Molicular diagnosis and haematobiochemical changes of theileriosis in crossbred cattle

Introduction

Bovine tropical theileriosis, a lymphoproliferative disease caused by Theileria annulata, remains one of India's most economically important diseases. The bont-legged tick Hyalomma anatolicum trans-stadially transmits the infection (Bhattacharyulu et al., 1975) [9] . The disease is endemic in tropical and subtropical areas of the world, with prevalence spanning Southern Europe and North Africa through the Middle East and Southern Russia into India and Southern China (Uilenberg, 1981; Dolan, 1989; Dumanli et al., 2005; Morrison and McKeever, 2006) [60, 19, 21, 44] . Around 250 million cattle worldwide are at risk of tropical theileriosis (Bishop et al. 2004) [11] , while in India, (approximately) 39 million crossbred cattle are at risk of T.annulata infection (Kolte et al., 2017) [34] . Due to the huge economic loss, to the tune of US\$ 384.3 million annually, theileriosis inflicts to the national economy, the disease has been listed among the top ten livestock diseases in India. (Minjauw and McLeod, 2003) [41] . High mortality and morbidity in all age groups of exotics and crossbreeds cattle are hallmarks of theileriosis (Jithendran, 1997; Bishop et al., 2004) [30,11] . The disease is clinically characterized by pyrexia, anorexia, enlarged superficial lymph nodes, tachycardia, dyspnoea, icterus and pallor of mucous membrane. Occasionally progressive anaemia, respiratory symptoms and gastrointestinal disorders, torticollis, convulsions and other nervous signs are also reported. Abortion, significant loss in milk production and death are other sequalae of this disease (Eamens et al., 2013) [13] . The case-fatality rate of the infection can be as high as 80% in the exotic breeds of cattle; however, the same is about 20% in the indigenous animals (Glass et al., 2003) [23,24] . The disease is prevalent in most parts of India (Sayin et al., 2003, Panda et al., 2011; Vahora et al., 2012; Chaudhri et al., 2013; Dharanesha et al., 2017) [54,65,61,14,18] . The diagnosis of T.annulata infection in bovines relies on traditional parasitological methods, including microscopy-based detection of piroplasms and macroschizonts in Giemsa-stained thin blood smears and lymph node biopsy, respectively (Aulakh and Singla, 2006) [6] . Even though the conventional tests are reliable and have high specificity, they fail to detect the pathogens in the asymptomatic carrier animals with low parasitaemia.

Further, it is difficult to differentiate the Theileria species based on the morphological features (Liu et al., 2010) [36] . The unrealistic time involved with the microscopical detection method, especially for the animals with low parasitaemia, renders it unsuitable for extensive studies. Furthermore, carrier animals are a potential source of infection for vectors and healthy susceptible populations and a means of spreading the disease to new areas (Callow, 1984; Bilgic et al., 2013) [13,10] . Hence, for sensitive diagnosis of the infection in carriers, molecular assays like PCR will be highly useful, detecting very low levels of parasitemia. The PCR has recently been the most widely used method for detecting T.annulata infection in cattle in epidemiological studies. Several investigations have shown that the technique is more sensitive and specific in identifying piroplasm-carrier animals than other traditional diagnostic techniques (d' Oliveira et al., 1995; Vatansever and Nalbantoglu, 2002; Paliwal et al., 2019; Mohmad et al., 2021) [20, 43, 47, 62] . The PCR also provided a more straightforward method to detect infection in the vector ticks for epidemiological studies as the staining of dissected salivary glands of the ticks is cumbersome, mainly when large sample sizes must be processed (Das and Ray, 2003. The study designed with the following objectives: to record the clinical signs and certain haemato-biochemical changes in crossbred cattle with Theileriosis, to diagnose the clinical Theileriosis in crossbred cattle by blood smear examination and to detect asymptomatic form of Theileria annulata with Polymerase Chain Reaction (PCR) [17] and to study the survival and prognostic findings in crossbred cattle with Theileriosis.

