A Real-Time PCR based assay for determining parasite to host ratio and parasitaemia in the clinical samples of Bovine Theileriosis

Debabrata Dandasena,Satparkash Singh,Paresh Sharma,Jaspreet Singh Arora,Shweta Murthy,Sonti Roy,G S Sreenivasamurthy,Vandna Bhanot,Vasundhra Bhandari

doi:10.1038/s41598-018-33721-3

Abstract

Theileria annulata is an intracellular parasite that causes active and latent forms of bovine theileriosis. Diagnosis of the disease is primarily based on traditional methods such as microscopy, however, PCR based methods have proven to be superior in the absence of clear disease symptoms. However, diagnosis is difficult in cases of lower parasitaemia by conventional PCR. Hence, a rapid and sensitive method which can detect early infection and low parasite load is required. Therefore, we have developed an absolute quantification based real-time PCR (qPCR) assay. Reference standard curve using recombinant plasmids of a host (hprt) and a parasite gene (tasp) was constructed, and the assay was initially standardised using in vitro T. annulata cell lines. Further, 414 blood samples from suspected theileriosis cases were also evaluated using qPCR. The assay can estimate host to parasite ratios, calculate parasitaemia and treatment effectiveness in the clinical cases of theileriosis. In comparison with the conventional PCR results, 44 additional positive cases were found. Therefore, the assay holds importance in a clinical setting due to its ability to quantify the parasite load in clinical samples. It may be further used in distinguishing active and latent theileriosis infections and detection of drug resistance in the field.

Highlights

Bovine theileriosis is caused by an apicomplexan parasite Theileria spp. which is an important tick-borne disease of livestock[1]
The diagnosis of theileriosis heavily relies on the microscopy, where Giemsa stain is used to check for Theileria infected multinucleated host cells (Koch’s bodies) and the piroplasm stage in the blood smear[14]
A quick, sensitive and specific diagnostic tool is a must for effective control of bovine theileriosis. quantification based real-time PCR (qPCR) has served as an efficient tool for detection and quantification of the parasites of various diseases

Summary

Result

Sensitivity, PCR Efficiency, and Standard curve analysis. A single copy gene specific (hypoxanthine phosphoribosyltransferase 1, hprt) to the host and the parasite (Theileria annulata surface protein, tasp) was used to quantify the host-parasite DNA30. The standard curve was plotted by serially diluting (1:10) the hprt and tasp plasmid constructs starting from the 106 to 10 GCN. A qPCR was first performed using the T. annulata cell lines against the hprt and tasp genes. The Cq values obtained were used to calculate the GCN of the host and parasite DNA in the cell lines in reference to the standard curve plotted using the hprt and tasp plasmid constructs. PCR using 18S rRNA and tasp gene were done for all samples to check for T. annulata infection. The real-time PCR analysis showed a reduction of parasite DNA from 72.54 ± 4.55% to 0.01 ± 0.003% after treatment, suggesting parasite clearance (Fig. 3A). Real-time PCR was performed on a DNA sample of cell lines treated and untreated with BPQ. After 72 hr, control cells exhibited 4.05 ± 0.29% parasite DNA, whereas, the treated cells had 1.55 ± 0.02% of parasitic DNA in them

Discussion

Materials and Methods