Bovine cerebral theileriosis: first molecular report in cross bred cattle calf in India

Abstract

Bovine tropical theileriosis caused by Theileria annulata, is a serious constraint to Indian dairy industry with more fatal infections in exotic cattle and substantial losses to cross-bred and indigenous zebu cattle. The present communication is to place on record the first report of molecular based confirmed case of cerebral theileriosis caused by T. annulata coupled with its morphological detection, clinical manifestations, haematological alterations and therapeutic management in a cross bred cattle calf from India. After preparation of peripheral thin blood smear from cross bred cattle calf at the site of collection and fixation with methanol, blood sample brought to Department of Veterinary Parasitology, College of Veterinary Science and A.H, Jabalpur and stained by standard protocol for Giemsa staining. Genomic DNA was isolated from the collected blood sample using QIAamp® DNA blood mini kit following the manufacturer’s recommendations and PCR was performed. The cross bred cow calf revealed high rise in temperature (105.5°F), increased heart rate, labored breathing with seromucous nasal discharge, enlargement of prescapular lymph node and animal exhibited tonic clonic convulsions in response to any sudden noise. Giemsa stained thin blood smear revealed intraerythrocytic piroplasm and Koch’sblue bodies of T. annulata within the cytoplasm of lymphocytes. The species of Theileria was confirmed by molecular amplification of genomic DNA as T. annulata.

Introduction

Bovine tropical theileriosis (BTT) caused by Theileria annulata, an intracellular apicomplexan parasite and trans-stadially transmitted by an acarine host, Hyalomma anatolicum anatolicum has become a disease of paramount importance causing heavy mortality in infected animals worldwide. The disease is a serious constraint to Indian dairy industry with fatal infections in exotic cattle and substantial mortality in cross-bred and indigenous zebu cattle. Theileriosis accounts for almost 70% bovine mortality with global losses to the tune of US $ 800 million per annum (Brown 1997) and BTT imposes production losses of about US$ 384.3 million on Indian livestock sector (Rajendran and Ray 2014). Bovine cerebral theileriosis also termed as turning sickness is an aberrant form of infection manifested rarely by typical turning and circling movements (Van Rensburg 1976; Van Amstel 1982; Saville 2002) while symptoms of depression, head pressing, opisthotonus, nystagmus, blindness, hyperesthesia, dysmetria, dyssynergia and terminal semicomatose status are also often observed from the cases reported from different parts of the world including India (Dabak et al. 2004; Sudan et al. 2012). Theileria parva, T. taurotragi and more rarely T. annulata are considered causative agents of the BCT (De Vos et al. 1981; Saville 2002; Sudan et al. 2012). In acute form of disease, animals succumb after 2–21 days but rarely in chronic case animal may survive up to 6 months (Van Amstel 1982; Lawrence et al. 2004). Previous worker in India cited the cases of T. annulata associated BCT based on morphology and clinical symptoms only (Sharma and Gautam 1973; Srivastava and Sharma 1976; Sudan et al. 2012). The present communication is the first report of molecular based confirmation of cerebral theileriosis case caused by T. annulata coupled with its morphological detection, clinical manifestation, haematological alterations and therapeutic management in a cross bred cattle calf from India.

Materials and methods

A two- months cross-bred cow calf weighing approximately 110 kg showed a high rise in temperature (105.5°F), increased heart rate 110 bpm, labored breathing with seromucous nasal discharge, and enlargement of prescapular lymph node and animal exhibited tonic clonic convulsions in response to any sudden noise. Faeces were hard and covered with bloody mucus. On enquiry from owner it was noted that the mother and her calf was brought from Jind (Haryana).

Peripheral thin blood smear was prepared from cross bred calf at the blood collection site and immediately fixed with methanol. Blood sample was brought to Department of Veterinary Parasitology, College of Veterinary Science and A.H, Jabalpur and blood smear stained by the standard protocol for Giemsa staining and examined under oil immersion of microscope (Gupta and Singla 2012). Number of piroplasm infected erythrocytes was counted out of 100 erythrocytes to know the level of parasitaemia (Ramazan and Ugur 2007).

Five ml of blood was collected from the jugular vein of infected calf, and two ml was transferred to a vial containing EDTA. The remaining 3 ml blood was transferred into another vial without anticoagulant to separate serum for biochemical examination. Total erythrocyte count, haemoglobin, packed cell volume, total leucocyte count and differential count were estimated (Schalm et al. 1975). Serum creatinine, total serum proteins, serum albumin, and aspartate transaminase were estimated using a semiautomatic analyzer, as per the manufacturer’s instructions and standard kits. Body hair coat was carefully examined to find out any ectoparasites and identified using standard keys (Taylor et al. 2007). After 5 days of treatment 5 ml of the calf's blood was drawn again, as previously described, in order to determine the haematobicochemical parameters and check for the presence of Thieleria annulata in the treated calf.

