Experimental Parasitology 139 (2014) 19–23
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Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Influence of experimental infection by Haemonchus contortus on acetylcholinesterase activity in lymphocytes of lambs Alexandre A. Tonin a,⇑, Aleksandro S. Da Silva b,⇑, Andressa S. Schafer c, Adelina R. Aires c, Camila B. Oliveira d, Daniela Zanini e, Maria R.C. Schetinger e, Vera M. Morsch e, Sonia T.A. Lopes a, Silvia G. Monteiro d, Marta L.R. Leal c a
Department of Small Animal, Universidade Federal de Santa Maria (UFSM), Brazil Department of Animal Science, Universidade do Estado de Santa Catarina, Brazil Laboratory of Animal Endocrinology and Metabology. Program of Post-Graduation in Veterinary Medicine, UFSM, Brazil d Department of Microbiology and Parasitology, UFSM, Brazil e Program of Post-Graduation in Toxicological Biochemistry, UFSM, Brazil b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sheep herds infected with
Haemonchus contortus in the hyperacute and acute phase of the disease. Experimental infection by H. contortus in lambs causes a reduction in the number of lymphocytes. Lambs infected with H. contortus showed a reduction in acetylcholinesterase activity in lymphocytes. The participation of the cholinergic system in the immune response is well documented. Lambs infected with H. contortus showed reduce of AChE activity in lymphocytes.
a r t i c l e
i n f o
Article history: Received 6 September 2013 Received in revised form 6 February 2014 Accepted 9 February 2014 Available online 19 February 2014 Keywords: Helminths Acetylcholine WBC Sheep
a b s t r a c t The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes of lambs experimentally infected by Haemonchus contortus. A total of 14 healthy lambs were used, divided into two groups of seven animals each. Group A (negative control) represented the uninfected animals, and Group B (positive control) was formed by animals infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the white blood cells (WBC) count, as well as the evaluation of AChE activity in lymphocytes. Parasitological stool exam (eggs per gram of feces – EPG) was performed on the same days to follow up the evolution of the infection. On day 15 PI it was verified negative EPG; however, on days 45 and 75 PI it was observed positive EPG only in the animals of group B. In the three evaluated periods was observed a lower number of leukocytes, associated with decreased lymphocytes and neutrophils in lambs infected by this gastrointestinal nematodes. Lambs infected with H. contortus showed significant (P < 0.01) lower AChE activity in lymphocytes compared uninfected. Statistically, there was a positive correlation (P < 0.05) between AChE activity in lymphocytes and number of lymphocytes (r = 0.69). The lymphocytes are cells with direct participation in the
⇑ Corresponding authors. E-mail addresses: [email protected] (A.A. Tonin), aleksandro_ss@yahoo. com.br (A.S. Da Silva). http://dx.doi.org/10.1016/j.exppara.2014.02.006 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.
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A.A. Tonin et al. / Experimental Parasitology 139 (2014) 19–23
cholinergic system; therefore, based on these results, it can be concluded that the experimental infection with H. contortus influences the number of lymphocytes, and consequently the AChE activity in these cells. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Sheep herds infected with Haemonchus contortus usually present weight loss, dehydration, diarrhea, anemia, lackluster wool and death in the hyperacute and acute phase of the disease. During the chronic phase, these signals are intensified, usually followed by severe swelling in the submandibular and ventral regions, due to the hypoalbuminemia (Freitas, 1982; Santa Rosa, 1996; Cavalcante et al., 2009; Fonseca et al., 2011). Although the infections in sheep are usually mixed (helminths of the Trichostrongylidae family), H. contortus has a higher prevalence and importance in this process (Fonseca et al., 2011). The immune response was involved to control infection by H. contortus, and that helminthes parasites generally stimulate a Th2-type response (Miller and Horohov, 2006). According to the literature, the existence of cholinergic receptors in the immune system cells is known, as well as the influence of cholinesterases in inflammation (Tayebati et al., 2002; Kawashima and Fujii, 2003; Kimura et al., 2003; Da Silva et al., 2011). Cholinesterases are involved in different vital functions in mammals, being the acetylcholinesterase (AChE: EC 3.1.1.7) and butyrylcholinesterase (BChE: EC 3.1.1.8) are enzymes that catalyze the hydrolysis of the acetylcholine (ACh), a key process in the regulation of the cholinergic system (Darvesh et al., 2003). The lymphocytes synthesize ACh, which is degraded by AChE, and in consequence of this, the complete cholinergic repertoire of immune cells was termed the lymphocytic cholinergic system (Kawashima and Fujii, 2003). The nictotinic cholinergic receptors (nAChR) can induce a rapid and transient Ca2+ influx in lymphocytes (Kimura et al., 2003) in inflammation. The regulation of inflammatory reactions occurs by activation of the a7 nAChR in macrophages, as cholinergic stimulation reduced pro-inflammatory cytokine production in a lethal endotoxemia model (Borovikova et al., 2000). Therefore, leukocytes and cholinesterase have concurrent participation during inflammation. The relation between cholinesterase and immune response mediated by lymphocyte is unknown by the infection of lambs H. contortus. However, study has shown a positive correlation between AChE activity in lymphocytes and lymphocytosis in rats experimentally infected with Trypanosoma evansi (Da Silva et al., 2011). Therefore, the aim of this study was to evaluate the AChE activity in lymphocytes of lambs experimentally infected with H. contortus. 2. Materials and methods Our experiment used 14 male lambs crossbred Corriedale x Texel, 5 months old and weighing in average 23 kg. They were kept in holding pens (one pen/group) during 30 days under a diet base of 10.7% protein (commercial feed – 30% and ryegrass hay – 70%) for the adaptation in the experimental environment. In this period, the animals received anthelmintic treatment based on Monepantel (Zolvix1Ò). The same diet was kept during the first 20 days of the experiment (post-infection); however at this period it was necessary to change the diet as a consequence of the severe disease evolution. From this period onward, the animals were fed hay ground ryegrass (70%), commercial ration (20%) and soybean meal (10%), providing a 13% protein diet base. Each animal 1
Novartis Healthcare A/S. Edvard Thomsens Vej 2300, Copenhague, Dinamarca.
