# Comparative molecular detection and phylogenetic analysis of Babesia canis vogeli in naturally infected dogs using two 18S rRNA primer sets in Khon Kaen, Thailand

## TL;DR

This study compares two DNA tests for detecting a tick-borne parasite in dogs in Thailand and finds one is more sensitive while the other detects additional parasites.

## Contribution

The study provides a direct comparison of two 18S rRNA primer sets for diagnosing Babesia canis vogeli in dogs, including their sensitivity and phylogenetic insights.

## Key findings

- Babf/Babc primer set detected all B. canis vogeli cases with 100% sensitivity.
- Bab7/Bab9 primer set also detected H. canis at a distinct amplicon size.
- Phylogenetic analysis showed minimal genetic variation among B. canis vogeli isolates from different regions.

## Abstract

Canine babesiosis, primarily caused by Babesia canis vogeli in Thailand, is a significant tick-borne disease of veterinary concern. Molecular diagnostics targeting the 18S rRNA gene have enhanced detection sensitivity and specificity compared to conventional methods. This study aimed to identify and characterize B. canis vogeli in naturally infected dogs in Khon Kaen, Thailand, to compare the diagnostic performance of two primer sets (Bab7/Bab9 and Babf/Babc), and to perform phylogenetic analysis of the isolates.

A total of 159 ethylenediaminetetraacetic acid blood samples from client-owned dogs presented to the Veterinary Teaching Hospital, Khon Kaen University, between July and October 2024, were examined. Samples underwent Giemsa-stained blood smear microscopy and PCR amplification of the 18S rRNA gene using both primer sets. Positive amplicons were sequenced and analyzed phylogenetically using the Maximum Likelihood method. Limit of detection (LOD), sensitivity, specificity, and positive predictive value (PPV) were calculated for each primer set using sequence-confirmed results as the reference.

Microscopy detected B. canis in 19/159 (11.9%) of samples, while PCR increased detection to 23/159 (14.47%). Babf/Babc detected all positive cases (100% sensitivity), while Bab7/Bab9 detected 95.65% of positives. Both primer sets achieved 100% specificity and PPV, with an equal LOD of 105 DNA copies. Bab7/Bab9 also amplified Hepatozoon canis at a distinct amplicon size (503 base pair). Sequence analysis confirmed all Babesia-positive samples as B. canis vogeli, showing 96.34%–100% identity with global isolates. Phylogenetic analysis grouped the sequences with B. canis vogeli from multiple geographic regions, revealing minimal intraspecific variation.

B. canis vogeli was the only subspecies identified in naturally infected dogs in Khon Kaen during the study period. Babf/Babc demonstrated superior diagnostic sensitivity for B. canis vogeli, whereas Bab7/Bab9 offered broader detection, including H. canis. Phylogenetic analysis revealed close genetic relationships with isolates worldwide. These findings support the use of Babf/Babc for specific diagnosis and Bab7/Bab9 for broader screening in endemic regions.