- Email: [email protected]
Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes
Accepted Manuscript Title: Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes Author: Shanker K. Singh Vivek K. Singh Brajesh K. Yadav Udayraj P. Nakade Priyambada Kumari Mukesh K. Srivastava Abhishek Sharma Soumen Choudhary Dilip Swain Satish K. Garg PII: DOI: Reference:
S0304-4017(16)30195-9 http://dx.doi.org/doi:10.1016/j.vetpar.2016.05.030 VETPAR 8030
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
26-1-2016 18-5-2016 23-5-2016
Please cite this article as: Singh, Shanker K., Singh, Vivek K., Yadav, Brajesh K., Nakade, Udayraj P., Kumari, Priyambada, Srivastava, Mukesh K., Sharma, Abhishek, Choudhary, Soumen, Swain, Dilip, Garg, Satish K., Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.05.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes Shanker K. Singh1,, Vivek K. Singh1, Brajesh K. Yadav1, Udayraj P. Nakade2, Priyambada Kumari3, Mukesh K. Srivastava1, Abhishek Sharma2, Soumen Choudhary2, Dilip Swain4 and Satish K. Garg2 1
Department of Veterinary Medicine, 2Department of Pharmacology and Toxicology,
4
Department of Animal Physiology, College of Veterinary Science and Animal
Husbandry, 3College of Biotechnology, DUVASU, Mathura – 281 001, U.P., India.
Corresponding author Email id: [email protected] 1
Graphical abstract
2
Highlights
Reduced cholinesterase activity is associated with pathogenesis of Trypanosoma evansi infection in buffaloes.
Reduction in the cholinesterase activity could be one of the strategy of T. evansi to evade the immune system of infected buffaloes
Alteration in the cholinesterase activity could be associated with T. evansi induced clinical signs in buffaloes
3
Abstract The present study aimed to investigate the association of cholinesterase activity with trypanosomosis in buffaloes. Thirty-three clinical cases of trypanosomosis in water buffaloes, found positive for trypomastigotes of T. evansi on blood smear examination, were divided into two groups based on clinical manifestations. Twenty diseased buffaloes revealing only common clinical signs were allocated to Group I, while the remaining 13 buffaloes showing common clinical manifestations along with neurological disturbances were allocated to Group II. Twelve clinically healthy buffaloes, free from any haemoprotozoa infection, were kept as healthy control (Group III). Blood samples were collected from buffaloes of all three groups to determine serum cholinesterase activity. Compared to buffaloes of healthy control group, cholinesterase activity in T. evansiinfected buffaloes of Group I and II was significantly (P<0.001) lower. However, no significant difference was observed in cholinesterase activity between the T. evansiinfected buffaloes exhibiting neurological disorders and no neurological disorders. Summing up, reduced cholinesterase activity seems to be associated with the pathogenesis of natural T. evansi infection and its clinical manifestations in buffaloes possibly by evading immune response. Further studies are warranted on association of cholinesterase activity in T. evansi-infected buffaloes with neurological disorders. Key Words: Buffaloes; Cholinesterase; Trypanosomosis; Neurological disorders 1. Introduction Trypanosoma evansi (T. evansi), belongs to the subgenus Trypanozoon, a group of Trypanosomes has a large diversity of mammalian hosts (Da Silva et al., 2010) and can affect both humans and animals (Desquesnes et al., 2013). Its infection is mainly
4
disseminated by Tabanid species (Tabanus sp., Chrysops sp. and Hematopota sp.) (Otto et al., 2010). T. evansi is considered as both a blood and tissue parasite owing to its ability to invade the nervous system not only in horses and dogs but also in cattle, buffaloes, deer and pigs (Rodrigues et al., 2009). Although trypanosomosis has long been recognized as an important disease in tropical and subtropical countries (Herrera et al., 2004; Da Silva et al., 2010), but the situation in India is quite different as pathogenic effects of trypanosomosis had been recorded long back in 1891 with very high mortality (>90%) as documented by Gill (1977) from different parts of India. T. evansi infection results in anemia, reproductive disorders, loss in body weight, milk and meat production and loss in draught power, and most often during chronic evolution, it leads to totally wasted animals (Desquesnes et al., 2013). Clinical signs of neurological disorders are reported in horses, camels, buffaloes, cattle, deer and cats infected by T. evansi (Desquesnes et al., 2013). In spite of enormous advancements in diagnosis, understanding of pathogenesis of experimental T. evansi infection and therapeutic approaches, mechanism of T. evansi-induced neurological disorders in buffaloes is yet to be unraveled especially in view of the fact that trypanosomes evade the immune defense mechanism of host, and thus it has become one of the major and puzzling issues in host-parasite interaction studies (Zambrano-Villa et al., 2002). Existence of cholinergic anti-inflammatory pathway is well documented (Pavlov and Tracey, 2006) and acetylcholine (ACh) plays an important role in attenuating the release of pro-inflammatory cytokines such as tumour necrosis factor (TNF), interleukin1 (IL-1), interleukin-6 (IL-6) and interleukin-18 (IL-18), without affecting the production of interleukin-10 (IL-10), an anti-inflammatory cytokine (Pavlov and Tracey, 2004).
