- Email: [email protected]
Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats
Research in Veterinary Science 88 (2010) 281–284
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats A.S. Da Silva a,*, R. Spanevello b, N. Stefanello a, P. Wolkmer a, M.M. Costa a, R.A. Zanette b, S.T.A. Lopes a, J.M. Santurio a, M.R.C. Schetinger a, S.G. Monteiro a a b
Universidade Federal de Santa Maria, Faixa de Camobi, Km 9, Campus Universitário, 97105-900 Santa Maria, RS, Brazil Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
a r t i c l e
i n f o
Article history: Accepted 28 August 2009
Keywords: Trypanosoma evansi Cat Acetylcholinesterase Butyrylcholinesterase
a b s t r a c t Changes in blood, plasma and brain cholinesterase activities in Trypanosoma evansi-infected cats were investigated. Seven animals were infected with 108 trypomastigote forms each and six were used as control. Animals were monitored for 56 days by examining daily blood smears. Blood samples were collected at days 28 and 56 post-inoculation to determine the activity of acetylcholinesterase (AChE) in blood and the activity of butyrylcholinesterase (BChE) in plasma. AChE was also evaluated in total brain. The activity of AChE in blood and brain, and the activity of BChE in plasma significantly reduced in the infected cats. Therefore, the infection by T. evansi influenced cholinesterases of felines indicating changes in the responses of the cholinergic system. Ó 2009 Elsevier Ltd. All rights reserved.
Trypanosoma evansi is a Salivarian trypanosomatid phylogenetically closely related to Trypanosoma brucei that displays a broad host range and geographical distribution in Latin America, Africa and Asia (Herrera et al., 2005). T. evansi causes an important horse disease called ‘‘Mal de Cadeiras’’, characterized by anemia, immunosuppression, emaciation, severe neurological signs and death of non-treated animals (Nunes and Oshiro, 1990; Franke et al., 1994; Silva et al., 1995; Herrera et al., 2005). Acetylcholinesterase (AChE: EC 3.1.1.7) and butyrylcholinesterase (BChE: EC 3.1.1.8) are enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine (ACh), a key process in the regulation of the cholinergic system (Darvesh et al., 2003). AChE is a specific choline esterase characterized by high concentrations in brain, nerve and red blood cells. BChE is a nonspecific choline esterase found in blood serum, pancreas, liver, and central nervous system (Kaplay, 1976). Changes in acetyltransferase and AChE activities (Brennessel et al., 1985) and increased dopamine and norepinephrine (Amole et al., 1989) are described associated with trypanosomiasis by Trypanosoma cruzi and T. brucei in mice. Clinical signs of neurological disorders are reported in horses, cattle and deer parasitized by T. evansi (Tuntasuvan et al., 1997, 2000; Rodrigues et al., 2005). Thus, the aim of this study was to investigate changes in the activity of blood and brain AChE and plasmatic BChE in cats infected by T. evansi. We chose to use cats in our study due to the chronic aspect of the disease in this specie (Choudhury and Misra, 1972), * Corresponding author. Tel./fax: +55 55 3220 8958. E-mail address: [email protected] (A.S. Da Silva). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.08.011
since it is in the chronic phase that neurological signs associated to brain lesions are seen in bovines and equines (Tuntasuvan et al., 1997; Rodrigues et al., 2005). Thirteen adult female Felis catus, weighing between 1912 and 2561 g were used. Animals were kept in individual cages with temperature and humidity controlled at 23 °C and 70%, respectively. They were fed with commercial ration and water ad libitum. All animals were submitted to a period of thirty days for adaptation. Hematological and biochemical examinations were performed three times at 15-day intervals. The evaluated patterns showed normal values (Bush, 2004). Cats were divided in two groups, a control group with six animals and an infected group with seven animals. They were inoculated intraperitoneally with 108 trypomastigote forms of a strain of T. evansi whereas the control group received a physiological solution. Parasitemia was estimated daily by microscopic examination of smears. The evolution of the disease was monitored by performing hematocrit measurements at 15-day intervals. Blood samples were collected in tubes with sodium heparin at days 28 and 56 postinoculation (PI) by jugular puncture after anesthesia with ketamine (0.08 mL kg 1) and xylazine (0.05 mL kg 1). Samples were diluted 1:50 (v/v) in lysis solution (0.1 mmol/L potassium/sodium phosphate buffer containing 0.03% Triton X-100) to determine AChE activity in blood. The BChE activity was measured in plasma after the blood was centrifuged for 10 min at 1000g. After sedation, animals were euthanized with T61 and brains of four T. evansiinfected cats and three control animals were removed, weighed and longitudinally sectioned. The right side of the brain was