consumed 1 kg of dry matter/day. Hematological (erythrogram and leukogram) and biochemical (hepatic and renal function) examinations were performed three times at 15-day intervals. After 30 days (day 0 of the experiment), the evaluated patterns showed normal values (Table 1), according to Feldman et al. (2000). Apparently the animals were healthy, and they had negative fecal exam for eggs, cysts and oocysts of parasites. The animals were divided into two groups with seven animals each: Group A was composed by of healthy animals (uninfected); In Group B, the animals were infected with H. contortus. Each animal from group B was infected orally with a total of 15,000 larvae (L3), divided in three moments of infection, 5000 larvae each time, at intervals of 3 days between infections (Rowe et al. 2008), obtained by coproculture technique (Roberts and O’Sullivan 1950). Blood was drawn through VacutainerÒ system in tubes with anticoagulant (EDTA) on the days 15, 45 and 75 post-infection (PI). Samples were stored in tubes containing anticoagulant in order to allow the separation of lymphocytes (4 mL) and WBC analysis (1 mL). Leukocytes count was done manually in a Neubauer chamber, modified (Feldman et al. 2000). Blood smears were prepared and stained by the Romanowski method for evaluation of differential leukocyte. The peripheral lymphocytes were isolated using Ficoll Hypaque density gradient as described by Böyum (1968). After separation, only samples with at least 95% of lymphocytes, as verified in the coulter STKS (Miami-USA) were used. After isolation of the lymphocytes, AChE activity was determined according to the method described by Fitzgerald and Costa (1993). Briefly, proteins of all samples were adjusted to 0.1–0.2 mg/mL, i.e., volume with similar numbers of lymphocytes. Volume of 0.2 mL intact cells were added to a solution containing 1.0 mM acetylthiocholine, 0.1 mM 5,50 -Dithiobis (2-nitrobenzoic acid), and 0.1 mM phosphate buffer (pH 8.0). Immediately before and after incubation for 30 min at 27 °C the absorbance was read on a spectrophotometer at 412 nm. AChE was calculated from the quotient between lymphocyte AChE activity and protein content and the results are expressed as lmol of AcSCh/h/mg of protein. Fecal samples for quantification of eggs per gram (EPG) were collected on 15, 45 and 75 PI, and processed according to the technique described by Gordon and Whitlock (1939). Five days after the end of experiment (day 80 PI), five animals from each group were euthanized (10 mg intravenous (IV) acepromazine; 2 g IV sodium thiopental; then 100 ml IV potassium chloride), and their parasite loads were determined (Ueno and Gonçalves, 1998). The euthanasia was necessary to confirm that the animals were infected (Group B) or negative (Group A), since the EPG, by itself, is not sufficient to validate the research. Normality test was performed on all the data. EPG and leukocytes data not present a normal distribution and they were logarithm before statistical analysis. Then, the AChE activity, leukocytes and EPG results were subjected to the Student test. The effect of AChE in lymphocytes on number of lymphocytes was analyzed by linear correlation. Values with probability (P) less than 5% were considered statistically different. 3. Results The animals, post-infection, were losing their physiological condition progressively, getting weak, lethargic, pale and reducing
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Table 1 Mean and standard deviation of hematologic parameters (Total leukocytes, neutrophils, lymphocytes, monocytes and eosinophils) in lambs uninfected and infected with gastrointestinal helminths on days 15, 45 and 75 post-infection. Parameters 3
Total Leukocytes (10 lL) *
4.000–12.000
Neutrophil (103 lL) * 700–6000
Lymphocytes (103 lL) * 2.000–9.000
Monocytes (103 lL) * 0–750
Eosinophils (103 lL) 0–1.000
*
# *
Day
Group A: control
Group B: infected
Probability (P)#
00 15 45 75 00 15 45 75 00 15 45 75 00 15 45 75 00 15 45 75
9.24 ± 1.30 9.86 ± 1.96 7.53 ± 1.24 9.25 ± 1.04 4.02 ± 0.79 4.17 ± 0.99 2.95 ± 0.75 3.53 ± 1.25 5.01 ± 1.24 5.37 ± 1.16 4.32 ± 1.23 5.49 ± 1.53 0.15 ± 0.12 0.19 ± 0.10 0.16 ± 0.07 0.09 ± 0.02 0.06 ± 0.14 0.13 ± 0.11 0.10 ± 0.08 0.14 ± 0.10
9.37 ± 1.82 7.44 ± 2.55 5.18 ± 1.08 5.55 ± 1.16 4.08 ± 0.95 2.31 ± 1.16 2.08 ± 0.62 2.03 ± 0.75 5.12 ± 1.50 4.79 ± 2.04 2.68 ± 0.80 3.31 ± 0.71 0.10 ± 0.08 0.15 ± 0.09 0.19 ± 0.11 0.09 ± 0.05 0.07 ± 0.18 0.19 ± 0.12 0.23 ± 0.19 0.12 ± 0.06