Materials and Methods

The study entitled Study on Diagnosis and Treatment of Theileriosis in Crossbred Cattle was conducted form August 2021 to December 2021 in the Department Veterinary Clinical Complex, College of Veterinary Science, Tirupati and surrounding Veterinary hospitals and Dairy farms in certain parts of Chittoor district, Andhra Pradesh. Ten apparently healthy crossbred cattle were selected as control group for obtaining normal data for comparison of parameters under study. Crossbred cattle presented to the large animal ward, Veterinary Clinical Complex, College of Veterinary Science, Tirupati, surrounding Veterinary Hospitals and Dairy farms in certain parts of Chittoor district with clinical signs suspected for theileriosis and asymptomatic form of theileriosis were taken up for the present study. Five ml blood was collected from twenty crossbred cattle with theileriosis. Five ml blood was collected once from ten apparently healthy crossbred cattle (Group I) for comparative study. Lymphnode aspiration was done for detection of schizonts in lymphocytes (Koch's blue bodies). Serum was separated immediately after clotting by centrifugation at 3000 rpm for 3 minutes). Cardiac Biomarker cardiac troponin-I was qualitatively assessed by using combo kit which was supplied by CTK Biotech, Inc, USA. The occurrence of Theileria annulata infections in crossbred cattle was recorded. The occurrence in relation to age, breed and gender were further analyzed. Clinical examination Crossbred cattle with symptoms suggestive of theileriosis like pyrexia, lymph node enlargement, anorexia, reduced milk yield, depression, anemia and icterus were subjected to thorough clinical examination and recorded in a prescribed proforma. Giemsa staining Peripheral blood smears were fixed with methanol for 1 minute and air dried. The smears were stained by Giemsa staining solution, 1:8 dilution, for 45 minutes, washed under running tap water, air dried and examined for Theileria annulata organisms under oil immersion objective of microscope. Field staining Two coplin jars were filled with Field's stain A and B solutions separately. Peripheral blood smears were fixed in methanol for one minute and air dried. This was followed by coupling of stain B and allowing it to act for 30 seconds. After washing the slides in running tap water, stain A was added and allowed to act for 30 seconds. The slides were further washed, air dried and observed under 100 X objective of a light microscope. Polymerase Chain Reaction Genomic DNA isolation by DNA blood mini kit (Qiagen) DNA isolation was done for 50 blood samples from the suspected cases for the detection of Theileria annulata. Organism Primer sequence pair Product size (bp) Reference Theileria annulata Tams 1 F: ATG CTG CAA ATG AGG AT Tspm 1 R: GGA CTG ATG AGA AGA CGA TGA G 785 Kirvar et al. (2000).

Evaluation of haemato-biochemical parameters

Haematological parameters such as haemoglobin, packed cell volume, total erythrocyte count, total leukocyte count and differential leukocyte count were estimated as per the standard procedures (Coles, 1986) [16] . Hb (g%) was estimated by acid hematin method, PCV (%) was estimated by microhematocrit method, TEC (106/µl) and TLC (103/µl) were carried out by haemocytometer method. Blood smear was stained with Leishman's method and DLC was carried out by battlement method (Omer et al., 2003) [45] . Serum biochemical parameters were estimated by semi auto analyzer using the Assay kits and techniques. Serum total protein Serum total protein was estimated by Biuret method, End point assay using Erba Mannheim ready to use kit and the values were expressed in g/dl (Modi et al., 2015) [42] . Serum albumin Serum albumin was estimated by Bromo cresol green (BCG), end point assay method using Erba Mannheim ready to use kit and the results were expressed in g/dl (Swami et al., 2019) [58] . Total bilirubin Total bilirubin was estimated by Jendrassik and Grof method, end point assay using Genix, Proton Biologicals India Pvt. ltd., ready to use kit and the results were expressed in mg/dl (Goel et al. 2017) [26] . AST Serum AST was estimated by International Fedaration of clinical Chemistry method (IFCC), Kinetic assay using Erba Mannheim ready to use kit and the results were expressed in IU/L (Al-Emarah et al., 2012) [4] . Glucose Serum Glucose was estimated by Trinder's method, end point assay using Erba Mannheim ready to use kit and the results were expressed in mg/dl (Gunes et al., 2016) [26] . Creatinine kinase -MB Serum Creatinine Kinase -MB was estimated by Modified International federation of chemical chemistry method (IFCC), kinetic assay using Biosystems ready use kits and the results were expressed in IU/l (Gupta et al., 2021) [28] . Cardiac Troponin -I Serum Cardiac Troponin -I was Qualitatively assessed by using the combo kit supplied by CTK Biotech, Inc, USA (Gupta et al., 2021) [28] .

Statistical analysis: Statistical analysis was performed by using one way ANOVA and correlation analysis by using software called IBM-SPSS (Software Packages for Social Sciences) version 22.0.

