Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle

Abstract

Background: The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered.

Results: We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca.

Conclusions: The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures.

Figure 1

Master curve generated by plotting the mean Cq values as a function of the known starting concentration of the standard dilutions. Ten-fold serial dilutions (10⁶ - 1 copy) of the linearized T. annulata recombinant plasmid were analyzed in 9 independent experiments with three replicate reactions per dilution step. Error bars represent standard deviations (SD) of Cq values from replicated standards. Confidence intervals (CI) of the means of Cq values from replicated standards are indicated by the dashed lines.

Figure 2

Graphical representation of individual parasitaemia (log₂ T. annulata /ml of blood) variations (black dots) throughout the study along with the corresponding PCV results (open dots) for each animal. Age of animals (in months) at first sampling was as follows: Animal #1, 92; Animal #2, 49; Animal #3, 79; Animal #4, 37; Animal #5, 47; Animal #6, 46; Animal #7, 121; Animal #8, 26.

Figure 3

Box Plot representing T. annulata per ml of blood in the animals grouped according to the sampling time. The boundary of the box closest to zero indicates the 25th percentile, the continuous line within the box marks the median, the dashed line marks the mean and the boundary of the box farthest from zero indicates the 75th percentile.