P > 0.05 P > 0.05 P < 0.05# P < 0.01# P > 0.05 P < 0.05# P < 0.05# P < 0.05# P > 0.05 P > 0.05 P < 0.05# P < 0.01# P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
In the same line, was considered significant difference between groups in the Student test, when presenting P < 0.05 or P < 0.01. Values reference according Feldman et al. (2000).
feed intake. The change of diet on day 20 p.i. allowed the lambs the maintenance of their basal physiologic status, since a better protein intake was provided. Therefore, the diet with an increased protein amount stabilized clinical signs of disease. WBC results are shown in Table 1. Significant (P < 0.05) WBC changes were observed in the infected lambs on days 15, 45 and 75 PI. The animals showed reduction in total leukocytes and neutrophils in infected animals at all times investigated. The reduced number of lymphocytes was observed in infected lambs on days 45 and 75 PI. Already the number of eosinophils and monocytes not differ between infected and control groups. The lambs infected with H. contortus showed significant (P < 0.01) reduction in AChE activity in lymphocytes compared uninfected at days 15, 45 and 75 PI (Fig. 1A). There was a positive correlation (P < 0.05) between AChE activity in lymphocytes and number of lymphocytes (r = 0.69) the time periods evaluated (Fig. 1B). The results of the EPG were presented in Fig. 2.The animals of Group A (negative control) showed negative EPG throughout the experiment; necropsy confirmed that the lambs were negative for helminths. The animal in group B (positive control) showed negative EPG on day 15 PI, but in the other two analyzes EPG was positive for eggs of H. contortus (Fig. 2). The mean parasite load in infected animals was of 2404 adult’s parasites of H. contortus. 4. Discussion The lambs experimentally infected with H. contortus showed a diminution in the number of leukocytes following the reduction in the number of neutrophils and lymphocytes, similar observed in by Leal et al. (2011). Since 1991 it is reported that infection with Haemonchus sp cause leukopenia by lymphopenia (Rahman and Collins, 1991). However, researchers reported an increase in total leukocytes in animals parasitized by gastrointestinal nematodes as a result of secondary bacterial infections (Feldman et al., 2000). Inoculation with H. contortus larvae induces T lymphocyte proliferation and the subsequent enlargement of abomasal lymph nodes and an increase in CD4+ lymphocytes in the abomasum wall and peripheral blood (Gill et al., 1993; Jacobs et al., 1995). Therefore, the reduction of peripheral lymphocytes at days 45 and 75 PI may be related with a local recruitment of this cell type in the
abomasal mucosa and abomasal lymph nodes. However, lambs that are three to 6 months old have fewer CD4+ lymphocytes in the abomasum wall related to diminished immune response against H. contortus (Watson et al., 1994). In contrast, a greater number of cd T lymphocytes have been observed in the abomasum wall of young sheep (Mackay and Hein, 1991), and cd T lymphocytes stimulated are able to produce IL-2, IFNc, and TNFa (Wood and Seow, 1996). Additionally, according to the literature, some factors causing immunosuppression in gastrointestinal nematodes are related to competition antigenic, stimulation of non-specific suppressor cells, competition with the surface of macrophages, lymphocytes modification of traffic through the parasitized tissues, production of factors that inhibit the immune response by parasites and polyclonal activation of lymphocytes (Soulsby, 1985), which may explain the results of the current study. It was found a positive correlation between AChE activity and the number of lymphocytes, i.e., these parameters were reduced in the infected animals when compared to lambs used as control. It is known that ACh is produced within the lymphocytes (Kawashima and Fujii, 2003). Therefore, reducing the number of lymphocytes certainly reduce the concentration of the free ACh, and hence a diminution in the AChE activity this cell (Kawashima and Fujii, 2003). Also we cannot rule out the possibility that the decrease of AChE may have an anti-inflammatory action in order to have more free ACh, to bind to lymphocytes and inhibit inflammation (Fujii et al., 2002, 2003; Das, 2007). ACh is a molecule involved in