282
A.S. Da Silva et al. / Research in Veterinary Science 88 (2010) 281–284
A
Trypomastigotes/field (1000x)
homogenized separately in buffer Tris–HCl 10 mmol, pH 7.2 with 160 mmol sucrose (1:10 w/v) to verify the activity of AChE. The samples were stored in tubes and frozen at 20 °C until analyzed. The left side of the brains and the spinal cord were stored in formol and histologically processed to investigate lesions in the central nervous. Smears were mounted and stained by the hematoxylin– eosin method. The AChE enzymatic assay in the whole blood was determined by the method of Ellman et al. (1961) modified by Worek et al. (1999). The specific activity of whole blood AChE was calculated from the quotient between AChE activity and hemoglobin content and the results were expressed as mU/l mol Hb. The same method was used for the determination of BChE activity in the plasma, except that the acetylcholine substrate was replaced by butyrylthiocholine and the results were expressed in the lmoles BcSCh/h/ mg of protein. The AChE enzymatic assay in the brain was determined by a modification of the spectrophotometric method of Ellman et al. (1961) as previously described by Rocha et al. (1993). All samples were carried out in duplicate or triplicate and the enzyme activity was expressed in lmoles AcSCh/h/mg of protein. The data were submitted to analysis of variance (ANOVA) followed by the Tukey’s test (P < 0.05). The procedure was approved by the Animal Welfare Committee of Federal University de Santa Maria (UFSM), number 23081.002891/2008-47. Examination of the peripheral blood smears showed a prepatency period between 24 and 48 h in the infected cats, with the peak of parasitemia recorded at day four PI. Thereafter, irregular waves of parasitemia were observed, ranging from zero to three trypomastigotes per microscopic field (Fig. 1A). In this period, all infected animals showed a drop in hematocrit (Fig. 1B) and weight
loss. Some cats presented neurological disorders such as instability and incoordination of the hind limbs. The animals also had anemia, which was previously described in other host species (De La Rue et al., 2000; Rodrigues et al., 2005). Uncoordinated gait and stiffening of the hind limbs observed in the cats of our study are often reported in horses (Rodrigues et al., 2005; Zanette et al., 2008). We observed a significant reduction in AChE activity (Fig. 2A) in the blood of cats infected by T. evansi at days 28 and 56 (P < 0.001). The BChE activity (Fig. 2B) also was reduced in the plasma of the parasitized animals at day 56 when compared to the control group (P < 0.05). Moreover, brain AChE activity (Fig. 2C) was reduced in the parasitized felines (P < 0.001). No difference was observed between infected and non-infected groups regarding the weight of the brain. The reduction of AChE and BChE might be a possible cause for these clinical signs, since these enzymes are essential in nerve impulse transmission where they break down acetylcholine that diffuses across synapse nerve during the nerve signal relay (Bartels et al., 2000). The histopathology showed that the clinical signs here observed were not related to the lesions in the central nervous system as reported in other species (Tuntasuvan et al., 1997; Rodrigues et al., 2005). Reduced AChE activity in blood and central nervous system may explain the motor incoordination and paralysis of the posterior observed in horses (Silva et al., 1995; Zanette et al., 2008) and in the cats of this study. This might be explained since this enzyme is one of the most efficient biological catalysts known and plays a key role in cholinergic neurotransmission by hydrolysing the transmitter acetylcholine, thus terminating its action (Soreq and Seidman, 2001; Mesulam et al., 2002). To examine the role of AChE in hematopoietic cell proliferation and differentiation, Soreq et al. (1994) administered a 15-mer
14 Cat 1
12
Cat 2
10
Cat 3
8
Cat 4
6
Cat 5 Cat 6
4
Cat 7
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3
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30
25
20
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35*
49*
Day Fig. 1. Parasitemia (A) and hematocrit levels (B) of T. evansi-infected cats at day 56 post-inoculation. Asterisk indicates statistical difference between infected and noninfected groups (*Tukey post hoc test P < 0.05).
