A novel modified-indirect ELISA based on spherical body protein 4 for detecting antibody during acute and long-term infections with diverse Babesia bovis strains

Chungwon J Chung,Carey L Bandaranayaka-Mudiyanselage,Carlos E Suarez,Ethan Adams,Grace Chung,Stephen S Lee,Susan J Waldron,Joanna Rzepka,Tj Heiniger,Chandima-Bandara Bandaranayaka-Mudiyanselage,Grace Yun

doi:10.1186/s13071-017-2016-9

Chungwon J Chung, Carey L Bandaranayaka-Mudiyanselage + Show 9 more

Open Access

https://doi.org/10.1186/s13071-017-2016-9

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Abstract

BackgroundCattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Since B. bovis is considered a persistent infection, developing a reliable, high-throughput assay that detects antibody during all stages of the infection could be pivotal for establishing better control protocols.MethodsA modified indirect enzyme-linked immunosorbent assay (MI-ELISA) was developed using the spherical body protein-4 (SBP4) of B. bovis to detect antibody against diverse strains through all infection stages in cattle. This SBP4 MI-ELISA was evaluated for sensitivity and specificity against field sera from regions with endemic and non-endemic B. bovis. Sera were also evaluated from cattle infected experimentally with various doses and strains during acute and persistent infection with parasitemia defined by nested PCR.ResultsThe format variables for SBP4 MI-ELISA were optimized and the cutoff for positive and negative interpretation was determined based on receiver operating characteristic curve analysis using B. bovis positive and negative sera tested in the reference immunofluorescence assay (IFA). The diagnostic specificity of the SBP4 MI-ELISA using IFA-negative sera collected from Texas was 100%, significantly higher than the cELISA (90.4%) based on an epitope in the rhoptry-associated protein-1 (RAP-1 cELISA). The diagnostic sensitivity of the SBP4 MI-ELISA was 98.7% using the IFA-positive sera collected from several areas of Mexico, in contrast to that of the RAP-1 cELISA at 60% using these same sera. In cattle infected with low and high doses of three B. bovis strains, the SBP4 MI-ELISA remained antibody positive for 11 months or more after initial detection at 10 to 13 days post-inoculation. However, the RAP-1 cELISA did not reliably detect antibody after eight months post-inoculation despite the fact that parasitemia was occasionally detectable by PCR. Furthermore, initial antibody detection by RAP-1 cELISA in low-dose infected animals was delayed approximately nine and a half days compared to the SBP4 MI-ELISA.ConclusionsThese results demonstrate excellent diagnostic sensitivity and specificity of the novel SBP4 MI-ELISA for cattle with acute and long-term carrier infections. It is posited that use of this assay in countries that have B. bovis-endemic herds may be pivotal in preventing the spread of this disease to non-endemic herds.

Highlights

Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission
Optimization of the spherical body protein-4 (SBP4) MI-ELISA and determination of the cutoff using Receiver operating characteristic (ROC) curve and scatter plot analysis The SBP4 MI-ELISA was optimized by evaluation of several format variables including the antigen coating procedure, serum incubation time, wash buffer, and concentration of horseradish peroxidase (HRP)/glutathione S-transferase (GST)-SBP4 conjugate used to detect antibody binding
The optimized format included the use of 50 μl undiluted serum to maximize assay simplicity. Using this optimized SBP4 MI-ELISA format, sample OD to negative control OD (S/N) ratios were determined for 1,253 sera that were already categorized as either B. bovis-immunofluorescence assay (IFA) positive or -IFA negative

Summary

Introduction

Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Friesian cattle vaccinated with an attenuated strain had evidence of parasitemia for up to 47 months as determined by sub-inoculation into splenectomized calves [4] These two experiments, though limited, provide strong evidence for long-term persistence of B. bovis infection in cattle. Another study monitored antibody responses based on the reference immunofluorescence assay (IFA) through 60 days postvaccination [7] In these two studies, both the cELISA and IFA were reliable for short-term monitoring of B. bovis-specific antibody responses; their reliability for monitoring longer post-infection periods has not been determined. Further defining long-term persistence of B. bovis infection and longevity of B. bovis-specific antibody responses in cattle after infection with various attenuated vaccine strains and pathogenic strains is crucial for developing better control measures

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