the regulation of immune function (Borovikova et al., 2000; Tayebati et al., 2002; Kawashima and Fujii, 2003), as it has antiinflammatory property due to the ability to inhibit or reduce the production of inflammatory mediators, such cytokines, serotonin, histamine, nitric oxide, lysosomal enzymes, prostaglandins and leukotrienes (Kawashima and Fujii, 2003; Das, 2007, 2012). As already mentioned, probably the decrease of AChE activity causes an increase of free ACh to bind to lymphocytes and inhibit inflammation (Descarries et al., 1997), which may have occurred with the lambs infected with H. contortus. ACh by binding to muscarinic ACh receptors (mAChRs) and nAChRs enhances lymphocyte cytotoxicity, increases their content of cGMP and inositol 1,4,5-triphosphate, and modulates DNA synthesis and cell proliferation, supporting the idea that the lymphocytic cholinergic system is involved in the regulation of immune function (Kawashima and
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Fig. 1. AChE activity in lymphocytes of lambs experimentally infected with gastrointestinal helminthes (A – ⁄P < 0.01). Positive correlation between the AChE activity and number of lymphocytes was observed (B – P < 0.05) on days 15, 45 and 75 post-infection.
Fig. 2. Mean and standard deviation of the number of eggs per gram of feces (EPG) of lambs infected with Haemonchus contortus compared to control on days 15, 45 and 75 post-infection. ⁄P < 0.01.
Fujii, 2003). In other infectious diseases is investigate the participation of cholinesterase in the immune response. For example, in infection by bacteria Leptospira interrogans serovar icterohaemorrhagiae was observed a situation similar to the present study, i.e., lymphopenia associated in reduced AChE activity (Silva et al., 2012). In contrast, in the infection by protozoan T. evansi was observed lymphocytosis associated to increase in AChE activity in lymphocytes (Da Silva et al., 2011), as an anti-inflammatory response against disease (Kawashima and Fujii, 2003; Das, 2012).
This study concludes that the lambs infected with H. contortus showed reduce of AChE activity in lymphocytes, and the AChE activity was correlated positively with reduced of the lymphocytes in lambs infected by this parasite. The reduced AChE activity probably was an attempt to regulate and/or modulate the concentration of ACh in lymphocytes and, consequently, the inflammation process. This information suggests this cholinesterase with participation in the pathogenesis and immune response of host, when parasitized by gastrointestinal nematode.
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Gill, H.S., Watson, D.L., Brandon, M.R., 1993. Monoclonal antibody to CD4+ T cells abrogates genetic resistance to Haemonchus contortus in sheep. Immunology 78, 43–49. Gordon, H.M., Whitlock, H.V., 1939. A new technique for counting nematode eggs in sheep faeces. J. Council Sci. Ind. Res. 12, 50–52. Jacobs, H.J., Ashman, K., Meeusen, E.N.T., 1995. Humoral and cellular responses following local immunization with a surface antigen of the gastrointestinal parasite Haemonchus contortus. Vet. Immunol. Immunopathol. 48, 323–332. Kawashima, K., Fujii, T., 2003. The lymphocytic cholinergic system and its contribution to the regulation of immune activity. Life Sci. 74, 675–696. Kimura, R., Ushiyama, N., Fujii, T., Kawashima, K., 2003. Nicotine-induced Ca2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line. Life Sci. 72, 2155. Leal, M.L.R., Nicolodi, P.R.S., Soares, J.F., Aires, A.R., Monteiro, S.G., Lopes, S.T.A., Ortolani, E.L., 2011. Hematological parameters of lambs infected experimentally with Haemonchus contortus and supplemented with selenium and vitamin E. Comp. Clin. Pathol. 20, 369–374. Mackay, C.R., Hein, W.R., 1991. Marked variations in cd T cell numbers and distribution throughout the life of sheep. Curr. Top. Microbiol. Immunol. 173, 107–111. Miller, J.E., Horohov, D.W., 2006. Immunological aspects of nematode parasite control in sheep. J. Anim. Sci. 84, E124–E132. Rahman, W.A., Collins, G.H., 1991. Changes in live weight gain, blood constituents and worm egg goats artificially infected with sheep-strain of Haemonchus contortus. Braz. Vet. J. 146, 543–550. Roberts, F.H.S., O’Sullivan, J.P., 1950. Methods for egg counts and larval cultures for strongyles infesting the gastrointestinal tract of cattle. Aust. J. Agric. Res. 1, 99– 102. Rowe, A., Gondro, C., Emery, D., Sangster, N., 2008. Genomic analyses of Haemonchus contortus infection in sheep: abomasal fistulation and two Haemonchus strains do not substantially confound host gene expression in microarrays. Vet. Parasitol. 