A.S. Da Silva et al. / Research in Veterinary Science 88 (2010) 281–284
A 250
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of the activity of this enzyme, because the evaluated liver enzymes, alanine aminotransferase and gamma-glutamyl transpeptidase, remained within the normal range during the entire experiment and were analyzed at days 28 and 56, what would characterize a chronic infection. ACh is a neurotransmitter and regulates the levels and activities of serotonin, dopamine and other neuropeptides and, thus, modulates both immune response and neurotransmission. Hence, both AChE and BChE by inactivating acetylcholine may enhance inflammation (Undurti and Das, 2007). This suggests that increased plasma and tissue activities of AChE and BChE seen in various clinical conditions could act as a marker of low-grade systemic inflammation (Undurti and Das, 2007). This is the first study to describe changes in the activity of the cholinergic system of T. evansi-infected animals. The chronic phase of the disease in cats reduces the levels of AChE in blood and brain, in addition to BChE in plasma. It is important to emphasize the similarity between the results of the central and peripheral cholinergic systems, which shows that the infection with T. evansi can directly or indirectly change the activities of AChE and BChE.
2
0
C
283
5 4 3 2 1
Brain* Control group
Infected group
Fig. 2. Acetylcholinesterase activity in blood (A), butyrylcholinesterase activity in plasma (B) and acetylcholinesterase activity in brain (C) of Trypanosoma evansiinfected cats. Asterisk indicates statistical difference between infected and noninfected groups (*Tukey post hoc test P < 0.05).
phosphorothioate oligonucleotide, antisense to the corresponding ACHE gene (AS-ACHE), to primary mouse bone marrow cultures. The researchers suggest that the hematopoietic role of AChE, anticipated to be inverse to the observed antisense effects, is to reduce proliferation of the multipotent stem cells committed to erythropoiesis, megakaryocytopoiesis, macrophage production, and to promote apoptosis in their progeny. Moreover, this finding may explain the tumorigenic association of perturbations in AChE gene expression with leukemia. Based on these data, one can suspect that the low AChE in cats may have contributed to the process of chronic anemia observed during the whole experiment in felines with T. evansi. Studies with T. evansi-infected animals show the disease presenting with liver damage (Aquino et al., 2002; Herrera et al., 2002). As the plasma BChE is synthesized in the liver (Wescoe et al., 1947), decreased liver function would result in reduction of the enzyme production (Singh et al., 1976; Lemberg and Macchi, 1981); another situation would be in acute infections (Whittaker, 1980). However, none of these hypotheses is related to inhibition
Amole, B., Sharpless, N., Wittner, M., Tanowitz, H.B., 1989. Neurochemical measurements in the brains of mice infected with Trypanosoma brucei (TREU 667). Annals of Tropical Medicine and Parasitology 83, 225–232. Aquino, L.P.C.T., Machado, R.Z., Alessi, A.C., Santana, A.E., Castro, M.B., Marques, L.C., 2002. Hematological, biochemical and anatomopathological aspects of the experimental infection with Trypanosoma evansi in dogs. Arquivos Brasileiro de Medicina Veterinária e Zootecnia 54, 8–18. Bartels, C.F., Xie, W., Miller-Lindholm, M.K., Schopfer, L.M., Lockridge, O., 2000. Determination of the DNA Sequences of acetylcholinesterase and butyrylcholinestersa from cat demonstration of the existence of both in cat plasma. Biochemical Pharmacology 60, 479–487. Brennessel, D.J., Wittner, M., Braunstein, V., Herbert, B., 1985. Acetylcholinesterase levels in skeletal muscle of mice infected with Trypanosoma