Genomic DNA extraction

Genomic DNA was extracted from blood samples as per procedure of DNA isolation kit (DNeasy blood and tissue kit, Qiagen) and stored at -20 °C till further use. Genomic DNA isolated from T. annulata infected cattle blood showing high parasitaemia was utilized as positive control. Genomic DNA was also isolated from the whole blood of infection-free, three-day old cow calf (both microscopically and PCR negative) and used as a negative control.

PCR assay

Molecular diagnosis of T. annulata was carried out by PCR as per the procedure described by d’Oliveira et al. (1995) by using the following primers: F-5’GTAACCTTTAAAAACGT3’ and R-5’GTTACGAACATGGGTTT3’specifically targeting merozoite surface antigen gene.

For the PCR assay, following components were used; 12.5 µL master mix (2x), 1 µL each (10 pmol/µL) of forward and reverse primers and 1 µL of template DNA and the volume was made up to 25µL with nuclease-free water. The cycling conditions were: initial denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min, and the final elongation was done at 72 °C for 10 min. The PCR product was checked for amplification by electrophoresis on a 1.5% agarose gel and visualized using gel documentation system (Syngene, UK).

Results

Clinical observation

Clinical examination of calf revealed pale and icteric mucous membrane of the conjunctiva. The animal was weak and emaciated. The muzzle was dry with seromucous nasal discharge from both the nostrils. The pre scapular lymph nodes were moderately swollen and enlarged. The rectal temperature was 105.5°F. Signs of respiratory distress were quite evident from labored breathing. The animal was repeatedly seen pressing head against the hard objects and exhibited tonic–clonic convulsions in response to sudden noise and occasional circling movements Microscopy.

Microscopic examination by Giemsa-stained blood thin blood smears revealed Koch’s blue bodies (KBB) in the cytoplasm of the lymphocytes and highly pleomorphic piroplasms in the RBCs identified as T.annulata (Fig. 1).

Fig. 1.

Koch’s blue body (Arrow head) in the lymphocytes and high level of T. annulata piroplasm (Arrow) in the RBC’s under oil immersion lens (× 1000)

PCR

Specific Primers directed amplification of PCR assay resulted in an amplicon size of 721 bp in 1.2% agarose gel correspond to T.annulata. Nonspecific amplification was not observed in the negative control sample (Fig. 2).

Fig. 2.

Agarose gel electrophoresis (1.5%) showing intact band of 721 bp fragment from genomic DNA of T. annulata Lane M: 100 bp DNA ladder, Lane1: Amplification of T. annulata genomic DNA from the blood of animal positive for infection (positive control) Lane C: Negative control (No template), Lane2: Positive processed field samples

Hematolobiochemical examination

The haematological values of cross bred cow calf before and after treatment of BCT were as follows: RBC (5.0 × 10⁶µLVs. 6.5 × 10⁶), Hb (9 g/dl Vs. 13.0 g/dl), WBC (9 × 10³/ µl Vs. 7 × 10³/µl), Lymphocytes (43% Vs. 60%); Neutrophils (53%Vs. 36%); Eosinophils (3% Vs. 2%) and Monocytes (1% Vs. 1%). Biochemical parameters of naturally infected calf with BCT were as follows: aspartate transaminase (60.50 IU/L); creatinine(1.25 mg/dl); total protein (6.5 g/dl); albumin (2.50 g/dl); globulin (4.17 g/dl) and whereas after treatment from theileriosis were as follows: aspartate transaminase (15.50 IU/L); creatinine(1.0 mg/dl); total protein(7.80 g/dl); albumin(3.25 g/dl); globulin(4.71 g/dl).