5
Concentration of ACh is regulated by cholinesterases e.g. acetylcholinesterase (AchE), which is expressed in nerve cells and erythrocytes, and butyrylcholinesterase (BuChE), which is found in plasma. Therefore, cholinesterase activity is considered as an indirect indicator of ACh levels. Alterations in acetyltransferase and acetylcholinesterase (AChE) activities (Brennessel et al., 1985) and increased dopamine and norepinephrine levels (Amole et al., 1989) have been associated with trypanosomosis in mice due to T. cruzi and T. brucei infections. Therefore, determination of cholinesterase activity in clinical cases of trypanosomosis will be of significance in further understanding the pathogenesis and host immune evasion mechanisms. Reduction in cholinesterase activity might be a possible cause of neurological disorders in trypanosomosis since this enzyme is essential in nerve impulse transmission during nerve signal relay (Bartels et al., 2000). 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. 2. 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,
6
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, 5 mL blood sample each was collected from all diseased buffaloes by jugular venipuncture before start of any therapy. Of this, 3 mL 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 acid
(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 5 mL 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
7
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 7.0 mg/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 1.0 µmol 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. . 3. 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
8
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). T helper (Th) cells play a central role in activation of immune system against infectious agents through secretion of lymphokines or cytokines. African trypanosomes target the Th cells and alter their activation, possibly for their own survival and perpetuity (Namangala, 2011). Trypanosoma b. brucei impairs both IL-2 production and IL-2 receptor expression by T cells and thereby depression of T-cell proliferative response in mice. Lymphocytes possess cholinergic components necessary to constitute an independent, non-neuronal cholinergic system (Fujii et al., 2008) and this lymphocytic cholinergic system could play a key role in modulation of immune functions via both muscarinic and nicotinic cholinergic receptors. In most trypanosome infection models, a strong Th1 protective response, mainly during early trypanosomosis has been reported (Mansfield and Paulnock, 2005; Namangala et al., 2009). Trypanosomes may evade such immune responses by inducing irrelevant immune responses during early stages of infection through enhancement of Th2 cytokines production and regulatory T-cell activation which results in increased production of IL-10 and TGF-β, both of which suppress early protective Th1 responses and hence favour parasite survival (Hertz et al., 1998; Tabel et al., 2008). Therefore, it is quite possible that T. evansi might be utilizing
9
vagal cholinergic pathway to impair Th1 immune response or to enhance Th2 antiinflammatory response in infected buffaloes possibly for their own survival and perpetuity. Mechanism of neurological signs in trypanosomosis is obscure. Recently, studies on experimental infections with T. evansi in various experimental animals have revealed remarkable reduction in cholinesterase activity (Da Silva et al., 2010, 2011b; Wolkmer et al., 2010, 2013). In the present study too, significant difference in cholinesterase activity was observed between T. evansi-infected and healthy buffaloes; thereby suggesting direct correlation between T. evansi infection and cholinesterase activity. But, cholinesterase activity was not found to be associated with neurological disorders as significant inhibition of cholinesterase was observed even in those clinical cases which did not exhibit any neurological signs. Neurological signs might be the consequence of necrotizing panencephalitis or meningoencephalitis (Rodrigues et al., 2009), ordue to alterations in actions of neurotransmitters (Da Silva et al., 2011a; Paim et al., 2011). Reduced cholinesterase activity in buffaloes in the present study is in agreement with the scientific reports demonstrating reduction in cholinesterase activity in T. evansi infected animals (Da Silva et al., 2010; Wolkmer et al., 2010). But are just contrary to the observations of Costa et al. (2012) who have reported remarkable increase in cholinesterase activity in blood of experimentally T. evansi infected rabbits on days 7 and 27 post-infection. These differences could be due to species difference between buffaloes and rabbits as the clinical signs and severity of disease is also different in buffaloes and rabbits as amongst the affected mammalian species, camels, horses, buffaloes and dogs have the most severe form of disease (Taylor and Authié, 2004).