Results and Discussion

In the present study crossbred cattle with clinical signs of theileriosis presented to the large animal ward, Veterinary Clinical Complex, College of Veterinary Science, Tirupati, surrounding Veterinary Hospitals and dairy farms in certain parts of Chittoor districts, Andhra Pradesh were screened. The occurrence of theileriosis in crossbred cattle was observed to be 14.2 per cent by blood smear and PCR. Investigation of age wise occurrence of theileriosis revealed that the crossbred cattle of 2-4 years age group were most susceptible (50.0%), followed by less than 1 year age group (25%) and more than 5 years age group (25.0%). These findings were in agreement with Alukh and Singla et al., (2006) [6] ; Panda et al. (2011) [65] ; Velusamy et al. (2014) [63] ; Kala et al. (2018) [32] and Sinha et al. (2021) [58] who opined that theileriosis was common among crossbred cattle that were of 2 -4 years of age group. Inverse age resistance and stronger passively acquired immunity were the contributing factors behind the lower incidence of haemoprotozoan parasites in younger age group of animals (Maharana et al. (2016) [38] . While, Khan et al. (2017) [33] reported higher prevalence in crossbred cattle above 5 years (29.90%), followed by calves below 1 years (27.15%) and in cattle 1-5 years age (23.06%). In the present study, Holstein Friesian crossbred cattle (50.0%) was most commonly affected with theileriosis followed by crossbred Jersey (30.0%) and Jersey cattle (20.0%). These findings were in agreement with previous studies (Terada et al., (1995) [63] ; Saleh and Mahran (2003) [52] ; Sayin et al. (2003) [54] ; Zahid et al. (2005) [64] ; Abdela and Bekele (2016) [1] ; Acharya et al. (2017) [2] and Goud and Vijayakumar [1] (2020) [27] . The percentage of occurrence of theileriosis in Holstein Friesian crossbred cattle might be related to more attention in the management of the crossbred cattle and development of no or less immunity as reported by McFadden et al. (2011) [40] and Kala et al. (2018) [32] . The present study revealed that female crossbred cattle (90.0%) were more commonly affected than male (10.0%). A female predominance in the present study concurs with earlier reports (Panda et al. (2011) [65] ; Abdela and Bekele (2016) [1] and Kala et al. (2018) [32] . Hence it is opined that the female predominance in the present study due to rearing female crossbred cattle for milk production in large numbers. Higher prevalence in female crossbred cattle population is also due to hormonal disturbances due to its use in milk production, which pose it to weakened immune system as reported by Sajid et al. (2009) [51] and Panda et al. (2011) [65] . This might be due to less market value of male crossbred cattle and also farmers are interested in raising female cattle as replacement stock due to introduction of artificial insemination technology in livestock production by owners who are not interested for rearing and spending on male calves as a result of which a greater number of blood samples from female crossbred cattle were presented for diagnosis. Similar observations were also recorded by Panda et al. (2011) [65] and Khan et al. (2017) [33] . Seasonal investigation on theileriosis in crossbred cattle revealed that the season played a significant role in the incidence of theileriosis in crossbred cattle. The highest incidence of disease was observed in the rainy season (62.0%) followed by winter season (38.0%). These might be due to abundance of ticks during the rainy season. The findings were in agreement with Kohli et al. (2014), Kala et al. (2018) [32] who reported that theileriosis most prevalent in rainy season. Our results were also in close agreement with a study, in which Panda et al. (2011) [65] ; Vahora et al. (2012) [61] ; Bhatnagar et al. (2015) [8] and Kala et al. (2018) [32] recorded higher incidence of theileriosis during rainy season. The most prominent clinical signs observed in crossbred cattle with theileriosis were anorexia, pyrexia, pale mucous membranes, milk yield, lymph node enlargement, depression, ectoparasite infestation, icterus, brown coloured urine, weight loss respiratory distress, diarrhoea and bloody faeces. The present findings were in accordance with earlier studies of Aulakh and Singla (2006) [6] ; Osman and Al-Gaabary (2007) [46] ; Col and Uslu (2007) [15] ; Hussein et al. (2007) [29] ; Masare et al. (2009) [39] ; Al-Hosary et al. (2010) [22] ; Qayyum et al. (2010) [48] ; Khan et al., (2011) [33] ; Sumathi and Veena (2012) [57] ; Singh et al. (2014); Ayadi et al. (2016) [7] and Singh et al. (2017). Anorexia could be attributed to persistence of fever. More over the enlargement of Lymphnode could be explained by lymphoid hyperplasia in early stages of the disease that occurs due to proliferation of schizonts inside the mononuclear cells and causes inflammatory reaction inside the infected lymphnodes (Anon (1997) [5] and Ugalmugle et al. (2010). In the present study pyrexia is due to the liberation of endogenous pyrogens attributed to cellular lysis and parasitemia leading to the stimulation of thermoregulatory center in the hypothalamus as reported by Glass et al. (2003) [24] . Increase in respiratory rate might be due to accumulation of edematous fluid inside the lung and thoracic cavity Muraguri et al. (2006). The depression and reduced milk yield may be due to inappetence, fever, anemia and deteriorating effect of parasite on body of the animal. Diarrhorea and bloody faeces seen in infected crossbred cattle can be explained as a result to inflammatory reaction and ulceration of abomasum and intestinal tract (Radostitis et al. (2007). In the present study the crossbred cattle showed pale mucus membranes with a similar finding in studies of Col and Uslu (2007) [15] ; Hussein et al. (2007) [29] ; Masare et al. (2009) [39] ; Singh et al. (2014); Madhukar et al. (2016) [37] ; Singh et al.