cruzi. The American Society of Tropical Medicine and Hygiene 34, 460–464. Bush, B.M., 2004. Interpretação de resultados laboratoriais para clínicos de pequenos animais. Roca, São Paulo. Choudhury, A., Misra, K.K., 1972. Experimental infection of T. evansi in the cat. Transactions of the Royal Society of Tropical Medicine and Hygiene 66, 672– 673. Darvesh, S., Hopkins, D.A., Geula, C., 2003. Neurobiology of butyrylcholinesterase. Nature Reviews Neuroscience 17, 131–138. De La Rue, M.L.L., Silva, R.A.M.S., Carli, G.A., 2000. Leucocytes and reticulocytes counts in acute infection of dogs with Tripanosoma evansi (Steel, 1885) Balbani, 1888. Revista Latinoamericana de Microbiologia 40, 163–166. Ellman, G.L., Coutney, K.O., Andres, V., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88–95. Franke, C.R., Greiner, M., Mehlitz, D., 1994. Investigations on naturally occurring Trypanosoma evansi infections in horses, cattle, dogs and capybaras (Hydrochaeris hydrochaeris) in Pantanal de Pocone´ (Mato Grosso, Brasil). Acta Tropica 58, 159–169. Herrera, H.M., Alessi, A.C., Marques, L.C., Santana, A.E., Aquino, L.P., Menezes, R.F., 2002. Experimental Trypanosoma evansi infection in the South American coati (Nasua nasua): hematological, biochemical and histopathological changes. Acta Tropica 81, 203–210. Herrera, H.M., Norek, A., Freitas, T.P.T., Rademaker, V., Fernandes, O., Jansen, N.M., 2005. Domestic and wild mammals infection by Trypanosoma evansi in a pristine area of the Brazilian Pantanal region. Parasitology Research 96, 121– 126. Kaplay, S.S., 1976. Acetylcholinesterase and butyrylcholinesterase of developing human brain. Biology of the Neonate 28, 65–73. Lemberg, A., Macchi, M.C., 1981. Usefulness of serum pseudocholinesterase isoenzymes in acute and chronic liver diseases and neoplasms (experimental and clinical studies). Acta Gastroenterology Latinoamericana 11, 125–132. Mesulam, M., Guillozet, A., Shaw, P., 2002. Acethylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyse acetylcholine. Neuroscience 110, 627–639. Nunes, V.L.B., Oshiro, E.T., 1990. Trypanosoma (Trypanozoon) evansi in the coati from the Pantanal region of Mato Grosso do Sul State, Brazil. Transactions of the Royal Society of Tropical Medicine and Hygiene 84, 692. Rocha, J.B.T., Emanuelli, T., Pereira, M.E., 1993. Effects of early undernutrition on kinetic parameters of brain acetylcholinesterase from adult rats. Acta Neurobiologiae Experimentalis 53, 431–437. Rodrigues, A., Fighera, R.A., Souza, T.M., Schild, A.L., Soares, M.P., Milano, J., Barros, C.S.L., 2005. Outbreaks of trypanosomiasis in horses by Trypanosoma evansi in
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the state of Rio Grande do Sul, Brazil: epidemiological, clinical, hematological, and pathological aspects. Pesquisa Veterinária Brasileira 25, 239–249. Silva, R.A.M.S., Barros, A.T.M., Herrera, H.M., 1995. Trypanosomosis outbreaks due to Trypanosoma evansi in the Pantanal, Brazi. A preliminary approach on risk factors. Revue D’Élevage et de Médicine Véterinaire dês Pays Tropicaux 48, 315– 319. Singh, D.S., Shukia, P.K., Gupta, J.P., Dubs, B., 1976. Serum cholinesterase in hepatic disorders. Journal Indian Medicine Association 66, 49–51. Soreq, H., Patinkin, D., Lev-Lehman, E., Grifman, M., Ginzberg, D., Ecksteint, F., Zakut, H., 1994. Antisense oligonucleotide inhibition of acetyicholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosis ex vivo. Proceedings of the National Academy of Sciences 91, 7907– 7911. Soreq, H., Seidman, S., 2001. Acetylcholinesterase-new roles for and old actor. Nature Reviews Neuroscience 2, 294–302. Tuntasuvan, D., Sarataphan, N., Nishikawa, H., 1997. Cerebral trypanosomiasis in native cattle. Veterinary Parasitology 73, 357–363.