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In conclusion, significantly reduced cholinesterase activity seems to be associated with pathogenesis of natural T. evansi infection and its clinical manifestations in buffaloes probably by evading immune response for their own survival and perpetuity. But based on this study, it is not possible to suggest the possible mechanism of cholinesterase inhibition and its association with neurological manifestations in buffaloes except for possible release of some endogenous mediator (s) or alterations in activity of neurotransmitters (Da Silva et al., 2011a; Paim et al., 2011) which may be responsible for neurological or non-neurological disorders. Further, possibility of promising therapeutic potential of cholinesterase reactivators in management of trypanosomosis in buffaloes and other species cannot be ruled out, but requires further studies. Acknowledgment: Authors are highly thankful to Honorable Vice-Chancellor of the University for providing the necessary facilities and funds. Reference Amole, B., Sharpless, N., Wittner, M., Tanowitz, H.B., 1989. Neurochemical measurements in the brains of mice infected with Trypanosoma brucei (TREU 667). Ann. Trop. Med. Parasitol. 83, 225–232. Aquino, L.P.C.T., Machado, R.Z., Alessi, A.C, Santana., A.E, Castro., M.B., Marques, L.C.,
Malheiros,
E.B.,
2002.
Hematological,
biochemical
and
anatomopathological aspects of the experimental infection with Trypanosoma evansi in dogs. Arq. Bras. Med. Vet. Zootec. 54(1), 8-18. Bartels, C.F., Xie, W., Miller-Lindholm, M.K., Schopfer, L.M., Lockridge, O., 2000. Determination
of
the
DNA
sequences
11
of
acetylcholinesterase
and
butyrylcholinesterse from cat demonstration of the existence of both in cat plasma. Biochem. Pharmacol. 60, 479–487. Brennessel, D.J., Wittner, M., Braunstein, V., Herbert, B., 1985. Acetylcholinesterase levels in skeletal muscle of mice infected with Trypanosoma cruzi. Am. J. Trop. Med. Hyg. 34, 460–464. Costa, M.M., Silva, A.S., Paim, F.C., França, R., Dornelles, G.L., Thomé, G.R., Serres, J.D., Schmatz, R., Spanevello, R.M., Gonçalves, J.F., Schetinger, M.R., Mazzanti, C.M., Lopes, S.T., Monteiro, S.G., 2012. Cholinesterase as inflammatory markers in a experimental infection by Trypanosoma evansi in rabbits. An. Acad. Bras. Cienc. 84(4), 1105-1113. Da Silva, A.S., Spanevello, R., Stefanello, N., Wolkmer, P., Costa, M.M., Zanette, R.A., Lopes, S.T.A., Santurio, J.M., Schetinger, M.R.C., Monteiro, S.G., 2010. Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats. Res. Vet. Sci. 88, 281–284. Da Silva, A.S., Monteiro, S.G., Gonçalves, J.F., Spanevello, R., Oliveira, C.B., Costa, M.M., Jaques, J.A., Morsch, V.M., Schetinger, M.R., Mazzanti, C.M., Lopes, S.T., 2011b. Acetylcholinesterase activity and lipid peroxidation in the brain and spinal cord of rats infected with Trypanosoma evansi. Vet. Parasitol. 175 (3-4), 237-244. Da Silva, A.S., Oliveira, C.B., Bertoncheli, C.M., Santos, R.P., Beckmann, D.V., Wolkmer, P., Gressler, L.T., Tonin, A.A., Graça, D.L., Mazzanti, A., Lopes, S.T., Monteiro, S.G., 2011a. Clinical signs and histopathology of brain, spinal cord and
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muscle
of
the
pelvic
limb
of
rats
experimentally
infected
with Trypanosoma evansi. Pathol. Res. Pract. 208(1), 39-44. Das, U.N., 2007. Acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation. Med. Sci. Monit. 13, 214-221. Desquesnes, M., Holzmuller, P., Lai, D.H., Dargantes, A., Lun, Z.R., Jittaplapong, S., 2013. Trypanosoma evansi and Surra: A Review and Perspectives on Origin, History, Distribution, Taxonomy, Morphology, Hosts, and Pathogenic Effects. BioMed. Res. Int. http://dx.doi.org/10.1155/2013/194176 Fujii, T., Takada-Takatori, Y., Kawashima, K., 2008. Basic and clinical aspects of nonneuronal acetylcholine: expression of an independent, non-neuronal cholinergic system in lymphocytes and its clinical significance in immunotherapy. J. Pharmacol. Sci. 106(2), 186-192. Gill, B., 1977. Trypanosomes and Trypanosomiasis of Indian Livestock, ICAR: Indian Council of Agricultural Research, New Delhi, India, 1st edition. Herrera, H.M., Davila, A.M.R., Norek, A., Abreu, U.G., Souza, S.S., D’Andrea, P.S., Jansen, A.M., 2004. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet. Parasitol. 125, 263–275. Hertz, C.J., Filutowicz, H., Mansfield, J.M., 1998. Resistance to the African trypanosomes is IFN-gamma dependent. J. Immunol. 161(12), 6775-6783. Hilali, M., Abdel-Gawad, A., Nassar, A., Abdel-Wahab, A., 2006. Hematological and biochemical changes in water buffalo calves (Bubalus bubalis) infected with Trypanosoma evansi. Vet. Parasitol. 139(1-3), 237-243. Kutty, K.M., 1980. Biological function of cholinesterase. Clin. Biochem. 13, 239-243.