154, 71–81. Santa Rosa, J., 1996. Enfermidade em caprinos: diagnóstico, patogenia, terapêutica e controle. Embrapa-SPI; Sobral: Embrapa-CNPC, Brasília, DF. Silva, C.B., Wolkmer, P., Da Silva, A.S., Paim, F.C., Tonin, A.A., Castro, V.S.P., Felin, D.V., Schmatz, R., Gonçalves, J.S., Badke, M.R.T., Morsch, V.M., Mazzanti, C.M., Lopes, S.T.A., 2012. Cholinesterases as markers of the inflammatory process in rats infected with Leptospira interrogans serovar Icterohaemorrhagiae. J. Med. Microbiol. 61, 278–284. Soulsby, E.J.L., 1985. Advances in immunoparasitology. Vet. Parasitol. 18, 303–319. Tayebati, S.K., El-Assouad, D., Ricci, A., Amenta, F., 2002. Immunochemical and immunocytochemical characterization of cholinergic markers in human peripheral blood lymphocytes. J. Neuroimmunol. 132, 147. Ueno, H., Gonçalves, P.C., 1998. Manual for Diagnosis of Helminthiasis in Ruminants. Press Color, Salvador, pp. 143. Watson, D.L., Colditz, I.G., Andrew, M., Gill, H.S., Altmann, K.G., 1994. Agedependent immune response in Merino sheep. Res. Vet. Sci. 57, 152–158. Wood, P.R., Seow, H.F., 1996. T cell cytokines and disease prevention. Vet. Immunol. Immunopathol. 54, 33–44.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Influence of experimental infection by Haemonchus contortus on acetylcholinesterase activity in lymphocytes of lambs Alexandre A. Tonin a,⇑, Aleksandro S. Da Silva b,⇑, Andressa S. Schafer c, Adelina R. Aires c, Camila B. Oliveira d, Daniela Zanini e, Maria R.C. Schetinger e, Vera M. Morsch e, Sonia T.A. Lopes a, Silvia G. Monteiro d, Marta L.R. Leal c a
Department of Small Animal, Universidade Federal de Santa Maria (UFSM), Brazil Department of Animal Science, Universidade do Estado de Santa Catarina, Brazil Laboratory of Animal Endocrinology and Metabology. Program of Post-Graduation in Veterinary Medicine, UFSM, Brazil d Department of Microbiology and Parasitology, UFSM, Brazil e Program of Post-Graduation in Toxicological Biochemistry, UFSM, Brazil b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Sheep herds infected with
Haemonchus contortus in the hyperacute and acute phase of the disease. Experimental infection by H. contortus in lambs causes a reduction in the number of lymphocytes. Lambs infected with H. contortus showed a reduction in acetylcholinesterase activity in lymphocytes. The participation of the cholinergic system in the immune response is well documented. Lambs infected with H. contortus showed reduce of AChE activity in lymphocytes.
a r t i c l e
i n f o
Article history: Received 6 September 2013 Received in revised form 6 February 2014 Accepted 9 February 2014 Available online 19 February 2014 Keywords: Helminths Acetylcholine WBC Sheep
a b s t r a c t The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes of lambs experimentally infected by Haemonchus contortus. A total of 14 healthy lambs were used, divided into two groups of seven animals each. Group A (negative control) represented the uninfected animals, and Group B (positive control) was formed by animals infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the white blood cells (WBC) count, as well as the evaluation of AChE activity in lymphocytes. Parasitological stool exam (eggs per gram of feces – EPG) was performed on the same days to follow up the evolution of the infection. On day 15 PI it was verified negative EPG; however, on days 45 and 75 PI it was observed positive EPG only in the animals of group B. In the three evaluated periods was observed a lower number of leukocytes, associated with decreased lymphocytes and neutrophils in lambs infected by this gastrointestinal nematodes. Lambs infected with H. contortus showed significant (P < 0.01) lower AChE activity in lymphocytes compared uninfected. Statistically, there was a positive correlation (P < 0.05) between AChE activity in lymphocytes and number of lymphocytes (r = 0.69). The lymphocytes are cells with direct participation in the
⇑ Corresponding authors. E-mail addresses: [email protected] (A.A. Tonin), aleksandro_ss@yahoo. com.br (A.S. Da Silva). http://dx.doi.org/10.1016/j.exppara.2014.02.006 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.