Tuntasuvan, D., Mimapan, S., Sarataphan, N., Trongwongsa, L., Intraraksa, R., Luckins, A.G., 2000. Detection of Trypanosoma evansi in brains of the naturally infected hog deer by immunohistochemistry. Veterinary Parasitology 87, 223–230. Undurti, N.D., 2007. Acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation. Medical Science Monitor 13, 214–221. Wescoe, W.C., Hunt, C.C., Riker, W.S., Litt, F.C., 1947. Regeneration rates of serum cholinesterase in normal individuals and in patients with liver damage. American Journal Physiology 149, 549–551. Whittaker, M., 1980. Plasma cholinesterase variants and the anaesthetist. Anaesthesia 35, 174–197. Worek, F., Mast, U., Kiderlen, D., 1999. Improved determination of acetylcholinestrase activity in human whole blood. Clinica Chimica Acta 288, 73–90. Zanette, R.A., Da Silva, A.S., Costa, M.M., Lopes, S.T.A., Santurio, J.M., Monteiro, S.G., 2008. Ocorrência de Trypanosoma evansi em eqüinos no município de Cruz Alta – RS, Brasil. Ciência Rural 38, 1468–1471.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Influence of Trypanosoma evansi in blood, plasma, and brain cholinesterase of experimentally infected cats A.S. Da Silva a,*, R. Spanevello b, N. Stefanello a, P. Wolkmer a, M.M. Costa a, R.A. Zanette b, S.T.A. Lopes a, J.M. Santurio a, M.R.C. Schetinger a, S.G. Monteiro a a b
Universidade Federal de Santa Maria, Faixa de Camobi, Km 9, Campus Universitário, 97105-900 Santa Maria, RS, Brazil Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
a r t i c l e
i n f o
Article history: Accepted 28 August 2009
Keywords: Trypanosoma evansi Cat Acetylcholinesterase Butyrylcholinesterase
a b s t r a c t Changes in blood, plasma and brain cholinesterase activities in Trypanosoma evansi-infected cats were investigated. Seven animals were infected with 108 trypomastigote forms each and six were used as control. Animals were monitored for 56 days by examining daily blood smears. Blood samples were collected at days 28 and 56 post-inoculation to determine the activity of acetylcholinesterase (AChE) in blood and the activity of butyrylcholinesterase (BChE) in plasma. AChE was also evaluated in total brain. The activity of AChE in blood and brain, and the activity of BChE in plasma significantly reduced in the infected cats. Therefore, the infection by T. evansi influenced cholinesterases of felines indicating changes in the responses of the cholinergic system. Ó 2009 Elsevier Ltd. All rights reserved.
Trypanosoma evansi is a Salivarian trypanosomatid phylogenetically closely related to Trypanosoma brucei that displays a broad host range and geographical distribution in Latin America, Africa and Asia (Herrera et al., 2005). T. evansi causes an important horse disease called ‘‘Mal de Cadeiras’’, characterized by anemia, immunosuppression, emaciation, severe neurological signs and death of non-treated animals (Nunes and Oshiro, 1990; Franke et al., 1994; Silva et al., 1995; Herrera et al., 2005). Acetylcholinesterase (AChE: EC 3.1.1.7) and butyrylcholinesterase (BChE: EC 3.1.1.8) are enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine (ACh), a key process in the regulation of the cholinergic system (Darvesh et al., 2003). AChE is a specific choline esterase characterized by high concentrations in brain, nerve and red blood cells. BChE is a nonspecific choline esterase found in blood serum, pancreas, liver, and central nervous system (Kaplay, 1976). Changes in acetyltransferase and AChE activities (Brennessel et al., 1985) and increased dopamine and norepinephrine (Amole et al., 1989) are described associated with trypanosomiasis by Trypanosoma cruzi and T. brucei in mice. Clinical signs of neurological disorders are reported in horses, cattle and deer parasitized by T. evansi (Tuntasuvan et al., 1997, 2000; Rodrigues et al., 2005). Thus, the aim of this study was to investigate changes in the activity of blood and brain AChE and plasmatic BChE in cats infected by T. evansi. We chose to use cats in our study due to the chronic aspect of the disease in this specie (Choudhury and Misra, 1972), * Corresponding author. Tel./fax: +55 55 3220 8958. E-mail address: [email protected] (A.S. Da Silva). 0034-5288/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2009.08.011
