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

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.

intensive, misleading (due to similar morphological features with other parasites like Babesia), and also require an expert technician 15,16 . However, the technique is not effective in early diagnosis due to low parasitaemia 16 .
Molecular diagnosis using conventional PCR based on several T. annulata specific genes, 18S rRNA, T. annulata merozoite surface protein (tams), T. annulata sporozoite surface protein (tasp) or Cytochrome III, and serological assays using TASP and TAMS antigens are still used for diagnostic purpose [16][17][18][19][20] . Recently, Real-time quantitative PCR based on the 18s rRNA and tams genes have been used for detection of the T. annulata parasites [21][22][23][24] . The surfacing problem of drug resistance against the current anti-Theileria drug buparvaquone (BPQ) also presses the need for a better diagnostic tool in evaluating treatment response 22,[25][26][27] . However, none of the current tools helps in identifying the host to parasite ratio, parasite burden, and chemotherapy response.
When compared to the other apicomplexan parasite like Plasmodium falciparum, which resides in RBC cell (nonnucleated), diagnosis of Theileria parasites poses challenge owing to their complex life cycle inside bovine leukocytes. The high sensitivity of the real-time PCR technique makes it an appropriate method for early disease diagnosis and parasite quantification 28,29 . In line with the current scenario, we have developed a qPCR assay which will help in identifying the parasite burden, host to parasite ratio and as well as treatment response in the field.

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 DNA 30 . The hprt and tasp were amplified using the gene-specific primers and cloned into the pBSK plasmid. The melting curve for the tasp and hprt showed a single peak at 78.72 °C and 84.57 °C, respectively for the reference plasmid DNA and the biological sample.
The PCR sensitivity was determined by serially diluted DNA from 10 ng to 1fg. Amplification was detected until the lowest DNA dilution of 1fg, and the Cq values ranged from 14.23 ± 0.28 (10 ng) to 30.94 ± 0.32 (1fg). The Gene Copy Number (GCN) of the two plasmids were calculated using the standard formula mentioned below in material and methods. The standard curve was plotted by serially diluting (1:10) the hprt and tasp plasmid constructs starting from the 10 6 to 10 GCN. A Cq value was obtained for each dilution, and three replicates were used for each dilution. The average Cq values were plotted against each respective GCN dilutions for both the plasmid constructs ( Table 1). The slope of the standard curve was found to be −3.04 for hprt plasmid and −3.12 for the tasp plasmid. The correlation coefficient (r 2 ) for the PCR reaction was found to be 0.99 while the PCR efficiency was 112% and 109% for the hprt and the tasp plasmid, respectively. A negative template control and uninfected cattle DNA samples were run to check for any contamination during the PCR runs.
Host-Parasite DNA Quantification. The host-parasite DNA was quantified from the in vitro T. annulata infected host leukocytes cell lines. 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. The GCN of the host was divided by a factor of 2 owing to its diploid genome. The GCN values were further used to calculate the host and parasite DNA ratio using the equation 2. In the 5 cell lines used for DNA quantification, the parasite DNA (%) was found to be ranging from 1.47 ± 0.53% to 5.94 ± 0.30% (Fig. 1).
Parasite load in the clinical cases of bovine theileriosis. The DNA was isolated from the whole blood collected from 414 cattle suspected for bovine theileriosis. PCR using 18S rRNA and tasp gene were done for all samples to check for T. annulata infection. Out of 414 samples, only 219 were PCR positive, however, when checked with real-time PCR 243 samples were found to be positive. Further, the C q values obtained for each sample was used for identifying the parasite load in the clinical samples using equation mentioned in material and method. The parasite load ranged from 3.18E + 05 to 2.54E + 10 (Fig. 2). The samples in which C q value ≥ 36.94 ± 0.25, i.e. the C q value of negative template control (NTC) were considered as negative.
Analysis of the Parasite DNA before and after drug treatment. To monitor the response of the chemotherapy, a blood sample was collected from infected cattle before and after 10 days after the treatment. Microscopy and, and real-time PCR analysis was done from the pre and post-treatment sample to determine the treatment efficacy. 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). Blood smears examination also revealed the absence of piroplasm in post-treatment as compared to pre-treatment blood sample. Similarly, real-time PCR was also performed on a DNA sample of cell lines treated and untreated with BPQ. DNA was isolated from the cell lines after 48 and 72 hrs of BPQ treatment to monitor the parasite growth. After, 48 hr the % parasite DNA Gene Copy Number hprt (Cq ± SD) tasp (Cq ± SD)