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A.A. Tonin et al. / Experimental Parasitology 139 (2014) 19–23
cholinergic system; therefore, based on these results, it can be concluded that the experimental infection with H. contortus influences the number of lymphocytes, and consequently the AChE activity in these cells. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction Sheep herds infected with Haemonchus contortus usually present weight loss, dehydration, diarrhea, anemia, lackluster wool and death in the hyperacute and acute phase of the disease. During the chronic phase, these signals are intensified, usually followed by severe swelling in the submandibular and ventral regions, due to the hypoalbuminemia (Freitas, 1982; Santa Rosa, 1996; Cavalcante et al., 2009; Fonseca et al., 2011). Although the infections in sheep are usually mixed (helminths of the Trichostrongylidae family), H. contortus has a higher prevalence and importance in this process (Fonseca et al., 2011). The immune response was involved to control infection by H. contortus, and that helminthes parasites generally stimulate a Th2-type response (Miller and Horohov, 2006). According to the literature, the existence of cholinergic receptors in the immune system cells is known, as well as the influence of cholinesterases in inflammation (Tayebati et al., 2002; Kawashima and Fujii, 2003; Kimura et al., 2003; Da Silva et al., 2011). Cholinesterases are involved in different vital functions in mammals, being the acetylcholinesterase (AChE: EC 3.1.1.7) and butyrylcholinesterase (BChE: EC 3.1.1.8) are enzymes that catalyze the hydrolysis of the acetylcholine (ACh), a key process in the regulation of the cholinergic system (Darvesh et al., 2003). The lymphocytes synthesize ACh, which is degraded by AChE, and in consequence of this, the complete cholinergic repertoire of immune cells was termed the lymphocytic cholinergic system (Kawashima and Fujii, 2003). The nictotinic cholinergic receptors (nAChR) can induce a rapid and transient Ca2+ influx in lymphocytes (Kimura et al., 2003) in inflammation. The regulation of inflammatory reactions occurs by activation of the a7 nAChR in macrophages, as cholinergic stimulation reduced pro-inflammatory cytokine production in a lethal endotoxemia model (Borovikova et al., 2000). Therefore, leukocytes and cholinesterase have concurrent participation during inflammation. The relation between cholinesterase and immune response mediated by lymphocyte is unknown by the infection of lambs H. contortus. However, study has shown a positive correlation between AChE activity in lymphocytes and lymphocytosis in rats experimentally infected with Trypanosoma evansi (Da Silva et al., 2011). Therefore, the aim of this study was to evaluate the AChE activity in lymphocytes of lambs experimentally infected with H. contortus. 2. Materials and methods Our experiment used 14 male lambs crossbred Corriedale x Texel, 5 months old and weighing in average 23 kg. They were kept in holding pens (one pen/group) during 30 days under a diet base of 10.7% protein (commercial feed – 30% and ryegrass hay – 70%) for the adaptation in the experimental environment. In this period, the animals received anthelmintic treatment based on Monepantel (Zolvix1Ò). The same diet was kept during the first 20 days of the experiment (post-infection); however at this period it was necessary to change the diet as a consequence of the severe disease evolution. From this period onward, the animals were fed hay ground ryegrass (70%), commercial ration (20%) and soybean meal (10%), providing a 13% protein diet base. Each animal 1
Novartis Healthcare A/S. Edvard Thomsens Vej 2300, Copenhague, Dinamarca.