since it is in the chronic phase that neurological signs associated to brain lesions are seen in bovines and equines (Tuntasuvan et al., 1997; Rodrigues et al., 2005). Thirteen adult female Felis catus, weighing between 1912 and 2561 g were used. Animals were kept in individual cages with temperature and humidity controlled at 23 °C and 70%, respectively. They were fed with commercial ration and water ad libitum. All animals were submitted to a period of thirty days for adaptation. Hematological and biochemical examinations were performed three times at 15-day intervals. The evaluated patterns showed normal values (Bush, 2004). Cats were divided in two groups, a control group with six animals and an infected group with seven animals. They were inoculated intraperitoneally with 108 trypomastigote forms of a strain of T. evansi whereas the control group received a physiological solution. Parasitemia was estimated daily by microscopic examination of smears. The evolution of the disease was monitored by performing hematocrit measurements at 15-day intervals. Blood samples were collected in tubes with sodium heparin at days 28 and 56 postinoculation (PI) by jugular puncture after anesthesia with ketamine (0.08 mL kg 1) and xylazine (0.05 mL kg 1). Samples were diluted 1:50 (v/v) in lysis solution (0.1 mmol/L potassium/sodium phosphate buffer containing 0.03% Triton X-100) to determine AChE activity in blood. The BChE activity was measured in plasma after the blood was centrifuged for 10 min at 1000g. After sedation, animals were euthanized with T61 and brains of four T. evansiinfected cats and three control animals were removed, weighed and longitudinally sectioned. The right side of the brain was
282
A.S. Da Silva et al. / Research in Veterinary Science 88 (2010) 281–284
A
Trypomastigotes/field (1000x)
homogenized separately in buffer Tris–HCl 10 mmol, pH 7.2 with 160 mmol sucrose (1:10 w/v) to verify the activity of AChE. The samples were stored in tubes and frozen at 20 °C until analyzed. The left side of the brains and the spinal cord were stored in formol and histologically processed to investigate lesions in the central nervous. Smears were mounted and stained by the hematoxylin– eosin method. The AChE enzymatic assay in the whole blood was determined by the method of Ellman et al. (1961) modified by Worek et al. (1999). The specific activity of whole blood AChE was calculated from the quotient between AChE activity and hemoglobin content and the results were expressed as mU/l mol Hb. The same method was used for the determination of BChE activity in the plasma, except that the acetylcholine substrate was replaced by butyrylthiocholine and the results were expressed in the lmoles BcSCh/h/ mg of protein. The AChE enzymatic assay in the brain was determined by a modification of the spectrophotometric method of Ellman et al. (1961) as previously described by Rocha et al. (1993). All samples were carried out in duplicate or triplicate and the enzyme activity was expressed in lmoles AcSCh/h/mg of protein. The data were submitted to analysis of variance (ANOVA) followed by the Tukey’s test (P < 0.05). The procedure was approved by the Animal Welfare Committee of Federal University de Santa Maria (UFSM), number 23081.002891/2008-47. Examination of the peripheral blood smears showed a prepatency period between 24 and 48 h in the infected cats, with the peak of parasitemia recorded at day four PI. Thereafter, irregular waves of parasitemia were observed, ranging from zero to three trypomastigotes per microscopic field (Fig. 1A). In this period, all infected animals showed a drop in hematocrit (Fig. 1B) and weight
loss. Some cats presented neurological disorders such as instability and incoordination of the hind limbs. The animals also had anemia, which was previously described in other host species (De La Rue et al., 2000; Rodrigues et al., 2005). Uncoordinated gait and stiffening of the hind limbs observed in the cats of our study are often reported in horses (Rodrigues et al., 2005; Zanette et al., 2008). We observed a significant reduction in AChE activity (Fig. 2A) in the blood of cats infected by T. evansi at days 28 and 56 (P < 0.001). The BChE activity (Fig. 2B) also was reduced in the plasma of the parasitized animals at day 56 when compared to the control group (P < 0.05). Moreover, brain AChE activity (Fig. 2C) was reduced in the parasitized felines (P < 0.001). No difference was observed between infected and non-infected groups regarding the weight of the brain. The reduction of AChE and BChE might be a possible cause for these clinical signs, since these enzymes are essential in nerve