Introduction

Ticks are a major problem in tropical and sub-tropical countries, including India, and so are tick-borne diseases. Theileriosis is the most predominant among these diseases, causing substantial economic losses to the livestock sector (Rashid et al. 2019). Several Theileria species can infect bovines, including Theileria annulata, T. orientalis, T. parva, and T. mutans. Tropical theileriosis (T. annulata) and east coast fever (T. parva) are important clinical manifestations of cattle, while T. orientalis (oriental theileriosis) causes benign and mild disease. However, recently, fatal outbreaks caused by T. orientalis have been documented in various parts of the world where it was previously considered non-pathogenic (McFadden et al. 2011; Thompson et al. 2020), including India (Aparna et al. 2011; Patial et al. 2021). If the animal is already infected with T. mutans or T. annulata, then the infection of benign Theileria species such as T. orientalis is much more pathogenic. (Junlong et al. 2015).

Two hundred fifty million cattle are affected globally by tropical theileriosis caused by T. annulata (Erdemir et al. 2007). In India, losses of approximately 8426.7 crore INR in the dairy sector have been attributed to Tropical theileriosis (Narladkar 2018). High-yielding crossbred cattle, exotic breeds, and young calves are highly susceptible to the clinical form of the disease and have a high mortality rate. In contrast, mild or subclinical form of disease prevails in indigenous breeds. In addition, sub-clinically infected or carrier animals often go undiagnosed and pose a potential risk of infection in susceptible animals. Subclinical theileriosis is associated with invisible production losses due to decreased milk production (Rashid et al. 2019; Singh et al. 2022), poor body scores, and increased disease susceptibility (Kolte et al. 2017).

Another important prevalent species of Theileria in India and other parts of Asia and Australia is T. orientalis which causes oriental theileriosis. It is a non-transforming species of Theileria and is known to cause mild or sometimes no disease. However, clinical diseases and outbreaks with high morbidity and severe clinical signs have been reported worldwide (McFadden et al. 2011; Kamau et al. 2011; Thompson et al. 2020). Clinical signs such as abortion, hemolytic anemia, jaundice, fever, and arthritis are observed in clinical infection of T. orientalis (Kamau et al. 2011; Patial et al. 2021). Economic loss in oriental theileriosis occurs because of decreased milk production and abortion (Perera et al. 2014; Gebrekidan et al. 2020). In addition, chronic subclinical infections with T. orientalis may also lead to anemia (Choi et al. 2016).

The diagnosis of theileriosis under field conditions primarily relies on clinical signs, history, tick infestation, and reported disease prevalence. Many laboratory diagnostic methods are available, ranging from simple microscopic examinations to complex serological and molecular techniques. Microscopic examination of lymph node biopsies and blood smears is the most common laboratory diagnostic technique (Chauhan et al. 2015). Although economical, it is not possible to detect subclinical carriers. In addition, expertise is required to identify organisms correctly. Moreover, it is difficult to distinguish between different Theileria species or to determine co-infection based on microscopic characteristics (Liu et al. 2010). Serological investigations, such as enzyme-linked immunosorbent assay and indirect fluorescent antibody tests, are more specific and sensitive than microscopic examination. However, these tests cannot differentiate between T. annulata and T. orientalis because of cross-reactivity between these species (Mans et al. 2015).

Molecular techniques such as PCR have the highest specificity and sensitivity among all diagnostic tests. In addition, PCR assays can easily differentiate closely related species. Furthermore, PCR is beneficial for detecting carrier animals, as parasitemia in such animals is reported to be above the detection limit of PCR (Mans et al. 2015). However, screening a large population for individual pathogens using PCR is laborious and resource-intensive. This limitation is overcome by multiplex PCR assays, in which multiple pathogens can be detected simultaneously in one reaction, thus saving time and money. Therefore, they are best suited for epidemiological investigation.

Therefore, in this study, we aimed to develop a multiplex PCR assay for the rapid and simultaneous detection of two important Theileria species, viz., Theileria annulata and Theileria orientalis.

