
 considered to be unexposed to Bartonella spp. IFA titers of ≥1:64 were
 considered positive in IFA tests. Bh = B. henselae; Bvb = B. vinsonii
 subsp. Berkhoffii; Bk= B. koehlerae R. rickettsii = Rickettsia rickettsii;
 E. canis = Ehrlichia canis; B. canis = Babesia canis; B. gibsoni= Babesia
 gibsoni. Five recombinant Bartonella henselae immunodominant proteins
 (rATP-β, rGroEL, rLemA, rSucB, and rVirB5) were tested in an ELISA format.
 Since rATP-β and rGroEL were the most reactive proteins, we used a
 combination of rATP-β and rGroEL (rATP-β plus r GroEL) to test 34 Group I
 (inadequate volume for 2/36 Group I sera) and 34 Group II control dogs by
 ELISA. ELISA optical density (OD) readings were measured at 450 nm. ELISA
 OD refers to the absorbance values measured in an ELISA test. The average
 OD value was calculated for each set of duplicate samples. ## Six Analysis
 files (rATP-β__analysis.csv, rATP-β_plus_rGroEL_analysis.csv,
 rGroEL_analysis.csv, rLemA_analysis.csv, rSucB_analysis.csv,
 rVirB5_analysis.csv) **All these analyses were accomplished using the
 following formulas:** True Positive Rate(TPR)= True Positive(TP)/(TP+
 False Negative(FN)) True Negative Rate(TNR)= True Negative(TN)/(TN+ False
 Positive(FP)) False Positive Rate(FPR)= FP/(FP+TN) False Negative
 Rate(FNR)= FN/(FN+TP) OD cutoff value in column A is a threshold value
 used to determine the TP,TN, FP, and FN. ## Sharing/Access information N/A
 ## Code/Software Python 3. 6.13 in Jupyterlab 3.2.1 R version 3.6.1 in R
 studio 1.1.456 \---------------------End of the
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