Giemsa stained thin blood smear revealed intraerythrocytic piroplasm and Koch blue bodies within the cytoplasm of lymphocytes confirmed as T. annulata (Fig.2). PCR assay further confirmed the presence of T. annulata (Fig. 3). 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 help 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 721bp 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). The most probable reason behind the BCT might be the release of toxin from tick having a suppressing effect on the reticuloendothelial system renders an animal more susceptible. 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 hrs with buparvaquone (@ 2.5 mg/kg b.wt. im) and supportive treatment for 5 days with oxytetracycline @10mg/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.