Material and methods

Positive control

A partial gene sequence of the major surface piroplasm protein (MPSP) cloned in the pBluescript II ks + vector synthesized by Bio Basic Inc., Canada, was used as a positive control for T. orientalis. The plasmid was reconstituted according to the manufacturer’s instructions and was inserted into competent DH5αcells. Similarly, for the positive control of T. annulata, the partial gene sequence merozoite-piroplasm surface antigen (TAMS1) cloned in the pBluescript II ks + vector (Bio Basic Inc., Canada) was purchased and processed further as mentioned above. DNA extracted from these DH5αcells was used as a positive control.

Primer design

After in silico screening and analysis of sequences from the GenBank® sequence database (http://www.ncbi.nlm.nih.gov/), two novel primer sets intended to identify the TAMS1 gene of T. annulata and MPSP gene of T. orientalis were designed using Primer3Plus software. Both primer sets had comparable annealing temperatures and were analyzed for self-annealing, hair loop formation, and self- and cross-dimer formation using the OligoAnalyzer software (https://www.idtdna.com/pages/tools/oligoanalyzer). The specificity of the primers was analyzed by BLASTn. The sequences of the primer sets and their corresponding product sizes are listed in Table 1.

Table 1 Specific primer designed for PCR assay
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Standardization of simplex and multiplex PCR

Simplex PCR assay was standardized for T. annulata and T. orientalis individually using the positive controls as a template. The PCR reaction mix in both cases had a total volume of 12.5µL consisting of 6.25µL of 2 × TopTaq PCR master mix (Qiagen, Germany), 0.4µL (10 µM) of each forward and reverse primers, and 1µL of template DNA and nuclease-free water to make up the final volume. The PCR was performed in Agilent SureCycler 8800 (USA). The gradient PCR was performed to optimize temperature (48–58 °C), primer concentration, and the number of cycles. For multiplex PCR assay, the reaction mix consisted of 6.25µL of 2 × TopTaq PCR master mix (Qiagen, Germany), 0.4µL (10µ M) of each forward and reverse primers for MPSP and TAMS1 gene, 1µL of template DNA, and 3.65 µL of nuclease-free water. Multiplex PCR was standardized to optimize the annealing temperature, thermo-cycling conditions, and primer concentration. PCR products were resolved in 1.8% agarose gel and visualized using a UV transilluminator (GeNei™ UVITEC imaging systems, Cambridge).

Determination of the minimum detection limits for simplex and multiplex PCR

The PCR amplicons of T. annulata and T. orientalis were purified from agarose gel using a gel purification kit (Qiagen, USA) and cloned into the pJET1.2 cloning vector. Plasmid DNA was extracted from the clones using a Zymo Plasmid Miniprep kit. The concentration of extracted DNA was estimated using a NanoDrop™ 2000 spectrophotometer. The estimation of the gene copy numbers was based on the assumption that the average weight of a base pair is 650 Da, thus calculating the number of plasmid copies using the following formula:

$$\mathrm{Copies}/\mathrm\mu\mathrm l=\frac{6.02\times10^{23}\;\left(\mathrm{copy}/\mathrm{mol}\right)\times\left[\mathrm{DNA}\;\mathrm{amount}\;\left(\mathrm g/\mathrm\mu\mathrm l\right)\right]}{\mathrm{DNA}\;\mathrm{length}\;\left(\mathrm{bp}\right)\times650\;\left(\mathrm g/\mathrm{mol}/\mathrm{bp}\right)}$$

To determine the sensitivity of simplex PCR for T. annulata and T. orientalis, the stock solution of purified recombinant plasmids containing the target sequence TAMS1 gene (pJET1.2/TAMS1) and MPSP gene (pJET1.2/MPSP) with the copy number 1010 were taken and tenfold dilution of these stock solution was made fold till copy number equals to 10. For determination of sensitivity of multiplex PCR assay, stock solutions of T. annulata and T. orientalis were mixed and diluted tenfold till copy number equals to 10.

Specificity assay for simplex and multiplex PCR

DNA was extracted from blood samples that were positive for Babesia spp., Anaplasma spp., and Trypanosoma spp. by microscopic examination. These positive DNA samples were used to determine the specificity of simplex PCR and multiplex PCR for T. orientalis and T. annulata. In addition, the cross-reactivity between T. orientalis and T. annulata was checked using their positive controls in both simplex and multiplex PCR.