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Mansfield, J.M., Paulnock, D.M., 2005. Regulation of innate and acquired immunity in African trypanosomiasis. Parasite Immunol. 27(10-11), 361-371. Namangala, B., 2011. How the African trypanosomes evade host immune killing. Parasite Immunol. 33(8), 430-437. Namangala, B., De Baetselier, P., Beschin, A., 2009. Both type-I and type-II responses contribute to murine trypano-tolerance. J. Vet. Med. Sci. 71(3), 313-318. Otto, M.A., Da Silva, A.S., Gressler, L.T., Farret, M.H., Tavares, K.C.S., Zanette, R.A., Miletti, L.C., Monteiro, S.G., 2010. Susceptibility of Trypanosoma evansi to human blood and plasma in infected mice. Vet. Parasitol. 168, 1–4. Paim, F.C., Da Silva, A.S., Wolkmer, P., Costa, M.M., Da Silva, C.B., Paim, C.B., Oliveira, M.S., Silva, L.F., Mello, C.F., Monteiro, S.G., Mazzanti, C.M., Lopes, S.T., 2011. Trypanosoma evansi: concentration of 3-nitrotyrosine in the brain of infected rats. Exp. Parasitol. 129 (1), 27-30. Pavlov, V.A., Tracey, K.J., 2004. Neural regulators of innate immune responses and inflammation. Cell. Mol. Life Sci. 61, 2322–2331. Pavlov, V.A., Tracey, K.J., 2006. Controlling inflammation: the cholinergic antiinflammatory pathway. Biochem. Soc. Trans. 34(6), 1037-1040. Rodrigues, A., Fighera, R.A., Souza, T.M., Schild, A.L., Barros, C.S., 2009. Neuropathology of naturally occurring Trypanosoma evansi infection of horses. Vet. Pathol. 46(2), 251-258. Singh, D.S., 1976. Serum cholinesterase in hepatic disorders. J. Indian Med. Assoc. 66, 49-51.
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Tabel, H., Wei, G., Shi, M., 2008. T cells and immunopathogenesis of experimental African trypanosomiasis. Immunol. Rev. 225, 128-139. Taylor, K., Authié E.M.L., 2004. Pathogenesis of animal trypanosomiasis. In: Maudlin I, Holmes PH and Miles MA. The trypanosomiasis. London. CABI publishing, p. 331-354. Wolkmer, P., Lopes, S.T., Franciscato, C., da Silva, A.S., Traesel, C.K., Siqueira, L.C., Pereira, M.E., Monteiro, S.G., Mazzanti, C.M., 2010. Trypanosoma evansi: cholinesterase activity in acutely infected Wistar rats. Exp. Parasitol. 125, 251255. Wolkmer, P., Silva, C.B., Paim, F.C., Duarte, M.M., Castro, V., Palma, H.E., França, R.T., Felin, D.V., Siqueira, L.C., Lopes, S.T., Schetinger, M.R., Monteiro, S.G., Mazzanti,
C.M.,
2013.
Pre-treatment
with
curcumin
modulates
acetylcholinesterase activity and proinflammatory cytokines in rats infected with Trypanosoma evansi. Parasitol. Int. 62(2), 144-149. Zambrano-Villa, S., Rosales-Borjas, D., Carrero, J.C., Ortiz-Ortiz, L., 2002. How protozoan parasites evade the immune response. Trends Parasitol. 18, 272–278.
15
B
2 4 3 .7 2 1 9 .1 2
400
300
200 A
5 9 .6 7 7 .9 8 A
6 4 .4 6 6 .1 2 100
12
13 p
II
I
II
(n
(n
=
=
20 = (n I p
G
G
ro
u
ro
p
u
u ro G
)
)
0
)
C h o l i n e s t e r a s e a c t i v it y ( u n i t/ L )
Fig. 1. A Trypanosoma evansi-infected buffalo exhibiting corneal opacity and tongue paresis
Fig. 2. Serum cholinesterase activity (Mean ± SE) in Trypanosoma evansi-infected buffaloes without neurological disorders (Group I), with neurological disorders (Group II) and healthy control buffaloes (Group III). A,BDifferent superscripts on bar diagram show significant (P<0.001) difference between the groups.
