Development and validation of a novel multiplex mPCR assay for the detection of Anaplasma, Babesia, and Theileria in captive wild ungulates

Wild animals are essential for preserving ecological balance and supporting economic stability. Nonetheless, they are at risk of extinction. Among the factors contributing to this endangerment are pathogens carried by blood-sucking arthropods, especially ticks. Hemoparasites such as Anaplasma, Babesia, and Theileria are spread to wildlife, livestock, and humans mainly by ticks. These intraerythrocytic parasites are traditionally identified by microscopic examination of blood smears stained with Giemsa. However, this method does not always accurately identify coinfections of hemoparasites. Moreover, detecting low levels of parasitism remains a challenge, making it unsuitable for timely diagnosis and predisposing wild animals to possible outbreaks, especially in captive settings. In this study, a multiplex polymerase chain reaction (mPCR) assay was developed to simultaneously detect Anaplasma, Babesia, and Theileria at the genus level in wild ungulates. Three pairs of specific, sensitive primers were designed based on the 16S ribosomal RNA gene (Anaplasma species) and the 18S ribosomal RNA gene (Babesia and Theileria) to generate target products of 318 bp for Anaplasma, 479 bp for Babesia , and 178 bp for Theileria. Blood samples were collected from a total of 60 captive Gazella bennettii (family Bovidae) and Axis porcinus (family Cervidae) from various zoos and wildlife parks in Punjab, Pakistan. To increase sample diversity and size, both families were included in the sampling process. A total of 40/60 (66.66%) samples tested positive, which were evaluated by PCR. Total genomic DNA was extracted for molecular detection by PCR. Three sets of newly designed primers based on genus were used to optimize multiplex PCR for melting temperature and crosslinking among primers. The multiplex PCR assay produced results consistent with monoplex PCR when using the newly designed primers. We detected hemoparasitic infections in 65% of samples, with infection rates of 25% for Anaplasma, 0% for Babesia, and 40% for Theileria. Downstream sequencing also confirmed these PCR results. This assay is a valuable complementary tool for routine, simultaneous, early, and accurate detection of Anaplasma, Babesia, and Theileria, supporting efforts to reduce indiscriminate antibiotic use, control antibiotic resistance, and protect endangered wild ungulates.