Indian Journal of Animal Research

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Clinico-morphological and Molecular Detection of Bovine Tropical Theileriosis in Cattle

Anil Kumar1,*, Rajesh Kumar2, Ramesh Tiwary2, Bhoomika3, G.D Singh1, Ajit Kumar4, Anil Gattani5, Pallav Shekhar6
1Department of Veterinary Clinical Complex, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
2Department of Veterinary Surgery and Radiology, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
3Department of Veterinary Public health, Bihar Veterinary College, Bihar Animal Sciences University Patna-800 014, Bihar, India.
4Department of Veterinary Parasitology, Bihar Veterinary College, Bihar Animal Sciences University Patna-800 014, Bihar, India.
5Department of Veterinary Biochemistry, College of Veterinary Science and Animal Husbandry, Jabalpur-482 001, Madhya Pradesh, India.
6Department of Veterinary Medicine, Bihar Veterinary College, Bihar Animal Sciences University, Patna-800 014, Bihar, India.
Background: Theileria spp. were targeted for detection and differentiation from clinical samples using PCR in the current investigation and the diagnostic sensitivity of this method was evaluated in comparison to microscopic observations. The clinical characteristics of bovine tropical theileriosis as clinical markers were also analysed based on the PCR data.

Methods: On the basis of clinical evaluation and microscopic inspection of blood smears, 200 blood samples from cattle suspected of having theileriosis were examined. To confirm the disease, the PCR method was employed by using 18S rRNA and Cyto b1 gene to confirm Theileria spp. and Theileria annulata respectively in clinical samples. 

Result: The results showed that microscopic examination detected 65/200 (32.50%), whereas, the PCR assay identified 120/200 (60.00%) for Theileria spp. Of the total 200 samples studied, 77 (38.50.3%) were positive for T. annulata specific PCR. Based on PCR confirmation of T. annulata infection in cattle, the predominant clinical signs were pyrexia, ticks’ infestations, anorexia, enlargement of lymph node, pale mucous membrane, lacrimation, coughing, drop in milk production and emaciation. The other clinical signs were observed as icterus, nasal discharge, salivation, melena and exophthalmia.
Bovine theileriosis is a widespread tick-borne disease of cattle in tropical and subtropical regions brought on by intracellular hemoparasites of the genus Theileria (Bursakov and Kovalchuk, 2019). Thileria annulata (T. annulata) widely considered to be more pathogenic causing Tropical Theileriosis and Theileria orientalis generally considered to cause Benign Theileriosis are the two main species found in India, that infect bovines and transmitted by Hyalomma anatolicum anatolicum and Haemophyssalis bispinosa respectively (Aparna et al., 2011).

Theileriosis is mostly diagnosed in the field based on clinical symptoms and a tick infestation on the diseased animals (Qayyum et al., 2010) and conventional laboratory techniques. The “Gold Standard” diagnostic test among conventional diagnostic methods is still the direct microscopic identification of theileria in blood smears and/or lymph node fluid smears. However, these tests are robust, have low sensitivity and are completely dependent on trained laboratory technicians. Giemsa stained thin blood smears are usually sufficient for detecting acute infections, as evidenced by Theileria schizonts in lymph node smears and piroplasms alone or in conjunction with schizonts in blood smears, but may not be in sub-clinical and carrier infections due to very low parasitemia (Salih et al., 2015). On blood slides, it is challenging to distinguish the morphology of Theileria species’ piroplasms (Almeria et al., 2001) especially due to mixed infections. Deoxyribonucleic acid (DNA)-DNA hybridization and the polymerase chain reaction (PCR) have combined to produce very sensitive and specific molecular diagnostic techniques for the detection and characterization of the organisms that cause theileriosis (Collins et al., 2002). Theileria annulata can be distinguished from other non-pathogenic Theileria species using PCR, which is thought to be a very sensitive and specific diagnostic method for pathogen identification and differentiation.

Aiming to identify and distinguish Theileria spp. from clinical samples using PCR assays, this study’s diagnostic sensitivity was compared to microscopic observations in light of the limitations of conventional diagnostic approaches. Based on PCR results, the clinical signs of bovine tropical theileriosis as clinical markers were also evaluated.
Selection of animals
 
The present study was held in the Department of Veterinary Medicine, BVC, BASU, Patna during March 2020 to February 2022. A total of 200 cattle of age ranging 3 months-6 years, either sex and irrespective of breed were selected based on the clinical signs suggestive of theileriosis like high fever of 104-107°F, swelling of parotid lymph nodes, pre-scapular and pre-femoral lymph nodes, pale mucous membrane, suspended rumination, bilateral nasal discharge, lacrimation and or which have the problem of tick infestation etc.
 
Collection of blood samples
 
The blood samples @ 3 ml from each suspected cases were collected aseptically from jugular vein and transferred in EDTA coated vial for preparation of thin blood smears and DNA extraction.
 