Collection of samples

Blood samples from cattle suspected of hemoprotozoan disease having any of the clinical signs, i.e., fever, enlarged superficial lymph nodes, tick infestation, and anemia, were used to validate and evaluate the efficacy of the designed multiplex PCR. Blood samples were collected from 216 suspected cattle from the Veterinary Clinical Complex, LUVAS, Hisar, and organized dairy farms near Hisar, Haryana, India.

Microscopic examination of blood smears

The blood smears were prepared by fresh whole blood and stained with Giemsa stain as per standard procedure. Each slide was examined for presence of Theileria piroplasm in erythrocytes and schizonts in lymphocytes.

Evaluation of multiplex PCR using field samples

The DNA was extracted from the blood using DNeasy blood and tissue kit (Qiagen, Germany) by following the method recommended by the manufacturer. The extracted DNA was then stored at − 20 °C until further used. First, simplex PCR was performed for each sample to detect presence of T. annulata and T. orientalis. Samples were then screened by multiplex PCR, and the results of all tests were compared.

Sequencing of samples

Sequencing was performed using the PCR amplicons from representative samples positive for T. annulata and T. orientalis, to further confirm the PCR results. PCR amplicons were purified for selected samples from agarose gel using a gel purification kit (Qiagen, USA) and sequenced using an array-based automatic capillary ABI 3130xL machine (Applied Biosystems™, USA).

Results

Standardization of simplex and multiplex PCR

PCR conditions were individually optimized for the T. annulata TAMS1 primer pair and the T. orientalis MPSP primer pair. The optimum annealing temperature was 58 °C based on the gradient PCR results for both primer pairs. The final optimized thermo-cycling conditions for both simplex PCR were as follows: initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s, and a final extension at 72 °C for 10 min. The TAMS1 and MPSP primer pairs amplified amplicons of predicted sizes of 229 bp and 466 bp, respectively.

The optimum conditions for multiplex PCR were initial denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 40 s and then a final extension at 72 °C for 10 min. In a multiplex PCR assay, the specific amplicons (229 bp and 466 bp for T. annulata and T. orientalis, respectively) belonging to individual species could be readily differentiated in a 1.8% agarose gel. No non-specific amplification was detected in the multiplex PCR assay.

Minimum detection limits of simplex and multiplex PCR

The simplex PCR for T. annulata was able to detect template up to 102 copies whereas the detection limit of simplex PCR for T. orientalis was 103 copies. The detection limit of multiplex PCR was 102 and 103 copies for T. annulata and T. orientalis, respectively.

Specificity assay for simplex and multiplex PCR

Each primer set was checked for cross-reactivity with other hemoprotozoa such as Babesia spp., Anaplasma spp., and Trypanosoma spp. Primers were species-specific, as no amplification was observed for DNA from other parasites (Fig. 1). In addition, T. annulata was not amplified by T. orientalis primers and vice versa. These results indicated that both primer sets were highly specific.

Fig. 1
figure 1

Specificity assay of Multiplex PCR for detecting T. annulata (229 bp) and T. orientalis (466 bp). Lane L: 100 bp ladder; Lane 1: positive control of T. annulata; 2: positive control of T. orientalis; 3: Positive sample for T. annulata and T. orientalis; 4: Positive sample for Babesia spp.; 5: Positive sample for Anaplasma spp.; 6: Positive sample for Trypanosoma spp., N: negative control

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Microscopic examination of blood smears

Blood smears of 216 animals were examined, out of which 77 (35.64%) animals were found positive for theileriosis. The piroplasm of various shapes such as round dot like, signet-ring shaped, oval, elongated, and rod-shaped were observed in erythrocytes. All the animals that were positive by blood smear examination were also positive in PCR examination. Microscopic smears for blood samples positive for PCR for T. annulata and T. orientalis were compared and are shown in Fig. 2 (T. annulata), Fig. 3 (T. orientalis), and Fig. 4 (mixed infection). From comparison, it was found difficult to distinguish the two infection and almost impossible to identify the mixed infections.

