RT PCR Assay to Determine Efficacy of Buparvaquone in Reducing Degree of Parasitemia of Theileria orientalis

Bovine theileriosis, a tick-borne haemoprotozoan disease of cattle is distributed all over the world. Tropical and sub-tropical regions, especially experience the loss of productivity in bovines due to tick and tick-borne diseases is mostly experienced by the tropical and subtropical countries due to the climatic conditions favourable for the propagation of ticks. The estimated annual loss in productivity worldwide ranges from 14 to 19 billion US dollars (De Castro 1997). The impact was found higher in developing countries due to unaffordable tick control and treatment costs. Tropical theileriosis due to Theileria annulata, the aetiological agent of tropical theileriosis’ is  recognised as pathogenic species since long owing to its  ability to multiply extensively during intraleukocyte development (Morrison 2015). Oriental Theilerosis has also been reported from different countries including India and Odisha (Sahoo et al., 2017) and is gaining significance since the last decade.

Since T. orientalis is non-lymphoproliferative, they are traditionally recognised as benign parasites of bovine. Outbreaks are mainly limited to immunosuppressed or stressed cattle, associated with poor farm management (Watts et al. 2016). However, the major economic losses include loss in milk production, abortions and severe morbidity. Clinical signs include fever, anaemia, in appetence, dyspnoea, swollen supra-scapular lymph node, and rarely haemoglobinuria is also observed (Fukushima et al., 2021). Examination of Blood smear, lymph node aspiration fluid examinations, and polymerase chain reaction (PCR) tests have been developed and standardised for detection of haemoprotozoans, with the PCR  tests  being the most sensitive and accurate amongst them (Perera et al., 2015).  Although Giemsa-stained blood and lymph smears are still the most often used diagnostic tools, particularly during the acute stage as generally enough infected cells can be seen through a microscope (Al-Hosary et al., 2020).  The lymph node biopsy smear examination had the lowest diagnostic efficacy as compared to microscopic examination of the peripheral blood smear  and found that, even at low parasitemia, PCR is a more precise and specific diagnostic technique for the identification of Theileria spp in cattle. (Farooq et al., 2019). The Multiplex PCR was found as useful approach to identify co-infection of tick-transmitted illnesses in cattle in endemic locations where, there are potential co-infections. (Kumar et al., 2021).

Buparvaquone is the most common drug for treatment of theileriosis due to its low toxicity and short plasma half-life of seven days. There is, however, a prepatent period of ten to thirteen days between the time a tick feeds on cow blood and the onset of fever and clinical symptoms. The invention of buparvaquone in 1980s has changed the treatment scenario, however it only reduces parasitaemia but animal remains as carrier (Dandsena et al., 2018). Despite the high prevalence of T. orientalis infection and the resulting economic losses in cattle of Odisha, no attempts have been made earlier to determine the efficacy of buparvaquone in lowering parasitemia of T. orientalis genotypes. The present study aims to assess the efficacy of Buparvaquone in reducing degree of parasitemia of Theileria orientalis by molecular method.

Source of sample
 
The blood samples in commercially available EDTA vials presented at Veterinary Clinical Complex, College of Veterinary Science and Animal Husbandry, Bhubaneswar, Odisha by the cattle owners from three different districts of Odisha (Cuttack, Puri and Khurda) for haemoparasite investigation were included in the study. The samples were collected from animals showing the signs of inappetence, fever, (>103°F), pale mucous membrane, dyspnoea and decreased milk yield were the basis for collection of samples from the period of April 2019 to July 2020.
 
Isolation of DNA from whole blood and qPCR
 
Genomic DNA extraction was carried out by using commercially available DNA mini kit (Qiagen, USA) from each blood sample. 200 µl of whole blood from each sample was utilized for DNA extraction by following the manufacturer’s instructions. The primers Tann and Tori were used for Theileria annulata and Theileria orientalis respectively (Table 1). Real-time Polymerised chain reaction was performed in a final reaction volume of 25 µl of reaction mixture containing 15 µl Taq-Man probe-based master mix (rotor gene Q), 1 µl forward and reverse primer each and 0.25 µl Taq-Man probe both for Theileria orientalis and Theileria annulata with cycling conditions as described in Table 2. The graphical representation in q PCR for Theileria annulata-FAM (Fluorescein, a dye label) channel was by green colour while for Theileria orientalis- JOE( Xanthene Fluorophore) channel, it was yellow colour.




 
Quantification of Theileria spp in blood samples
 
In 9 consecutive plate runs with three replicate reactions per dilution step, tenfold serial dilutions (101-109) of the linearised Theileria spp recombinant plasmid were studied. The results of these dilution series were used to determine the analytical sensitivity and linearity of the procedure for real-time PCR quantification. The analytical sensitivity of the assay, also known as the limit of detection (LoD), was defined as the lowest concentration (target gene copy) that can be detected. These serial dilutions were used to estimate the number of test runs (number per response) where 95 percent of test runs yielded positive results.
 
Calculation of parasitic load
 
The standard deviation (SD) of the quantification cycle values determined for the ten-fold serial dilutions of plasmid PCR standards was used to indicate intra-assay and inter-assay reproducibility of the Theileria spp in TaqMan qPCR assay. The amount of parasitemia was determined by converting Cq values into an estimate of copy number (Q) per reaction tube.102 means lowest parasite concentration, 109 means highest parasite concentration.

By nanodrop spectrophotometer the concentration of genomic DNA was detected. Two standard curves, one for Theileria annulata and one for Theileria orientalis were created. Calculation of the gene copy number of plasmids was done by given formula (Dandsena et al. 2018).