Microscopic examination of blood smears
 
Blood smears were stained by Giemsa’s staining technique. The smears were examined microscopically for the presence of Theileria piroplasms in erythrocytes and Koch’s blue bodies in lymphocytes. The presence of even a single piroplasm was considered positive for Theileria spp. Before considering it negative, at least 50 microscopic fields per slide were observed. The morphological characteristics of Theileria were identified according to key described by Soulsby (1982).
 
DNA isolation and PCR Amplification
 
Genomic DNA from each blood sample was isolated by using the Wizard® Genomic DNA Purification Kit (Promega, USA), as per the manufacturer’s instructions and stored at - 20°C till future use. The different sets of primers used in the study were custom synthesized from Eurofins Genomics Pvt. Ltd. (India) (Table 1) for the amplification of targeted genes of Theileria spp. both on genus and species-specific grounds. The PCR was performed as per the method described previously (Cao et al., 2013) with slight modification briefly, 25 μl reaction mixture containing 12.5 μl of MAX PCR Master Mix (2´Premix) (Takara), 2.0 μl (10 pmol/ μl) of each primers, 2 ìl of the DNA template and nuclease free water (Thermo Scientific, USA) to make total volume up to 25 μl. Positive control and negative control have been included in each run. The cycling condition for Theileria genus was, Initial denaturation at 95°C for 5 minutes and 35 cycles of denaturation; 1 min.  at 94°C, annealing; 1 min. at 60°C and extension 90 seconds at 72°C and final extension for10 minutes at 72°C. The cycling condition for T. annulata was initial denaturation at 95°C for 2 minutes and 35 cycles of: denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, extension at 72°C for 1 minute and final extension at 72°C for 10 minutes. The amplified PCR product was checked by electrophoresis on 1.5% agarose gel stained with ethidium bromide in horizontal electrophoresis apparatus (Thermo Scientific, China) and viewed in gel documentation system (VILBER).

Table 1: Theileria genus specific, species specific and self-designed primers.



Table 2: Animals positive for the presence of Theileria sp. by microscopic examination (ME) of blood smears and by PCR for Theileria spp. and Theileria annulata.


 
Data analysis
 
Chi square test was performed to test the association between blood smear examination and PCR.
The results of present investigation showed that microscopic examination detected 65/200 (32.50%), whereas, the PCR assay identified 120/200 (60.00%) for theileria spp. Of the total 200 samples studied, 77 (38.50.3%) were positive for T. annulata specific PCR (Table 2). Under microscopic examination, low to high numbers of polymorphous theileria parasites were observed mostly as annular or round form and some were detected as in oval, coma or dot form inside the red blood cells of the stained blood smears (Fig 1) and also in few cases Koch’s blue bodies  inside lymphocytes were observed (Fig 2).

Fig 1: (GSTBS ´ 100) showing signet ring shaped piroplasms of Theileria spp.



Fig 2: Photomicrograph (GSTBS ´ 100) showing Koch’s blue bodies of Theileria spp. in lymphocytes.



The results of present study are in agreement with those of Kumar et al., (2022). Theileria genus specific 18S rRNA gene was used (Fig 3), because it has been shown to be an effective marker for investigation in Theileria spp. (Cao et al., 2013). PCR assay targeting cytob1 gene was used to amplify the single and specific 312 bp fragment to diagnose the tropical theileriosis (Fig 4). Bilgic et al., (2010) reported that cytochrome b gene is highly sensitive in detection of T. annulata infections in cattle and cytob1 gene is highly specific for the detection of T. annulata parasites at the level of low parasitaemia, especially in carrier cattle and can also discriminate T. annulata from non-pathogenic Theileria species and other haemoparasites (Bilgic et al., 2013). This demonstrates the effectiveness of the PCR technique for theileriosis confirmation and supports the use of genus- and species-specific primers in our research.

Fig 3: Showing the gel electrophoresis image of 18srRNA amplification of Theileria genus, where Lane 5 negative sample; Lane 11 negative control; Lane 12 positive control; other lane positive samples; lane M: 100 bp ladder.



Fig 4: Showing the gel electrophoresis image of Cytob1 gene amplification of T. annulata, where Lane 1-10 positive sample; Lane 11 negative control; Lane 12 positive control; lane M: 100 bp ladder.



Previous research has conclusively demonstrated that, when used to diagnose Babesia species and T. annulata, PCR-based methods are more sensitive than other diagnostic techniques (Kundave et al., 2017).

