High resolution melting analysis of the 18S rRNA gene for the rapid diagnosis of bovine babesiosis

Abstract

BackgroundBovine babesiosis is caused by protozoan parasites of the genus Babesia and presents a wide spectrum of clinical manifestations. Disease severity depends on the type of Babesia species infection. Generally, B. bovis and B. bigemina are considered as the causative agents of bovine babesiosis; in addition, Babesia ovata and B. major are a group of benign bovine piroplasms. Therefore, species identification is important for diagnosis, epidemiological investigations and follow-up management.MethodsReal-time PCR combined with high resolution melting (RT-PCR-HRM) analysis was used to detect and discriminate four Babesia species infective to cattle, including Babesia bovis, B. bigemina, B. major and B. ovata. The melting profiles and melting temperatures (Tm) of the amplicon targeting 18S rRNA revealed differences that can discriminate the four Babesia spp. Sensitivity and specificity of the analytical method were evaluated using 50 blood samples collected from experimentally infected cattle and 240 blood samples from areas where bovine babesiosis is an issue.ResultsRT-PCR-HRM analysis allowed to detect and discriminate four Babesia spp. (B. bovis, B. bigemina, B. major and B. ovata), which were responsible for bovine babesiosis in China. The protocol was validated with DNA samples from experimentally infected cattle and field infection in cattle.ConclusionsOur results indicate that RT-PCR-HRM is a fast and robust tool for the simultaneous detection and discrimination of four Babesia species that are responsible for bovine babesiosis in China. This approach is applicable for both field and experimental samples, thus it could be useful in epidemiological investigations and diagnoses of bovine babesiosis.

Systematic Review of Ticks and Tick-Borne Pathogens of Small Ruminants in Pakistan

“…However, microscopic and serological methods are of limited value due to several limitations, including lower sensitivity and specificity, cross-reactivity, inability to detect carrier infections, and the requirement of expertise and time [ 140 , 144 , 145 ]. These limitations have been overcome through the use of highly sensitive molecular methods, including conventional PCR (cPCR), quantitative PCR (qPCR), nested PCR (nPCR), reverse line blotting (RLB), loop-mediated isothermal amplification (LAMP), high-resolution melting (HRM) assays, high-throughput microfluidics-based real-time PCR and the next-generation sequencing (NGS) [ 105 , 146 , 147 , 148 , 149 , 150 ].…”

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia

“…In parasitology, HRM analysis is extensively applied for protozoan parasites. For example, based on the 18S ribosomal gene, it allowed the rapid diagnosis of bovine babesiosis [ 29 ]. It was also evaluated to monitor Plasmodium falciparum treatment efficacy [ 30 ] and to detect and differentiate among Theileria annulata , Theileria orientalis , and Theileria sinensis species [ 31 ].…”

High Resolution Melting (HRM) approach-based strategy for simultaneous differentiation of five Babesia species response for human babesiosis

“…Alhough our primers may match the target gene of some piroplasma, such as Thelieria orientalis, Thelieria does not infect humans, so it is no problem to identify human infection with Babesia. The most important thing is that the principle of qPCR-HRM identi cation and detection is based on the difference of single base in ampli cation fragments of different insect species, so different microbes can form different melting curves, different Tm values, and different GCP [55]. Therefore, even if we detect microbes with similar genes to Babesia, our detection methods for Babesia also have high speci city.…”