16
S0304-4017(16)30195-9 http://dx.doi.org/doi:10.1016/j.vetpar.2016.05.030 VETPAR 8030
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
26-1-2016 18-5-2016 23-5-2016
Please cite this article as: Singh, Shanker K., Singh, Vivek K., Yadav, Brajesh K., Nakade, Udayraj P., Kumari, Priyambada, Srivastava, Mukesh K., Sharma, Abhishek, Choudhary, Soumen, Swain, Dilip, Garg, Satish K., Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.05.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Potential association of reduced cholinesterase activity with Trypanosoma evansi pathogenesis in buffaloes Shanker K. Singh1,, Vivek K. Singh1, Brajesh K. Yadav1, Udayraj P. Nakade2, Priyambada Kumari3, Mukesh K. Srivastava1, Abhishek Sharma2, Soumen Choudhary2, Dilip Swain4 and Satish K. Garg2 1
Department of Veterinary Medicine, 2Department of Pharmacology and Toxicology,
4
Department of Animal Physiology, College of Veterinary Science and Animal
Husbandry, 3College of Biotechnology, DUVASU, Mathura – 281 001, U.P., India.
Corresponding author Email id: [email protected] 1
Graphical abstract
2
Highlights
Reduced cholinesterase activity is associated with pathogenesis of Trypanosoma evansi infection in buffaloes.
Reduction in the cholinesterase activity could be one of the strategy of T. evansi to evade the immune system of infected buffaloes
Alteration in the cholinesterase activity could be associated with T. evansi induced clinical signs in buffaloes
3
Abstract The present study aimed to investigate the association of cholinesterase activity with trypanosomosis in buffaloes. Thirty-three clinical cases of trypanosomosis in water buffaloes, found positive for trypomastigotes of T. evansi on blood smear examination, were divided into two groups based on clinical manifestations. Twenty diseased buffaloes revealing only common clinical signs were allocated to Group I, while the remaining 13 buffaloes showing common clinical manifestations along with neurological disturbances were allocated to Group II. Twelve clinically healthy buffaloes, free from any haemoprotozoa infection, were kept as healthy control (Group III). Blood samples were collected from buffaloes of all three groups to determine serum cholinesterase activity. Compared to buffaloes of healthy control group, cholinesterase activity in T. evansiinfected buffaloes of Group I and II was significantly (P<0.001) lower. However, no significant difference was observed in cholinesterase activity between the T. evansiinfected buffaloes exhibiting neurological disorders and no neurological disorders. Summing up, reduced cholinesterase activity seems to be associated with the pathogenesis of natural T. evansi infection and its clinical manifestations in buffaloes possibly by evading immune response. Further studies are warranted on association of cholinesterase activity in T. evansi-infected buffaloes with neurological disorders. Key Words: Buffaloes; Cholinesterase; Trypanosomosis; Neurological disorders 1. Introduction Trypanosoma evansi (T. evansi), belongs to the subgenus Trypanozoon, a group of Trypanosomes has a large diversity of mammalian hosts (Da Silva et al., 2010) and can affect both humans and animals (Desquesnes et al., 2013). Its infection is mainly
4
disseminated by Tabanid species (Tabanus sp., Chrysops sp. and Hematopota sp.) (Otto et al., 2010). T. evansi is considered as both a blood and tissue parasite owing to its ability to invade the nervous system not only in horses and dogs but also in cattle, buffaloes, deer and pigs (Rodrigues et al., 2009). Although trypanosomosis has long been recognized as an important disease in tropical and subtropical countries (Herrera et al., 2004; Da Silva et al., 2010), but the situation in India is quite different as pathogenic effects of trypanosomosis had been recorded long back in 1891 with very high mortality (>90%) as documented by Gill (1977) from different parts of India. T. evansi infection results in anemia, reproductive disorders, loss in body weight, milk and meat production and loss in draught power, and most often during chronic evolution, it leads to totally wasted animals (Desquesnes et al., 2013). Clinical signs of neurological disorders are reported in horses, camels, buffaloes, cattle, deer and cats infected by T. evansi (Desquesnes et al., 2013). In spite of enormous advancements in diagnosis, understanding of pathogenesis of experimental T. evansi infection and therapeutic approaches, mechanism of T. evansi-induced neurological disorders in buffaloes is yet to be unraveled especially in view of the fact that trypanosomes evade the immune defense mechanism of host, and thus it has become one of the major and puzzling issues in host-parasite interaction studies (Zambrano-Villa et al., 2002). Existence of cholinergic anti-inflammatory pathway is well documented (Pavlov and Tracey, 2006) and acetylcholine (ACh) plays an important role in attenuating the release of pro-inflammatory cytokines such as tumour necrosis factor (TNF), interleukin1 (IL-1), interleukin-6 (IL-6) and interleukin-18 (IL-18), without affecting the production of interleukin-10 (IL-10), an anti-inflammatory cytokine (Pavlov and Tracey, 2004).