In the present study, 65 (32.50%) and 120 (60%) samples were found positive for Theileria spp. by microscopic examination and PCR, respectively. Chi-square statistical analysis revealed a significant (p<0.05) difference in detection sensitivity when PCR assay was compared with microscopic examination. The PCR included those 55 samples which were found negative by Giemsa’s staining (Table 3). Considering microscopic blood smear examination as the gold standard method, the sensitivity of PCR was found to be 100% in clinically suspected animals. The present study findings are in agreement with Charaya et al., (2016), Rajkumar et al., (2020) and Ullah et al., (2021) who reported a significantly higher sensitivity of PCR technique in detection of Theileria spp. as compared to microscopic blood smear examination and the technique also allowed for specific discrimination between pathogenic and non-pathogenic theilerias which cannot be accomplished by traditional diagnosis by microscopic observation (Almeria et al., 2001). 

Table 3: 2´2 Contingency table for comparison of blood smear and PCR.



Due to artefacts, incorrect staining, inexperience, loss of the piroplasmic form as a result of hemolysis and inadequate sensitivity, the microscopic examination of blood smears revealed false negative results (Chauhan et al., 2015) and lack of discrimination of other morphologically related parasites if mixed infections (Ullah et al., 2021). This makes it quite evident that PCR assays are better to microscopy examinations.

Out of 200 clinically examined animals in our study, 77 (38.50%) animals tested positive for T. annulata on PCR assays and displayed clinical indications of disease (Table 4).  The predominant clinical signs in cattle suffering from tropical theileriosis were pyrexia (100%), ticks infestations (92.20%), Anorexia (80.51%), enlargement of lymph node (75.32%), pale mucous membrane (70.12%), lacrimation (68.83%), coughing (59.74%), drop in milk production (49.35%) and emaciation (41.55%).

Table 4: Clinical signs in cattle infected with T. annulata.



The other clinical signs were observed as icterus (24.67%), nasal discharge (23.40%), salivation (22.10%), melena (15.58%) and exophthalmia (11.68%). Clinical indicators of T. annulata infection include anaemia, wasting, jaundice and enlargement of superficial lymph nodes (Ma et al., 2020). In the present study, 92.20 percent of theileriosis affected animals had tick infestation which is comparable to the results obtained by Khawale et al., (2020), who had observed 92.53 per cent of theileriosis affected animals with tick infestation, which are a major risk factor for the spread of theileriosis (Khattak et al., 2012). Variation in clinical signs shown by the animals in the present study might be attributed to various housing and management practices along with the degree of infection. In the early stages of the disease, due to increase in microschizont proliferation inside lymphocytes and inflammatory reactions in the infected lymph nodes, lymphoid hyperplasia superficial lymph nodes is observed (Al-Emarah et al., 2012).

In the present study, hyperthermia ranged from 103°F to 105°F was observed in cattle affected with bovine tropical theileriosis. The diverse nature of clinical symptoms of Theleriosis are due to high levels of inflammatory cytokines (TNF-α, IL-1 and IL-6), produced by infected mononuclear cells (Col and Uslu, 2006). The clinical signs like anorexia, emaciation and melena in the present study might be due to the increased cytokines. Overproduction of TNF-α and lymphocyte infiltration are thought to be contributing factors for ophthalmopathy in calf theileriosis (Shanker et al., 2013).

A significant loss in milk production and milk composition has been reported because of Theileriosis (Memon et al., 2017; Perera et al., 2014).

Pale mucus membranes indicator of anaemia, might be due to removal of the parasitized erythrocytes by reticulo-endothelial system (Farooq et al., 2019), persistent blood loss due to permanent blood sucking ticks (Durrani et al., 2008), reduced erythrogenesis due to TNF-α (Boulter and Hall, 2000) or due to erythrophagocytosis (Modi et al., 2015).

The respiratory symptoms like nasal discharge, respiratory discomfort and cough, could be attributed to advanced cases of severe pulmonary edema caused by released vasoactive substances from collapsing alveolar cells (Abdel-Hamied et al., 2020).

Therefore, thorough inspection of diseased cattle based on clinical signs especially of swollen lymph nodes, pallor mucous membranes, pyrexia, coughing respiratory distress, lacrimation and exophthalmia, as major clinical markers might be used in clinical diagnosis of T. annulata infection in the field conditions.
PCR assay demonstrated higher sensitivity for diagnosing Bovine theileriosis than microscopy. Theileria annulata may be distinguished from non-pathogenic Theileria species and other hemoparasites using the 18S rRNA gene and Cyto b1 gene based PCR assay, which can also be used to monitor the effectiveness of pharmacological treatments. Despite the lack of diagnostics in the field, considering these clinical manifestations, particularly swollen lymph nodes, pallor of the mucous membranes, pyrexia, coughing, respiratory distress, lacrimation and exophthalmia as the major clinical markers could help in making a clinical diagnosis of theileriosis in cattle. 
The author acknowledges the support and cooperation of the Dean, BVC, Patna and Director Research, Bihar Animal Sciences University (BASU), Patna for providing necessary funds and facilities for completing the research work.
None

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