(2017) and Goud and Vijayakumar (2020) [27] . They explained that the paleness of mucus membranes exhibited the development of anemia and reduction of the haemoglobin concentration and total erythrocytic count was due to destruction and removal of infected erythrocytes by reticuloendothelial system. Persistent loss of blood causing by persistent blood sucking ticks could play a role as well. The removal of piroplasams infected erythrocytes by macrophages and in the organs of reticuloendothelial system has been suggested as a cause of anemia (Singh et al. (2001). The normocytic normochromic anemia observed in crossbred cattle with T.annulata attributed to the toxic metabolites of which has harmful effects on the bone marrow as they interferes the process of erythropoiesis. Boulter and Hall (2000) [12] , mentioned that Tumor Necrosis Factor α has been implicated in the pathogenesis of anemia in theileriosis by suppressing haemopoietic progenitus. In infected crossbred cattle icterus mucus membranes which are a yellowish discoloration examined on eye conjunctiva which is a result of increased level of total bilirubin. The jaundice of conjunctival mucus membranes is similarly found by Col and Uslu (2007) [15] ; Hussein et al. (2007) [29] ; Masare et al. (2009) [39] ; Ugalmugle et al. [37] and Singh et al. (2017).The icterus observed in affected animals may be due to the reason that, infected and even uninfected red blood cells were destroyed mainly in the liver and spleen, resulting in progressive haemolytic anaemia, the number of infected erythrocytes increases drastically and phagocytosis by reticuloendothelial cells of parasitized erythrocytes lead to development of haemolytic anaemia and icterus as reported by Radwan et al. (2013) [50] . Leukogram showed significant decrease in total leukocyte count and Neutrophils while the lymphocytes showed significant increase in comparison with control group. Such changes in the leukogram might be attributed to persistent harmful effect of T.annulata effect on haemopoietic organ especially on bone marrow and their interference with the process of leukogenesis and due to proliferation of lymphocytes in the lymphoid organ as defensive response to invading parasites. Relative increase number of lymphocytes and monocytes reflects compensating mechanism as target cells in response to their invasion with T.annulata as reported by Sarma et al. (2016) [53] . Similar results were observed in theileriosis infected crossbred cattle Lamiaa et al. (1997) [35] . In the current work the mean values of total protein, albumin, glucose values revealed significant decrease in crossbred cattle infected with theileriosis when compared with control group. In contrary, Total serum globulin, total bilirubin and AST showed significance increase in mean values of the infected crossbred cattle. These results were in agreement with Omer et al. (2003) [45] ; Col and Uslu (2007) [15] ; Hussein et al. (2007) [29] ; Alam and Nasr (2011) [3] and Modi et al. (2015) [42] . The above significant biochemical changes probably indicate inflammatory -changes in hepatic cells which in turn effect their functions (Singh et al. (2001). Changes in protein may be due to harmful effect of theleria toxic metabolites on liver cells. These results were supported by Stockman et al. (2000) and Yeruham et al. (2003). Low serum total protein concentration in crossbred cattle naturally infected with T.annulata was probably due to hypoalbunemia arising from liver failure as reported by Omer et al. (2003) [45] . Hyper gamma globunemia was mentioned as a major cause of increase in globulin level as consequence of immune response against infection with theileriosis Singh et al. (2001) and Fujinaga (1981). Positive results with qualitative cardiac troponin I test and significant increase of Creatinine Kinase MB levels in two infected crossbred cattle might be due to anemia and parasitemia which contributed to the pathophysiology of myocardial damage as reported by Fartashvad et al. (2013) and Sarma et al. (2016) [53] . Increase in AST level in theileriosis infected crossbred cattle indicate the hepatic tissue damage that included coagulation necrosis, damage of hepatic cords with heavy infiltration of lymphocytes in peripheral areas indicating severe damage to hepatobiliary system due to hypoxia resulting from anemia