5
Concentration of ACh is regulated by cholinesterases e.g. acetylcholinesterase (AchE), which is expressed in nerve cells and erythrocytes, and butyrylcholinesterase (BuChE), which is found in plasma. Therefore, cholinesterase activity is considered as an indirect indicator of ACh levels. Alterations in acetyltransferase and acetylcholinesterase (AChE) activities (Brennessel et al., 1985) and increased dopamine and norepinephrine levels (Amole et al., 1989) have been associated with trypanosomosis in mice due to T. cruzi and T. brucei infections. Therefore, determination of cholinesterase activity in clinical cases of trypanosomosis will be of significance in further understanding the pathogenesis and host immune evasion mechanisms. Reduction in cholinesterase activity might be a possible cause of neurological disorders in trypanosomosis since this enzyme is essential in nerve impulse transmission during nerve signal relay (Bartels et al., 2000). 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. 2. 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,
6
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, 5 mL blood sample each was collected from all diseased buffaloes by jugular venipuncture before start of any therapy. Of this, 3 mL 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 acid
(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 5 mL 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
7
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 7.0 mg/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 1.0 µmol 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. . 3. 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
8
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). T helper (Th) cells play a central role in activation of immune system against infectious agents through secretion of lymphokines or cytokines. African trypanosomes target the Th cells and alter their activation, possibly for their own survival and perpetuity (Namangala, 2011). Trypanosoma b. brucei impairs both IL-2 production and IL-2 receptor expression by T cells and thereby depression of T-cell proliferative response in mice. Lymphocytes possess cholinergic components necessary to constitute an independent, non-neuronal cholinergic system (Fujii et al., 2008) and this lymphocytic cholinergic system could play a key role in modulation of immune functions via both muscarinic and nicotinic cholinergic receptors. In most trypanosome infection models, a strong Th1 protective response, mainly during early trypanosomosis has been reported (Mansfield and Paulnock, 2005; Namangala et al., 2009). Trypanosomes may evade such immune responses by inducing irrelevant immune responses during early stages of infection through enhancement of Th2 cytokines production and regulatory T-cell activation which results in increased production of IL-10 and TGF-β, both of which suppress early protective Th1 responses and hence favour parasite survival (Hertz et al., 1998; Tabel et al., 2008). Therefore, it is quite possible that T. evansi might be utilizing
9
vagal cholinergic pathway to impair Th1 immune response or to enhance Th2 antiinflammatory response in infected buffaloes possibly for their own survival and perpetuity. Mechanism of neurological signs in trypanosomosis is obscure. Recently, studies on experimental infections with T. evansi in various experimental animals have revealed remarkable reduction in cholinesterase activity (Da Silva et al., 2010, 2011b; Wolkmer et al., 2010, 2013). In the present study too, significant difference in cholinesterase activity was observed between T. evansi-infected and healthy buffaloes; thereby suggesting direct correlation between T. evansi infection and cholinesterase activity. But, cholinesterase activity was not found to be associated with neurological disorders as significant inhibition of cholinesterase was observed even in those clinical cases which did not exhibit any neurological signs. Neurological signs might be the consequence of necrotizing panencephalitis or meningoencephalitis (Rodrigues et al., 2009), ordue to alterations in actions of neurotransmitters (Da Silva et al., 2011a; Paim et al., 2011). Reduced cholinesterase activity in buffaloes in the present study is in agreement with the scientific reports demonstrating reduction in cholinesterase activity in T. evansi infected animals (Da Silva et al., 2010; Wolkmer et al., 2010). But are just contrary to the observations of Costa et al. (2012) who have reported remarkable increase in cholinesterase activity in blood of experimentally T. evansi infected rabbits on days 7 and 27 post-infection. These differences could be due to species difference between buffaloes and rabbits as the clinical signs and severity of disease is also different in buffaloes and rabbits as amongst the affected mammalian species, camels, horses, buffaloes and dogs have the most severe form of disease (Taylor and Authié, 2004).
