Apparently no study has been undertaken on
Apparently, no study has been undertaken on alterations in cholinesterase activity and its correlation with neurological disorders in clinical case of trypanosomosis in buffaloes. Therefore, the present study was undertaken to evaluate the alterations in cholinesterase activity in clinical cases of trypanosomosis in buffaloes carrying natural infection of T. evansi.
Materials and methods Thirty-three water buffaloes (Bubalus bubalis) naturally suffering with trypanosomosis, confirmed positive for trypomastigotes of T. evansi on blood smear examination, were included in the present study and divided into two groups based on clinical signs. Twenty diseased buffaloes revealing only common clinical manifestations like pyrexia, inappetence, cachexia, corneal opacity (Fig. 1.), pallid mucous membrane, ventral and limb oedema but no neurological disturbances were included in Group I, while the remaining 13 buffaloes exhibiting common clinical manifestations along with neurological disturbances e.g. hyperesthesia, allotriophagy, head-butting, uncoordinated gait, circling movement, tilting of head, twitching of muzzle, facial paresis, tongue paresis (Fig. 1.), frequent passage of small amount of urine, and constricted pupil constituted the Group II. All diseased buffaloes were presented by their owners in Veterinary Clinics of College of Veterinary Science and Animal Husbandry, Mathura, India, for treatment. In addition, 12 clinically healthy buffaloes free from any haemoprotozoan infection including Trypanosoma, based on blood smear examination, were also included in the study and these constituted the Group III and referred as healthy control. With the consent of animal owners, 5mL blood sample each was collected from all diseased buffaloes by jugular venipuncture before start of any therapy. Of this, 3mL blood was transferred into a tube containing clot activators and used for harvesting serum while the remaining blood was transferred into a tube containing disodium ethylenediaminetetraacetic PFI 3 (EDTA) and used for routine haematological examinations (data not shown). For preparation of blood smear, a drop of blood sample was obtained by aseptic pricking of ear tips of diseased buffaloes and spread over a clean slide to prepare the blood smear. Similarly 5mL blood sample each was also collected from healthy buffaloes (Group III) and used for routine haematological examinations and serum harvesting. Blood smears were prepared from healthy buffaloes too. Diagnosis of T. evansi infection was based on detection of trypomastigotes in blood smears stained with the Wright technique. Harvested serum samples were transferred into cryovials and stored at −20°C until estimation of the cholinesterase activity. Thorough clinical examination of the diseased buffaloes was made to record the clinical manifestations. All infected buffaloes were treated by administering diminazene aceturate, a trypanocidal drug, at the dose rate of 7.0mg/kg bodyweight by intramuscular route and other supportive therapy. Serum cholinesterase activity was estimated by an optimized version of Ellman method by using cholinesterase activity assay kit (Sigma-Aldrich) following the procedure as described by the manufacturer. Cholinesterase activity was expressed as units/L (One unit of cholinesterase is the amount of enzyme that catalyzes the production of 1.0μmol of thiocholine per minute at room temperature at pH 7.5). Statistical differences between the data of different groups were determined using one-way analysis of variance (ANOVA) followed by the Tukey’s test. The level of statistical significance for all the comparisons made was established at P<0.05.
Results and discussion Serum cholinesterase activity was significantly (P<0.001) lower in T. evansi infected buffaloes of both Groups I and II compared to that in healthy control buffaloes of Group III as shown in Fig. 2. But, no significant difference was observed in cholinesterase activity between the buffaloes of Group I showing no neurological signs, and those of Group II showing neurological manifestations. Therefore, results of our study evidently suggest that T. evansi significantly reduces cholinesterase activity, which possibly may play a key role in the pathogenesis of trypanosomosis by evading immune response in diseased buffaloes. Reduced cholinesterase activity indirectly results in overproduction of ACh in infected buffaloes. Since BChE is the main cholinesterase in serum which is synthesized in liver (Kutty, 1980) and thus alterations in hepatic function would result in reduced activity of this enzyme in blood (Singh, 1976). Albeit, we have not evaluated the liver functions in the present study, but several researchers have demonstrated alterations in hepatic functions in T. evansi-infected animals (Aquino et al., 2002, Hilali et al., 2006). Therefore, possibility of reduced cholinesterase activity in T. evansi-infected buffaloes due to hepatic damage cannot be ruled out apart from the involvement inflammatory processes in trypanosomosis as cholinesterase participates in regulation of immune response (Das, 2007).