and jaundice (Singh et al. Alam and Nasr (2011) [3] and Al-Emarah et al. (2012) [4] . In the present study lower levels of blood glucose, might be attributed to anorexia and metabolic disturbances of diseased animals (Pandey et al. (1987). The findings were in line with the Col and Uslu (2007) [15] they reported that the decreased serum glucose concentration due to utilization of glucose by theileriosis in the blood and hepatic disfunction as a result of theileria infection. In the present study microscopic examination of 141 giemsa stained peripheral blood smears from crossbred cattle 15 crossbred cattle were positive for T.annulata (10.56%) infection the findings were in agreement with previous reports of Sharma and Sohanpal ( 2001 2016) [1] and Acharya et al. (2017) [2] . Out of 50 blood samples 5 were positive for asymptomatic form of T.annulata infection by PCR with an expected amplification size of 785 bp. The present findings were in agreement with the studies of Quyyum et al. (2010); (Abdela and Bekele, 2016) [1] Acharya et al. (2017) [2] and Goud and Vijayakumar (2020) [27] . The PCR is superior method over the traditional method (Blood smear) to diagnose the asymptomatic form of [48] ; Nair et al., (2011); Abdela and Bekele (2016) [1] and Acharya et al. (2017) [2] . So, all these forms could be detected in giemsa staining smears maximum of signet ring forms, this clearly indicate that the prevalence of pathogen has been unaltered in the state to despite extensive treatment and control strategies adopted especially in dairy cattle sector. Babesia organisms are round to pyriform, amoeboid form occurring in the erythrocytes as reported by Soulsby (2012) [55] . Even though the routine diagnosis of T.annulata infection is based on light microscopy, this method is not sensitive enough or sufficiently specific to detect chronic carriers. So molecular techniques such as PCR enables sensitive and specific detection of T.annulata. Hence, the results of the present study highlighted the need to rule out the existence of carrier status in dairy cattle, before introducing any stressful condition like transportation or vaccination. It is confirmed that PCR test is more sensitive in detecting low grade of infection in carrier animals as compared with microscopic examination. Similar observations were made by d'Oliviera et al. (1995). Appropriate tick control strategies are also to be adopted for containing infection at reduced level in herds in endemic areas. In the present study 2 Holstein Friesian crossbred cattle with theileriosis infection died due to severe anemia and involvement of myocardium. These findings were in agreement with Razavi et al. (2015); Gupta et al. (2018) [27] and Gupta et al. (2021) [28] . The present study revealed severe anemia with involvement of myocardium are poor prognostic indicators in two crossbred cattle with theileriosis. In the present study clinical cases showed signs of improvement after 3rd day of treatment in both the groups. After 14th day of treatment clinical signs resolved and the clinical condition of the animals improved and disappearance of T.annulata organisms from blood. So, the animals returned to feeding and rumination. It was observed that there was significant increase in Haemoglobin, PCV, TEC values in both the groups after 14 days post treatment. The post treatment mean values of Total protein, serum albumin, blood glucose significantly increased and post treatment mean values total bilirubin and AST were significantly decreased in both the groups. These findings were in agreement with earlier studies of Omer et al. (2003) [45] ; Aulakh and Singla (2006) [6] ; Col and Uslu (2007) [15] ; Durrani et al. (2010) [22]: Quyyum et al. (2010); Panda et al. (2011) [65] ; Modi et al. (2015) [42] ; Gunes et al. (2016) [26] ; Kachhawa et al. (2016) [31] ; Khan et al. (2017) [33]: Singh et al. (2017); Gupta et al. (2018) and Swami et.al (2019) [58] . In the present study 2 clinical cases of crossbred cattle with theileriosis died before attempting the treatment the blood samples biochemical profile from these two cattle revealed the elevation in CKMB and positive for qualitative test cardiac troponin I indicates myocardial degeneration in crossbred cattle Fartashvand et al. (2013) and Gupta et al (2018) [27] .

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