Abstract
Canine monocytic ehrlichiosis caused by Ehrlichia canis is one of the leading tick-borne diseases of dogs, particularly in tropical countries. A highly sensitive and specific diagnostic method is essential for early detection to facilitate treatment. This study was conducted to develop E. canis loop-mediated isothermal amplification (LAMP) assay, a highly sensitive yet simple molecular technique, targeting the citrate synthase (gltA) gene of E. canis. Canine blood samples were subjected to conventional PCR targeting E. canis gltA. After analysis of the sequences of PCR amplicons, LAMP primers were generated. The optimum temperature and time for the LAMP assay were determined using eight samples—after which, the effectiveness and reproducibility of LAMP were verified by testing 40 samples, which included PCR-positive and negative samples. The detection limit was also established. The optimal condition for the assay was 61 °C for 60 min. Compared to PCR, the LAMP assay had a relative sensitivity and specificity of 92.5 and 100%, respectively. Statistical analysis using McNemar’s test showed that the E. canis LAMP assay has no significant difference with PCR. Therefore, the LAMP assay developed in this study may be used as an alternative to PCR in the detection of E. canis.
Highlights
Canine monocytic ehrlichiosis (CME) is caused by Ehrlichia canis, an obligate intracellular tick-borne pathogen of dogs primarily transmitted by the brown dog tick Rhipicephalus sanguineus [1,2]
Our recent study suggested that CME is the leading TBD of dogs in the Philippines [7]
Aside from causing significant morbidities and fatalities in dogs, E. canis is recognized as a potential zoonotic pathogen after being reported in some cases in humans [22,23,24]
Summary
Canine monocytic ehrlichiosis (CME) is caused by Ehrlichia canis, an obligate intracellular tick-borne pathogen of dogs primarily transmitted by the brown dog tick Rhipicephalus sanguineus [1,2]. Recent studies utilizing PCR showed that E. canis is a leading tick-borne pathogen of dogs in Southeast. PCR is a highly sensitive molecular technique capable of detecting E. canis DNA as early as 4–10 days post-infection [13]. PCR is mostly applied in research since it is time-consuming, sophisticated, and labor-intensive [14] It has limited use in clinical settings due to the need for expensive equipment and adequate skills. Some studies reported LAMP development for the detection of E. canis that targeted the heat shock operon groESL gene [18] and p30 gene [14,19]. The use of LAMP in the detection of E. canis in the Philippines has not been investigated. The development of a LAMP assay targeting the citrate synthase gene (gltA) for detecting. The detection method mentioned may be possibly adapted in veterinary clinics in the Philippines and other countries
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