impulse transmission where they break down acetylcholine that diffuses across synapse nerve during the nerve signal relay (Bartels et al., 2000). The histopathology showed that the clinical signs here observed were not related to the lesions in the central nervous system as reported in other species (Tuntasuvan et al., 1997; Rodrigues et al., 2005). Reduced AChE activity in blood and central nervous system may explain the motor incoordination and paralysis of the posterior observed in horses (Silva et al., 1995; Zanette et al., 2008) and in the cats of this study. This might be explained since this enzyme is one of the most efficient biological catalysts known and plays a key role in cholinergic neurotransmission by hydrolysing the transmitter acetylcholine, thus terminating its action (Soreq and Seidman, 2001; Mesulam et al., 2002). To examine the role of AChE in hematopoietic cell proliferation and differentiation, Soreq et al. (1994) administered a 15-mer
14 Cat 1
12
Cat 2
10
Cat 3
8
Cat 4
6
Cat 5 Cat 6
4
Cat 7
2 0 0
1
2
3
4
5
6
9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 54 56
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Hematocrit (%)
35
30
25
20
15 0
7*
21*
35*
49*
Day Fig. 1. Parasitemia (A) and hematocrit levels (B) of T. evansi-infected cats at day 56 post-inoculation. Asterisk indicates statistical difference between infected and noninfected groups (*Tukey post hoc test P < 0.05).
A.S. Da Silva et al. / Research in Veterinary Science 88 (2010) 281–284
A 250
mU/l mol Hb
200 150 100 50 0
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56*
Day 5
µmoles BcSCh/h/mg of protein
B
4 3
References
1
28
56*
Day
6
µmoles AcSCh/h/mg of protein
of the activity of this enzyme, because the evaluated liver enzymes, alanine aminotransferase and gamma-glutamyl transpeptidase, remained within the normal range during the entire experiment and were analyzed at days 28 and 56, what would characterize a chronic infection. ACh is a neurotransmitter and regulates the levels and activities of serotonin, dopamine and other neuropeptides and, thus, modulates both immune response and neurotransmission. Hence, both AChE and BChE by inactivating acetylcholine may enhance inflammation (Undurti and Das, 2007). This suggests that increased plasma and tissue activities of AChE and BChE seen in various clinical conditions could act as a marker of low-grade systemic inflammation (Undurti and Das, 2007). This is the first study to describe changes in the activity of the cholinergic system of T. evansi-infected animals. The chronic phase of the disease in cats reduces the levels of AChE in blood and brain, in addition to BChE in plasma. It is important to emphasize the similarity between the results of the central and peripheral cholinergic systems, which shows that the infection with T. evansi can directly or indirectly change the activities of AChE and BChE.
2
0
C
283
5 4 3 2 1
Brain* Control group
Infected group
Fig. 2. Acetylcholinesterase activity in blood (A), butyrylcholinesterase activity in plasma (B) and acetylcholinesterase activity in brain (C) of Trypanosoma evansiinfected cats. Asterisk indicates statistical difference between infected and noninfected groups (*Tukey post hoc test P < 0.05).
phosphorothioate oligonucleotide, antisense to the corresponding ACHE gene (AS-ACHE), to primary mouse bone marrow cultures. The researchers suggest that the hematopoietic role of AChE, anticipated to be inverse to the observed antisense effects, is to reduce proliferation of the multipotent stem cells committed to erythropoiesis, megakaryocytopoiesis, macrophage production, and to promote apoptosis in their progeny. Moreover, this finding may explain the tumorigenic association of perturbations in AChE gene expression with leukemia. Based on these data, one can suspect that the low AChE in cats may have contributed to the process of chronic anemia observed during the whole experiment in felines with T. evansi. Studies with T. evansi-infected animals show the disease presenting with liver damage (Aquino et al., 2002; Herrera et al., 2002). As the plasma BChE is synthesized in the liver (Wescoe et al., 1947), decreased liver function would result in reduction of the enzyme production (Singh et al., 1976; Lemberg and Macchi, 1981); another situation would be in acute infections (Whittaker, 1980). However, none of these hypotheses is related to inhibition
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