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In conclusion, significantly reduced cholinesterase activity seems to be associated with pathogenesis of natural T. evansi infection and its clinical manifestations in buffaloes probably by evading immune response for their own survival and perpetuity. But based on this study, it is not possible to suggest the possible mechanism of cholinesterase inhibition and its association with neurological manifestations in buffaloes except for possible release of some endogenous mediator (s) or alterations in activity of neurotransmitters (Da Silva et al., 2011a; Paim et al., 2011) which may be responsible for neurological or non-neurological disorders. Further, possibility of promising therapeutic potential of cholinesterase reactivators in management of trypanosomosis in buffaloes and other species cannot be ruled out, but requires further studies. Acknowledgment: Authors are highly thankful to Honorable Vice-Chancellor of the University for providing the necessary facilities and funds. Reference Amole, B., Sharpless, N., Wittner, M., Tanowitz, H.B., 1989. Neurochemical measurements in the brains of mice infected with Trypanosoma brucei (TREU 667). Ann. Trop. Med. Parasitol. 83, 225–232. Aquino, L.P.C.T., Machado, R.Z., Alessi, A.C, Santana., A.E, Castro., M.B., Marques, L.C.,
Malheiros,
E.B.,
2002.
Hematological,
biochemical
and
anatomopathological aspects of the experimental infection with Trypanosoma evansi in dogs. Arq. Bras. Med. Vet. Zootec. 54(1), 8-18. Bartels, C.F., Xie, W., Miller-Lindholm, M.K., Schopfer, L.M., Lockridge, O., 2000. Determination
of
the
DNA
sequences
11
of
acetylcholinesterase
and
butyrylcholinesterse from cat demonstration of the existence of both in cat plasma. Biochem. Pharmacol. 60, 479–487. Brennessel, D.J., Wittner, M., Braunstein, V., Herbert, B., 1985. Acetylcholinesterase levels in skeletal muscle of mice infected with Trypanosoma cruzi. Am. J. Trop. Med. Hyg. 34, 460–464. Costa, M.M., Silva, A.S., Paim, F.C., França, R., Dornelles, G.L., Thomé, G.R., Serres, J.D., Schmatz, R., Spanevello, R.M., Gonçalves, J.F., Schetinger, M.R., Mazzanti, C.M., Lopes, S.T., Monteiro, S.G., 2012. Cholinesterase as inflammatory markers in a experimental infection by Trypanosoma evansi in rabbits. An. Acad. Bras. Cienc. 84(4), 1105-1113. Da Silva, A.S., Spanevello, R., Stefanello, N., Wolkmer, P., Costa, M.M., Zanette, R.A., Lopes, S.T.A., Santurio, J.M., Schetinger, M.R.C., Monteiro, S.G., 2010. Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats. Res. Vet. Sci. 88, 281–284. Da Silva, A.S., Monteiro, S.G., Gonçalves, J.F., Spanevello, R., Oliveira, C.B., Costa, M.M., Jaques, J.A., Morsch, V.M., Schetinger, M.R., Mazzanti, C.M., Lopes, S.T., 2011b. Acetylcholinesterase activity and lipid peroxidation in the brain and spinal cord of rats infected with Trypanosoma evansi. Vet. Parasitol. 175 (3-4), 237-244. Da Silva, A.S., Oliveira, C.B., Bertoncheli, C.M., Santos, R.P., Beckmann, D.V., Wolkmer, P., Gressler, L.T., Tonin, A.A., Graça, D.L., Mazzanti, A., Lopes, S.T., Monteiro, S.G., 2011a. Clinical signs and histopathology of brain, spinal cord and
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muscle
of
the
pelvic
limb
of
rats
experimentally
infected
with Trypanosoma evansi. Pathol. Res. Pract. 208(1), 39-44. Das, U.N., 2007. Acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation. Med. Sci. Monit. 13, 214-221. Desquesnes, M., Holzmuller, P., Lai, D.H., Dargantes, A., Lun, Z.R., Jittaplapong, S., 2013. Trypanosoma evansi and Surra: A Review and Perspectives on Origin, History, Distribution, Taxonomy, Morphology, Hosts, and Pathogenic Effects. BioMed. Res. Int. http://dx.doi.org/10.1155/2013/194176 Fujii, T., Takada-Takatori, Y., Kawashima, K., 2008. Basic and clinical aspects of nonneuronal acetylcholine: expression of an independent, non-neuronal cholinergic system in lymphocytes and its clinical significance in immunotherapy. J. Pharmacol. Sci. 106(2), 186-192. Gill, B., 1977. Trypanosomes and Trypanosomiasis of Indian Livestock, ICAR: Indian Council of Agricultural Research, New Delhi, India, 1st edition. Herrera, H.M., Davila, A.M.R., Norek, A., Abreu, U.G., Souza, S.S., D’Andrea, P.S., Jansen, A.M., 2004. Enzootiology of Trypanosoma evansi in Pantanal, Brazil. Vet. Parasitol. 125, 263–275. Hertz, C.J., Filutowicz, H., Mansfield, J.M., 1998. Resistance to the African trypanosomes is IFN-gamma dependent. J. Immunol. 161(12), 6775-6783. Hilali, M., Abdel-Gawad, A., Nassar, A., Abdel-Wahab, A., 2006. Hematological and biochemical changes in water buffalo calves (Bubalus bubalis) infected with Trypanosoma evansi. Vet. Parasitol. 139(1-3), 237-243. Kutty, K.M., 1980. Biological function of cholinesterase. Clin. Biochem. 13, 239-243.
