New sensitive real-time PCR targeting p28 gene for detection of Ehrlichia canis in blood samples from dogs

Patrícia Gonzaga Paulino,Huarrisson Azevedo Santos,Carlos Luiz Massard,Olga Lucia Herrán Ramírez ,Tays Araújo Camilo ,Renata Lins Da Costa ,Nathália Alves Senne ,Miguel Angelo Leite Mota

doi:10.1590/0103-8478cr20200891

Patrícia Gonzaga Paulino, Huarrisson Azevedo Santos + Show 6 more

Open Access

https://doi.org/10.1590/0103-8478cr20200891

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Abstract

ABSTRACT: This study aims to describe a new detection method of a quantitative real-time polymerase chain reaction (qPCR) targeting the 28 kDa outer membrane protein gene (p28) as well as to compare this method with a conventional PCR (cPCR), which targets the same gene, in order to evaluate the performance of the technique designed in this study in detecting Ehrlichia canis (E. canis). Optimum oligonucleotides concentrations were reached, and the analytical sensitivity and specificity of the qPCR were performed. A total of 218 dogs’ whole blood samples were conventionally collected for this study. The DNA was extracted from each sample. Subsequently, the samples were tested by an established cPCR and the new qPCR to compare each technique’s performances. This new qPCR method for the molecular detection of E. canis presented a detection limit of ten copies of the fragment and was considered specific for E. canis according to analytical specificity analyses performed in vitro and in silico. The standard curve revealed 100% efficiency and a coefficient of determination (R2) equivalent to 99.8%. Among the samples examined by qPCR, 24.31% were considered positive, significantly greater than those detected by cPCR (15.13%). The qPCR technique reached a higher sensitivity than the cPCR when targeting the p28 gene in detecting E. canis. The qPCR standardized in this study is an efficient method for confirming canine monocytic ehrlichiosis (CME) diagnosis and might provide the parasitemia monitoring during the disease treatment.

Highlights

Ehrlichia canis is an obligatory intracellular hemoparasite that possesses major significance in veterinary medicine, namely in the tropical countries where this infection occurs with more frequency (AGUIAR et al, 2020)
The disease produced by E. canis is called canine monocytic ehrlichiosis (CME)
The present study aims to develop a sensitive quantitative real-time polymerase chain reaction (qPCR) method targeting the p28 gene that may be used for specific detection of E. canis in clinical samples from naturally infected dogs as well as to compare this qPCR with a conventional PCR for targeting the p28 gene of E. canis, as previously described by Nakaghi et al (2010)

Summary

Introduction

Ehrlichia canis is an obligatory intracellular hemoparasite that possesses major significance in veterinary medicine, namely in the tropical countries where this infection occurs with more frequency (AGUIAR et al, 2020). The vector Rhipicephalus sanguineus sensu lato (s.l.) must actively contribute toward raising the infection rates in tropical areas (VIEIRA et al, 2011). This pathogen has zoonotic potential, representing a public health concern (NICHOLSON, 2010). V.51, n.12, subclinical phases (WANER et al, 1997; DE CASTRO et al, 2004; MYLONAKIS et al, 2004). These phase differences are not explicit in dogs with naturally occurring disease (HARRUS et al, 2012)

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