Discussion
A quick, sensitive and specific diagnostic tool is a must for effective control of bovine theileriosis. qPCR has served as an efficient tool for detection and quantification of the parasites of various diseases. In the present study, we have developed a diagnostic assay which will help us in the diagnosis and quantification of T. annulata parasite and also monitor treatment effectiveness. Although the assay has limitations and could not differentiate between infected and vaccinated animals. Due to insufficient studies on the differences among vaccinated and T. annulata infected animals, until a date no molecules/gene have been identified which can differentiate between the two groups. The real-time PCR is based on the absolute quantification method using recombinant plasmids corresponding to single copy genes specific to the parasite, (tasp) and the host (hprt). In a previously published report, 18S rRNA based qPCR for detection and quantification of T. annulata was used, however, owing to its multi-copies in the single genome and sequence similarity among various Theileria and Babesia parasites, it may pose several disadvantages and limitations in testing clinical samples 30,31 . We have utilised parasite gene tasp, which is preferred over other determinants such as 18S rRNA, TAMS gene of T. annulata for diagnosis 23,[31][32][33] . We have developed a qPCR assay, in which reference standard curves were generated for calculating host and parasite ratios in the clinical samples.
This approach has been utilised earlier on four T. parva cell lines, however, there is no such report on T. annulata 30 . In the cell lines analysed, we have found the % parasite DNA varied from 1.47% to 5.94%, which is in line with earlier studies on T. parva cell lines which showed it to be 0.9% to 3%. Less amount of % parasite DNA in the cell lines corresponds to the fact that parasite genome is quite smaller in comparison to bovine genome 30 . The  advantage of calculating the host-parasite ratios will provide useful insights to understand host-pathogen biology and assist in various experimental approaches, e.g. use of sample with higher parasite load for performing whole genome sequencing of the parasite Further, we have also performed qPCR on 414 bovine blood samples. 44 additional samples were diagnosed as infected by qPCR in comparison to conventional PCR due to presence of low parasitemia. The parasite load in the clinical samples was found to be ranging from 3.18E + 05 to 2.54E + 10 parasites/mL of blood. Ros-Garcia et al., 2012 reported the development of a real-time PCR assay for quantification of parasite load, however, it cannot calculate the host-parasite ratios 21 . However, with our assay, we can calculate both the host-parasite ratios and as well as parasitaemia in clinical samples. The assay also provides an additional benefit of quantifying parasite load in clinical samples, which would pave a way to distinguish between active and carrier animals. However, studies on more sample number and its correlation with the clinical profile will be required to establish the same.
BPQ was selected for treatment of theileriosis due to low toxicity and a long plasma half-life of 7 days 34 . However, a prepatent period of 10-13 days is observed from tick feeding on to cattle blood to onset of fever and clinical symptoms. If the diagnosis followed by the treatment is delayed, it results in animal death. Therefore, timely diagnosis, as well as monitoring treatment response are crucial for disease control and drug resistance. Earlier report has observed parasite clearance after 8-10 days of BPQ treatment 35 . Therefore, we monitored the parasite load in cattle before and after 10 days of treatment and observed a marked decrease in the parasitaemia indicating the treatment was effective. Similarly, the same approach was tested in T. annulata cell lines incubated with the BPQ for 48 to 72 hrs. In all the cases, we observed a decrease in the % parasite DNA, indicating parasite death.
This assay holds importance in calculating the ratio of the parasite DNA in the bovine host which will further help in answering various research questions. Further, it can lead to monitoring the decrease in parasite load in clinical cases to assess the animal clinical state and treatment effectiveness.