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Mansfield, J.M., Paulnock, D.M., 2005. Regulation of innate and acquired immunity in African trypanosomiasis. Parasite Immunol. 27(10-11), 361-371. Namangala, B., 2011. How the African trypanosomes evade host immune killing. Parasite Immunol. 33(8), 430-437. Namangala, B., De Baetselier, P., Beschin, A., 2009. Both type-I and type-II responses contribute to murine trypano-tolerance. J. Vet. Med. Sci. 71(3), 313-318. Otto, M.A., Da Silva, A.S., Gressler, L.T., Farret, M.H., Tavares, K.C.S., Zanette, R.A., Miletti, L.C., Monteiro, S.G., 2010. Susceptibility of Trypanosoma evansi to human blood and plasma in infected mice. Vet. Parasitol. 168, 1–4. Paim, F.C., Da Silva, A.S., Wolkmer, P., Costa, M.M., Da Silva, C.B., Paim, C.B., Oliveira, M.S., Silva, L.F., Mello, C.F., Monteiro, S.G., Mazzanti, C.M., Lopes, S.T., 2011. Trypanosoma evansi: concentration of 3-nitrotyrosine in the brain of infected rats. Exp. Parasitol. 129 (1), 27-30. Pavlov, V.A., Tracey, K.J., 2004. Neural regulators of innate immune responses and inflammation. Cell. Mol. Life Sci. 61, 2322–2331. Pavlov, V.A., Tracey, K.J., 2006. Controlling inflammation: the cholinergic antiinflammatory pathway. Biochem. Soc. Trans. 34(6), 1037-1040. Rodrigues, A., Fighera, R.A., Souza, T.M., Schild, A.L., Barros, C.S., 2009. Neuropathology of naturally occurring Trypanosoma evansi infection of horses. Vet. Pathol. 46(2), 251-258. Singh, D.S., 1976. Serum cholinesterase in hepatic disorders. J. Indian Med. Assoc. 66, 49-51.
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Tabel, H., Wei, G., Shi, M., 2008. T cells and immunopathogenesis of experimental African trypanosomiasis. Immunol. Rev. 225, 128-139. Taylor, K., Authié E.M.L., 2004. Pathogenesis of animal trypanosomiasis. In: Maudlin I, Holmes PH and Miles MA. The trypanosomiasis. London. CABI publishing, p. 331-354. Wolkmer, P., Lopes, S.T., Franciscato, C., da Silva, A.S., Traesel, C.K., Siqueira, L.C., Pereira, M.E., Monteiro, S.G., Mazzanti, C.M., 2010. Trypanosoma evansi: cholinesterase activity in acutely infected Wistar rats. Exp. Parasitol. 125, 251255. Wolkmer, P., Silva, C.B., Paim, F.C., Duarte, M.M., Castro, V., Palma, H.E., França, R.T., Felin, D.V., Siqueira, L.C., Lopes, S.T., Schetinger, M.R., Monteiro, S.G., Mazzanti,
C.M.,
2013.
Pre-treatment
with
curcumin
modulates
acetylcholinesterase activity and proinflammatory cytokines in rats infected with Trypanosoma evansi. Parasitol. Int. 62(2), 144-149. Zambrano-Villa, S., Rosales-Borjas, D., Carrero, J.C., Ortiz-Ortiz, L., 2002. How protozoan parasites evade the immune response. Trends Parasitol. 18, 272–278.
15
B
2 4 3 .7 2 1 9 .1 2
400
300
200 A
5 9 .6 7 7 .9 8 A
6 4 .4 6 6 .1 2 100
12
13 p
II
I
II
(n
(n
=
=
20 = (n I p
G
G
ro
u
ro
p
u
u ro G
)
)
0
)
C h o l i n e s t e r a s e a c t i v it y ( u n i t/ L )
Fig. 1. A Trypanosoma evansi-infected buffalo exhibiting corneal opacity and tongue paresis
Fig. 2. Serum cholinesterase activity (Mean ± SE) in Trypanosoma evansi-infected buffaloes without neurological disorders (Group I), with neurological disorders (Group II) and healthy control buffaloes (Group III). A,BDifferent superscripts on bar diagram show significant (P<0.001) difference between the groups.
16