consumed 1 kg of dry matter/day. Hematological (erythrogram and leukogram) and biochemical (hepatic and renal function) examinations were performed three times at 15-day intervals. After 30 days (day 0 of the experiment), the evaluated patterns showed normal values (Table 1), according to Feldman et al. (2000). Apparently the animals were healthy, and they had negative fecal exam for eggs, cysts and oocysts of parasites. The animals were divided into two groups with seven animals each: Group A was composed by of healthy animals (uninfected); In Group B, the animals were infected with H. contortus. Each animal from group B was infected orally with a total of 15,000 larvae (L3), divided in three moments of infection, 5000 larvae each time, at intervals of 3 days between infections (Rowe et al. 2008), obtained by coproculture technique (Roberts and O’Sullivan 1950). Blood was drawn through VacutainerÒ system in tubes with anticoagulant (EDTA) on the days 15, 45 and 75 post-infection (PI). Samples were stored in tubes containing anticoagulant in order to allow the separation of lymphocytes (4 mL) and WBC analysis (1 mL). Leukocytes count was done manually in a Neubauer chamber, modified (Feldman et al. 2000). Blood smears were prepared and stained by the Romanowski method for evaluation of differential leukocyte. The peripheral lymphocytes were isolated using Ficoll Hypaque density gradient as described by Böyum (1968). After separation, only samples with at least 95% of lymphocytes, as verified in the coulter STKS (Miami-USA) were used. After isolation of the lymphocytes, AChE activity was determined according to the method described by Fitzgerald and Costa (1993). Briefly, proteins of all samples were adjusted to 0.1–0.2 mg/mL, i.e., volume with similar numbers of lymphocytes. Volume of 0.2 mL intact cells were added to a solution containing 1.0 mM acetylthiocholine, 0.1 mM 5,50 -Dithiobis (2-nitrobenzoic acid), and 0.1 mM phosphate buffer (pH 8.0). Immediately before and after incubation for 30 min at 27 °C the absorbance was read on a spectrophotometer at 412 nm. AChE was calculated from the quotient between lymphocyte AChE activity and protein content and the results are expressed as lmol of AcSCh/h/mg of protein. Fecal samples for quantification of eggs per gram (EPG) were collected on 15, 45 and 75 PI, and processed according to the technique described by Gordon and Whitlock (1939). Five days after the end of experiment (day 80 PI), five animals from each group were euthanized (10 mg intravenous (IV) acepromazine; 2 g IV sodium thiopental; then 100 ml IV potassium chloride), and their parasite loads were determined (Ueno and Gonçalves, 1998). The euthanasia was necessary to confirm that the animals were infected (Group B) or negative (Group A), since the EPG, by itself, is not sufficient to validate the research. Normality test was performed on all the data. EPG and leukocytes data not present a normal distribution and they were logarithm before statistical analysis. Then, the AChE activity, leukocytes and EPG results were subjected to the Student test. The effect of AChE in lymphocytes on number of lymphocytes was analyzed by linear correlation. Values with probability (P) less than 5% were considered statistically different. 3. Results The animals, post-infection, were losing their physiological condition progressively, getting weak, lethargic, pale and reducing
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Table 1 Mean and standard deviation of hematologic parameters (Total leukocytes, neutrophils, lymphocytes, monocytes and eosinophils) in lambs uninfected and infected with gastrointestinal helminths on days 15, 45 and 75 post-infection. Parameters 3
Total Leukocytes (10 lL) *
4.000–12.000
Neutrophil (103 lL) * 700–6000
Lymphocytes (103 lL) * 2.000–9.000
Monocytes (103 lL) * 0–750
Eosinophils (103 lL) 0–1.000
*
# *
Day
Group A: control
Group B: infected
Probability (P)#
00 15 45 75 00 15 45 75 00 15 45 75 00 15 45 75 00 15 45 75
9.24 ± 1.30 9.86 ± 1.96 7.53 ± 1.24 9.25 ± 1.04 4.02 ± 0.79 4.17 ± 0.99 2.95 ± 0.75 3.53 ± 1.25 5.01 ± 1.24 5.37 ± 1.16 4.32 ± 1.23 5.49 ± 1.53 0.15 ± 0.12 0.19 ± 0.10 0.16 ± 0.07 0.09 ± 0.02 0.06 ± 0.14 0.13 ± 0.11 0.10 ± 0.08 0.14 ± 0.10
9.37 ± 1.82 7.44 ± 2.55 5.18 ± 1.08 5.55 ± 1.16 4.08 ± 0.95 2.31 ± 1.16 2.08 ± 0.62 2.03 ± 0.75 5.12 ± 1.50 4.79 ± 2.04 2.68 ± 0.80 3.31 ± 0.71 0.10 ± 0.08 0.15 ± 0.09 0.19 ± 0.11 0.09 ± 0.05 0.07 ± 0.18 0.19 ± 0.12 0.23 ± 0.19 0.12 ± 0.06
P > 0.05 P > 0.05 P < 0.05# P < 0.01# P > 0.05 P < 0.05# P < 0.05# P < 0.05# P > 0.05 P > 0.05 P < 0.05# P < 0.01# P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05 P > 0.05
In the same line, was considered significant difference between groups in the Student test, when presenting P < 0.05 or P < 0.01. Values reference according Feldman et al. (2000).