Discussion

Giemsa stained thin blood smear revealed intraerythrocytic piroplasm and Koch’s blue bodies within the cytoplasm of lymphocytes confirmed as T. annulata (Fig. 1). PCR assay further confirmed the presence of T. annulata (Fig. 2). The level of parasitaemia was very high (60%) and presence of schizonts in blood smear indicated the acute phase of disease. Further presence of schizont infected cells in blood are indicative to schizont transforming species. In case of T. parva, schizont stage is pathogenic causing lymphodestruction while in case of T. annulata, both schizont and piroplasm stages are pathogenic (Mehlhorn and Schein 1984). Otherwise, presence of only piroplasm stage in blood is indicative of benign form of theileriosis (Sivakumar et al. 2014). In the present case, occurrence of maximum number of annular form of piroplasmic stages along with presence of schizonts, which are difficult to demonstrate in chronic theileriosis and BCT, was indicative of T. annulata infection (Bader et al. 1986),

Species level differentiation of various Theileria species based on schizonts either by ultrastructure or by light microscope is quite impossible. Additionally, it is very tough for species level identification based upon piroplasm shape and size (Schein et al. 1978).

Serological methods have inherent limitations of cross reactivity (Passos et al. 1998). Nucleic acid based assays like PCR allow diagnosis of parasite at levels far below the detection limit of the frequently used parasitological techniques and helps in the confirmation of parasite up to species level by employing species specific primers (d’Oliveria et al. 1995; Farooq et al. 2019). Therefore, PCR assay was employed in the present case for species level identification of the Theileria responsible for cerebral form of theileriosis.

Primers derived from the gene encoding the 30-kDa major merozoite surface antigen of T. annulata were used in the present study. A 721 bp fragment was amplified from the genomic DNA extracted from the cross-bred calf blood. PCR assay confirmed the identity of T. annulata as the etiological agent of BCT in the present study.

Based on morphological characters collected ticks from the calf were identified as Hyalomma anatolicum anatolicum which transmits T. annulata trans-stadially and it is considered as world’s most damaging ticks (Giles et al. 1978). The tick is established in a majority of the middle East and South Asian countries, including India (Bhatia and Shah 2001). Following a tick infestation, a significant amount of a toxin was released, making the reticuloendothelial system's defence function less efficient, allowing blood parasites to do their harm to the host (Thomas and Neitz 1958). It could also be due to an autoimmune disorder induced by parasites leading to intravascular agglutination of lymphoblasts (Van Rensburg 1976). More stress due to translocation may have altered the immune function and response to infection by the calf as calf along with mother was transported (Martin et al. 2011). Possible trophism of the parasite towards the central nervous system leads to lesion in endothelium responsible for lymphoblastic accumulation and venous thrombi (Bader et al. 1986). However, the severity of theileriosis depends on the inter play of parasite, vertebrate host, tick vector and environmental factors (Clift et al. 2020).

The infected calf was treated twice at the interval of 72 h with buparvaquone (@ 2.5 mg/kg b.wt. im) and supportive treatment for 5 days with oxytetracycline @10 mg/kg b.wt im, 3 mL meloxicam with paracetamol im, 3 mL vitamin B complex and rumentas bolus 1 BD for 5 days. Blood sample was found negative after 5 days for T. annulata after screening with blood smear examination and PCR. The adjunction of antioxidants and antitheilerial agents may play a crucial role in salvaging the animals from lethal theileriosis.

Haematological values of Hb, PCV, and TEC were lower which may be due to various factors like severe damage caused by organisms inside the erythrocyte during their multiplication (Ganguly et al. 2015); destruction of erythrocyte infected with Theileria schizonts owing to immune mediated erythrophagocytosis (Uilenberg 1981); removal of piroplasm infected erythrocytes by reticuloendothelial system (Campbell and Spooner 1999); increased level of activated complement products (Omer et al. 2002) and destruction of oxidized erythrocyte by oxygen free radicals responsible for anaemia (Mbassa et al. 1994). Decreased value of serum total protein, albumin and globulin might be due to diseased lymph node resulting in extra-vascular proteinaceous fluid in body cavities (Stockham et al., 2000) in addition to liver failure (Omer et al.2003). Increased level of AST is indicative of necrosis as it is involved in amino acid and carbohydrate metabolism (Murray et al. 2000) whereas raised value of creatinine suggests muscle damage due to anemic conditions (Kaneko 1989). Though present case was of typical bovine cerebral theileriosis, however it was difficult to understand the precise mechanism of T. annulata induced BCT to mitigate the new threats for livestock. Nervous form is not a consistent feature of theileriosis in cattle hence, it is imperative if cattle is showing nervous symptoms along with anemia then same should be diagnosed to rule out the possibility of T. annulata infection. The present communication places on record the first report of molecular based confirmation of bovine cerebral theileriosis caused by T. annulata in a cross bred cattle calf from India.

Funding

This work was supported by Dean, College of Veterinary Science and Animal Husbandry, Jabalpur, India.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.