Fig. 2
figure 2

Geimsa stained blood smear of sample with infection of T. annulata (100 × magnifications)

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Fig. 3
figure 3

Geimsa stained blood smear of sample with infection of T. orientalis (100 × magnifications)

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Fig. 4
figure 4

Geimsa stained blood smear of sample with mixed infection of T. annulata and T. orientalis (100 × magnification)

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Evaluation of field sample using multiplex PCR

DNA samples from 216 cattle were examined using both simplex and multiplex PCR (Fig. 5). On examination by simplex PCR for T. annulata, 128 (59.3%) animals out of 216 were found positive for T. annulata. In simplex PCR for T. orientalis, 19 (8.8%) animals out of 216 were found positive for T. orientalis. Multiplex PCR showed that out of 216 animals, 131 (60.64%) were found to be infected, of which 112 were infected with T. annulata, five were infected with T. orientalis, and 14 had mixed infection. The results of multiplex PCR were similar to those of simplex PCR except for two animals that were positive for T. annulata in simplex PCR while negative in multiplex PCR (Table 2). This is the first report of T. orientalis in Haryana, India.

Fig. 5
figure 5

Multiplex PCR for detection of T. annulata (229 bp) and T. orientalis (466 bp) of blood samples of cattle. Lane L: 100 bp ladder (GeneDirex); N: negative control; P: positive control; Lane 1 to 11: samples

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Table 2 Result of simplex and multiplex PCR assay of screened blood samples of cattle
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Sequencing of samples

Representative samples were sequenced and submitted to NCBI (accession no. ON248942 for T. orientalis, and ON248941 for T. annulata). These nucleotide sequences were analyzed using the NCBI BlastN software. The submitted sequence of T. orientalis showed 98.95% identity with isolate of cattle from China (MT517305.1) and 97–98.95% similarity with other T. orientalis sequences. The submitted sequence of TAMS1 gene of T. annulata showed 97.9% with T. annulata isolates of cattle from Ankara, Turkey (AJ276654.1), and 95–97% similarity with other T. annulata sequences.

Discussion

Owing to the high tick vector population in tropical and subtropical regions, haemoprotozoan diseases are a major concern for dairy animals in these regions (Li et al. 2016; Fukushima et al. 2021; Krishnamoorthy et al. 2021). However, due to climatic changes and global warming, the vectors such as ticks have expanded to new regions, thus increasing the risk of haemoprotozoan diseases in these areas (Medlock et al. 2013; Rojas-Downing et al. 2017). Theileriosis is one such haemoprotozoan disease responsible for huge economic losses in the dairy industry (Narladkar 2018). There are several species of Theileria infecting bovines, but T. annulata is the most widely prevalent and causes severe clinical infections. Therefore, epidemiological studies and preventive strategies primarily focused on the T. annulata. But in past few decades, outbreaks and clinical reports of T. orientalis had been reported worldwide particularly from Australia and Asia including India (Kamau et al. 2011; Aparna et al. 2011; Kakati et al. 2015). Thus, these areas have prevalence of both the species, which necessitates the inclusion of T. orientalis also in the epidemiological studies, vaccination for formulation of better prevention, and control strategies for theileriosis.

The conventional approach of diagnosis by microscopic examination is neither sensitive (Nayel et al. 2012) nor can it differentiate between these two species (Bilgiç et al. 2013). Similarly, serological tests have the problem of cross-reactivity between different species of Theileria (Renneker et al. 2009) and also have chances of false-negative results in chronic infections as immune responses in such cases wane away over time (Ranjan et al. 2015). Molecular tests such as PCR are highly sensitive and specific and multiplexing can reduce the cost and time of diagnosis for multiple pathogens, especially in epidemiological studies where many samples are to be analyzed (El Damaty et al. 2021; Tuli et al. 2015). Many scientific reports of multiplex PCR for detecting different hemoprotozoa such as Theileria, Babesia, Anaplasma, and Trypanosoma are available (Bilgiç et al. 2013; Ashuma et al. 2014; Ganguly et al. 2020; Charaya et al. 2021). However, literature describing multiplex PCR assays for diagnosing different species of Theileria in cattle is very scarce (Orkun et al. 2012; Junlong et al. 2015). Therefore, in this study, multiplex PCR was standardized for rapid differential diagnosis of T. annulata and T. orientalis. The multiplex PCR standardized in the present study can differentially detect both the species of Theileria in less than 5 h of receiving a blood sample.