  

 
Plotting standard curve for Theileria spp
 
The isolated plasmid DNA was diluted with nuclease-free water to a final concentration of 100 ng/µl (copy 1010) before being serially diluted seven times by ten-fold dilution to a final concentration of 10 fg/µl (copy 103). Real-time Polymerised chain reaction was performed by using three different concentrations for quantification. 12.5 µl master mix (Rotor Gene Q), 1 µl of primers both forward and reverse, 0.3 µl probe, 5 µl DNA and 5.2 µl RT-PCR grade nuclease free water was used to make it 25 µl for both Theileria annulata and Theileria orientalis.

Detection Theileria spp by real time PCR
 
Bovine theileriosis caused by T. annulata and T. orientalis are transmitted by ixodid ticks that significantly affects animal productivity. On eastern coast of India, the hot and humid climatic condition encourage vector multiplication and thereby results in higher incidence of such ailment. For effective control of bovine theileriosis, a rapid, sensitive and specific diagnostic method is required. Multiplex qPCR has proven to be an effective approach for detecting and quantifying parasites (Perera et al., 2015). Using a variety of molecular markers including major piroplasm surface protein (MPSP), 11 genotypes of T. orientalis complex have been identified (Gebrekidan et al. 2020). 

Out of 55 samples, 42 (76.36%) samples were found positive for Theileria spp, of which, 11 (26.11%) samples were positive to Theileria annulata, 24 (57.14%) samples to Theileria orientalis and 7 (16.66%) samples were positive for mixed infections. Rest 13 (23.63%) blood samples were tested negative for any of the Theileria spp (Fig 1).


 
Concentration of Theileria orientalis in affected cattle
 
A standard linear curve was plotted taking ten-fold serial dilution of plasmid DNA for seven times with maximum (100 ng/µl or copy 10¹⁰) and minimum (10 fg/µl or copy 10³) concentrations of T. orientalis. The concentration of haemoparasites in blood samples tested were deduced in clinically affected cows that ranged from 2.85×10³ to 1.51×10⁵ parasites per ml of blood. Parasitic load for T. orientalis in clinically affected cases were measured against the above standard curve that ranged from 1.94×10⁴ to 5.88×10⁵ parasites per ml of blood (Fig 2 a and b). The prevalence study revealed the dominance of T. orientalis over T. annulata. The higher presence of T. orientalis could be due to the possible role of the most abundant tick vector in India, Rhipicephalus sp in transmission (Patial et al., 2021). Parasitemia of T. orientalis assessed through real time PCR in lactating cows of Odisha by RT PCR targeting MPSP gene from T. orientalis cloned into pUC57 estimated the parasitic load to be ranging from  6.9-16.8×103/µl of blood (Sahoo et al., 2020). Detection of T. orientalis from Haemaphyslis longicornis ticks using qPCR assay in South Australia reported genotypic concentration of different strains ranging from 3.5×10¹-3.6 x10⁴ per ml of blood (Hammer et al., 2016). The detection of T. orientalis conducted by hydrolysis quantitative probe assay and quantified parasitemia (>3×10⁵ GC/µl) showed strong correlation with clinical signs (Boegma et al. 2015).
 


Efficacy of buparvaquone on Theileria spp.
 
In the 1980s, the development of new chemotherapeutic medicines like buparvaquone (BPQ) expanded therapy options. Due to its low toxicity and seven day plasma half-life, buparvaquone (BPQ) is the medicine of choice for treating theileriosis. However, point mutations in the parasite’s mitochondrial cytochrome b gene have resulted in resistance, which has become a source of concern (Mhadhbi et al., 2015). Ten cows with clinical signs of fever (>104°F), anaemia, dyspnoea, salivation, swelling of pre-scapular lymph node, decreased milk yield and positive for T. orientalis (through molecular test) were treated with buparvaquone (Butalex) @ 2.5 mg/kg bw at an interval of 48 hrs. The degree of parasitemia decreased 10 days post-treatment from 4.04×10⁴ to 2.21×10² per ml of blood indicating significant reduction in parasitemia (Table 3). However, none of treated animal showed complete reduction of the T. orientalis within such period as confirmed by and quantified by real time PCR for presence of T. orientalis. While previous studies have documented efficacy of the drug against T.annulata, the efficacy studies against T.orientalis is scanty. Previous studies detected a significant drop in parasite DNA from 72.54+4.55% to 0.01+0.003%. during RT-PCR assay indicates its efficacy in treatment of T. annulata with buparvaquone (Dandsena et al., 2018). In the present investigation, reduction of the parasite load of T. orientalis after treatment with two doses of buparvaquone at an interval of 48 hours indicates efficacy of the drug at the recommended dose and frequency. There has been previous reports of treatment of single dose of buparvaquone and repeat dose of buparvaqoune with oxytetracycline, where remission of clinical signs were observed (Goud et al., 2020). The results of the study will help support the clinician to implement suitable therapeutic approach against prevailing theileriosis. Therapeutic trial on T. annulata could not be included because of less number of positive cases. However, studies can be planned against mixed infection with T. annulata.

The study concluded that Buparvaquone (Bulatex) administered to clinically affected cows @2.5 mg/kg bw intramuscularly twice at an interval of 48 hrs proved efficacious in reducing T. orientalis concentration and complete disappearance of clinical signs ten days post treatment. Further studies with different treatment regimen and associated risk factors can be undertaken in different regions to throw more light on level of drug resistance or efficacy rate.

All authors declared that there is no conflict of interest.