Materials and Methods
Sample Collection, DNA Isolation, and Theileria Specific PCR. Blood samples were collected from cattle belonging to different endemic regions of India. Samples were collected and preserved in EDTA by a trained veterinarian. A total of 414 blood samples were collected from suspected cases of theileriosis (showing clinical symptoms) from different states, Andhra Pradesh, Telangana, Punjab, and Haryana. Study design and reporting follow the Standards for the Reporting of Diagnostic accuracy (STARD-2015: http://www.stard-statement.org/) (S1 Flow Diagram).
Genomic DNA was isolated from 2 ml of blood samples using previously published DNA isolation protocol 1 . Quality and integrity of the DNA were checked using Nanodrop and by running the genomic DNA on 0.8% agarose gel. After the quality check DNA samples were stored at −80 °C until further experiments. T. annulata specific 18S rRNA and tasp gene primers were used for conventional and qPCR ( Table 1).
The PCR conditions for the primers mentioned above are as follows:  The primer sequences are given in Table 1. The amplified products were cloned into a pBSK plasmid using the TA cloning method as described earlier (https://www.thermofisher.com/in/en/home/life-science/cloning/ ta-cloning-kits.html). Later pBSK was transformed into the Top10 cells competent E. coli cells, and cloned plasmids were selected by growing cells in an ampicillin medium. Cloned plasmids were confirmed by restriction digestion using XcmI and Sanger sequencing using gene-specific primers. The NCBI Nucleotide database was queried to confirm the gene sequences. qPCR SYBR Green Based Assay. Primers for use with intercalating dye-based qPCR were designed using Primer 3 software for tasp and hprt genes. The analysis was carried out on an Applied Biosystems 7500. Melting curve analysis was done for both the primers showing single and specific peaks. 6 Tenfold serial dilution (10 6 to 10 GCN) of the pBSK-hprt and pBSK-tasp plasmids were used for generating the standard curve (Table 2).
In vitro culture of the T. annulata infected bovine lymphocyte cell line. T. annulata infected leukocyte culture were established by isolating the PBMCs from the clinically infected cattle 1 . Briefly, isolated PBMCs were cultured in the RPMI 1640 medium with 10% FBS and Pen/Strep (100 µg/ml) solution at 37 °C with 5% CO 2 . T. annulata infected bovine cells transform and continue to grow in vitro condition for an infinite time. T. annulata parasites inside the bovine cells were confirmed by PCR using tasp specific primers.
Monitoring treatment response using qPCR assay. First, the efficacy of BPQ was checked on T. annulata cell line. 2.5 × 10 5 cells/ml of T. annulata cell line was dispensed in a 6-well tissue culture plate. Further, BPQ was added at the concentration of 50 ng/ml to the test wells, and DNA was isolated from the wells after 48 and 72 hr of incubation. Cells without BPQ were treated as control, and gDNA was isolated. Similarly, the treatment response in a T. annulata infected cattle was also checked by collecting blood samples before and after 10 days of the BPQ treatment. All the experiments were done in triplicate. qPCR was done to check the parasitaemia in the cell line before and after treatment. GCN calculation. The gene copy of the plasmids was calculated using the below-mentioned equation 30 .  The cloned tasp and hprt gene in a pBSK cloning vector (pBSK-tasp & pBSK-hprt) were 10 fold serially diluted up to 6 dilutions ranging from 10 6 to 10 copy/µl. The standard curve was plotted as copy number vs. C q value for each of the dilutions in triplicates for pBSK-tasp and pBSK-hprt respectively.
Estimation of Host-Parasite DNA ratio. The host to parasite DNA ratio was calculated using equation 2 as mentioned below. As the bovine cells are diploid, the number of bovine cells were considered to be half the GCN of the hprt gene. As the parasite is in the haploid state while surviving inside host cells, the number of parasite cells is considered equivalent to GCN of the tasp gene 30 . The GCN was then used to calculate the amount of host and parasite DNA in the tested sample. Ethical approval and informed consent. Oral consent was taken from the farm owners before drawing blood from animals. There is no specific law in India which requires permission from the ethics committee for collecting less than 5 ml of blood. Further, blood samples were collected by professional veterinarians.

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.