Highly specific and sensitive primers are required for efficient PCR. Primers based on Tams1 and MPSP genes were used for T. annulata and T. orientalis, respectively. Both primers were specific as they showed no amplification for other hemoprotozoa and one another in the specificity assay. TAMS1 gene is widely used for the detection of T. annulata. Kirvar et al. (Kirvar et al. 2000) also reported that primers designed from the TAMS1 gene showed no cross-reactivity with other hemoprotozoa. The present and previous studies have shown that there is no cross-reactivity of the TAMS1 gene T. annulata with other Theileria species, making it a suitable candidate for multiplex PCR (Orkun et al. 2012; Kundave et al. 2018; Ganguly et al. 2020). Similarly, the MPSP gene is also widely described for molecular identification of T. orientalis (Ota et al. 2009; Kamau et al. 2011). The specificity of the multiplex PCR was further elucidated by the fact that amplicons of only the expected size were observed in the analysis of field samples.

The standardized multiplex PCR assay in the present study had a very low minimum detection limit for both species; therefore, the assay had high sensitivity. The high sensitivity of this assay ensures that it can efficiently detect subclinical infections (Khatoon et al. 2015). The detection of subclinical carriers is of utmost importance in theileriosis control as they serve as source of infection for healthy animals (Abdela and Bekele 2016). Additionally, subclinical infections are linked to anemia, poor immunity, and production loss (Kolte et al. 2017).

The prevalence by microscopic examination (35.64%) was very low when compared to that of multiplex PCR (60.64%). The lower sensitivity of blood smear examination was reported by other workers also (Nourollahi-Fard et al. 2015; Kundave et al. 2015; Charaya et al. 2016). The sensitivity of the test was lower than PCR because of the low parasitemia in chronic and subclinical carrier animals (Nayel et al. 2012). There is lot of polymorphism in piroplasmic form of Theileria which creates confusion and requires a great expertise to correctly identify the organism (Acharya et al. 2017). The distinction of the two species was not possible by microscopic examination; thus, the prevalence of specific species of theielria and mixed infections cannot be determined. In the present study also, microscopic smears for blood samples positive for PCR for T. annulata and T. orientalis were compared; it was difficult to distinguish the two infection and almost impossible to identify the mixed infections.

On screening 216 animals using simplex PCR, 128 (59.25%) were positive for T. annulata, while 19 (8.79%) were positive for T. orientalis. The results of multiplex PCR were almost the same as those of simplex PCR except for two cases, which were positive for T. annulata with simplex PCR while negative with multiplex PCR. This minute difference may be because parasitemia might be very low in those cases, as the detection ability of multiplex PCR is compromised by the extremely low level of parasitemia (Ganguly et al. 2020). The multiplex PCR used in the present study was as efficient as the simplex PCR, highlighting its potential usefulness in epidemiological investigations.

The results indicated that the prevalence of T. annulata was higher than that of T. orientalis in the study region. A high prevalence of T. annulata has been previously reported in the studied geographical area (Ganguly et al. 2020; Charaya et al. 2021). However, there have been no previous studies regarding the prevalence of T. orientalis in the studied geographical area. To the best of our knowledge, this is the first report of T. orientalis in Haryana, India. T. orientalis is generally considered mild or virulent, but recent outbreaks with high mortality and morbidity from different parts of the country suggest the importance of epidemiological investigation of the organism. The present study also indicated that mixed infections are also prevalent. Multiple species infections of parasites can alter their pathogenicity and disease pattern and can also lead to cross-immunity (Awad et al. 2020). Competition between the two species of parasites in the same host leads to genetic changes in both parasites (Bashey 2015). Thus, periodic molecular epidemiology of both Theileria spp. is necessary in areas such as India where both species co-exist. The multiplex PCR assay standardized in the present study could potentially be useful in such investigations.

Conclusion

T. annulata and T. orientalis are both important species of Theileria that affect cattle and both these species co-exist in the same geographical area. Thus, to formulate better prevention and control strategies, the epidemiology of both the species should be investigated. The multiplex PCR assay developed in the present study can rapidly and efficiently detect both the Theileria species, i.e., T. annulata and T. orientalis. The assay is highly specific and sensitive. Thus, it may be useful in epidemiological studies.