feed intake. The change of diet on day 20 p.i. allowed the lambs the maintenance of their basal physiologic status, since a better protein intake was provided. Therefore, the diet with an increased protein amount stabilized clinical signs of disease. WBC results are shown in Table 1. Significant (P < 0.05) WBC changes were observed in the infected lambs on days 15, 45 and 75 PI. The animals showed reduction in total leukocytes and neutrophils in infected animals at all times investigated. The reduced number of lymphocytes was observed in infected lambs on days 45 and 75 PI. Already the number of eosinophils and monocytes not differ between infected and control groups. The lambs infected with H. contortus showed significant (P < 0.01) reduction in AChE activity in lymphocytes compared uninfected at days 15, 45 and 75 PI (Fig. 1A). There was a positive correlation (P < 0.05) between AChE activity in lymphocytes and number of lymphocytes (r = 0.69) the time periods evaluated (Fig. 1B). The results of the EPG were presented in Fig. 2.The animals of Group A (negative control) showed negative EPG throughout the experiment; necropsy confirmed that the lambs were negative for helminths. The animal in group B (positive control) showed negative EPG on day 15 PI, but in the other two analyzes EPG was positive for eggs of H. contortus (Fig. 2). The mean parasite load in infected animals was of 2404 adult’s parasites of H. contortus. 4. Discussion The lambs experimentally infected with H. contortus showed a diminution in the number of leukocytes following the reduction in the number of neutrophils and lymphocytes, similar observed in by Leal et al. (2011). Since 1991 it is reported that infection with Haemonchus sp cause leukopenia by lymphopenia (Rahman and Collins, 1991). However, researchers reported an increase in total leukocytes in animals parasitized by gastrointestinal nematodes as a result of secondary bacterial infections (Feldman et al., 2000). Inoculation with H. contortus larvae induces T lymphocyte proliferation and the subsequent enlargement of abomasal lymph nodes and an increase in CD4+ lymphocytes in the abomasum wall and peripheral blood (Gill et al., 1993; Jacobs et al., 1995). Therefore, the reduction of peripheral lymphocytes at days 45 and 75 PI may be related with a local recruitment of this cell type in the
abomasal mucosa and abomasal lymph nodes. However, lambs that are three to 6 months old have fewer CD4+ lymphocytes in the abomasum wall related to diminished immune response against H. contortus (Watson et al., 1994). In contrast, a greater number of cd T lymphocytes have been observed in the abomasum wall of young sheep (Mackay and Hein, 1991), and cd T lymphocytes stimulated are able to produce IL-2, IFNc, and TNFa (Wood and Seow, 1996). Additionally, according to the literature, some factors causing immunosuppression in gastrointestinal nematodes are related to competition antigenic, stimulation of non-specific suppressor cells, competition with the surface of macrophages, lymphocytes modification of traffic through the parasitized tissues, production of factors that inhibit the immune response by parasites and polyclonal activation of lymphocytes (Soulsby, 1985), which may explain the results of the current study. It was found a positive correlation between AChE activity and the number of lymphocytes, i.e., these parameters were reduced in the infected animals when compared to lambs used as control. It is known that ACh is produced within the lymphocytes (Kawashima and Fujii, 2003). Therefore, reducing the number of lymphocytes certainly reduce the concentration of the free ACh, and hence a diminution in the AChE activity this cell (Kawashima and Fujii, 2003). Also we cannot rule out the possibility that the decrease of AChE may have an anti-inflammatory action in order to have more free ACh, to bind to lymphocytes and inhibit inflammation (Fujii et al., 2002, 2003; Das, 2007). ACh is a molecule involved in the regulation of immune function (Borovikova et al., 2000; Tayebati et al., 2002; Kawashima and Fujii, 2003), as it has antiinflammatory property due to the ability to inhibit or reduce the production of inflammatory mediators, such cytokines, serotonin, histamine, nitric oxide, lysosomal enzymes, prostaglandins and leukotrienes (Kawashima and Fujii, 2003; Das, 2007, 2012). As already mentioned, probably the decrease of AChE activity causes an increase of free ACh to bind to lymphocytes and inhibit inflammation (Descarries et al., 1997), which may have occurred with the lambs infected with H. contortus. ACh by binding to muscarinic ACh receptors (mAChRs) and nAChRs enhances lymphocyte cytotoxicity, increases their content of cGMP and inositol 1,4,5-triphosphate, and modulates DNA synthesis and cell proliferation, supporting the idea that the lymphocytic cholinergic system is involved in the regulation of immune function (Kawashima and
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Fig. 1. AChE activity in lymphocytes of lambs experimentally infected with gastrointestinal helminthes (A – ⁄P < 0.01). Positive correlation between the AChE activity and number of lymphocytes was observed (B – P < 0.05) on days 15, 45 and 75 post-infection.
Fig. 2. Mean and standard deviation of the number of eggs per gram of feces (EPG) of lambs infected with Haemonchus contortus compared to control on days 15, 45 and 75 post-infection. ⁄P < 0.01.
Fujii, 2003). In other infectious diseases is investigate the participation of cholinesterase in the immune response. For example, in infection by bacteria Leptospira interrogans serovar icterohaemorrhagiae was observed a situation similar to the present study, i.e., lymphopenia associated in reduced AChE activity (Silva et al., 2012). In contrast, in the infection by protozoan T. evansi was observed lymphocytosis associated to increase in AChE activity in lymphocytes (Da Silva et al., 2011), as an anti-inflammatory response against disease (Kawashima and Fujii, 2003; Das, 2012).
This study concludes that the lambs infected with H. contortus showed reduce of AChE activity in lymphocytes, and the AChE activity was correlated positively with reduced of the lymphocytes in lambs infected by this parasite. The reduced AChE activity probably was an attempt to regulate and/or modulate the concentration of ACh in lymphocytes and, consequently, the inflammation process. This information suggests this cholinesterase with participation in the pathogenesis and immune response of host, when parasitized by gastrointestinal nematode.
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