Development and validation of enzyme-linked immunosorbent assays for the serodiagnosis of canine Bartonelloses
Data files
Nov 04, 2025 version files 41.59 KB
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Dogs_ELISA_OD_data.csv
12.99 KB
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rATP-β__analysis.csv
4.18 KB
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rATP-β_plus_rGroEL_analysis.csv
4.03 KB
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README.md
3.52 KB
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rGroEL_analysis.csv
3.96 KB
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rLemA_analysis.csv
4.40 KB
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rSucB_analysis.csv
4.21 KB
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rVirB5_analysis.csv
4.30 KB
Abstract
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.
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).
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
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