- Email: [email protected]
Involvement of cholinergic and purinergic systems during the inflammatory response caused by Aeromonas hydrophila in Rhamdia quelen
Microbial Pathogenesis 99 (2016) 78e82
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Involvement of cholinergic and purinergic systems during the inflammatory response caused by Aeromonas hydrophila in Rhamdia quelen Matheus D. Baldissera a, *, Carine F. Souza b, Pedro Henrique Doleski a, Guerino B. Júnior b, Agueda C. de Vargas c, Bernardo Baldisserotto b, ** a b c
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Department of Preventive Veterinary Medicine, Universidade Federal de Santa Maria, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2016 Received in revised form 4 August 2016 Accepted 8 August 2016 Available online 9 August 2016
The aim of this study was to evaluate the cholinergic (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) and purinergic (adenosine deaminase (ADA)) systems in head kidney, spleen, total blood and serum samples in experimentally infected fish with A. hydrophila, and the involvement of these systems during the inflammatory process. Silver catfish (Rhamdia quelen) juveniles were divided into two groups with seven fish each: uninfected (negative control) and infected (positive control). On day 2 post-infection, animals were euthanized and the head kidney, spleen, total blood and serum were collected. AChE and ADA activities in head kidney and spleen decreased in infected animals compared to uninfected animals, as well as AChE in total blood and seric ADA activities. BChE activity was not expressed in the evaluated tissues. Therefore, our results lead to the hypothesis that cholinergic and purinergic systems play an important role on the immune response against A. hydrophila with an antiinflammatory effect. In summary, AChE and ADA activities reduced probably in order to protect against tissue inflammatory damage caused by infection. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Adenosine deaminase Acetylcholine Fish Infectious diseases
1. Introduction Aquaculture is one of the fastest growing food and food producing sectors due to increased demand for fish and seafood, providing one-third of seafood consumed worldwide in the last decade [1,2]. However, the intensification of aquaculture has increased the risk of infectious diseases, such as those caused by Aeromonas hydrophila, that are recognized as one the most serious challenges in the fish production, limiting the development of aquaculture and causing important economic losses [3,4]. Aeromonas hydrophila is an important aquatic bacterium pathogen of freshwater aquaculture, the causative agent of epizootic ulcerative syndrome [5]. The main symptoms of this pathogen are ulcerations, hemorrhagic focus, infectious abdominal dropsy and
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] [email protected] (B. Baldisserotto). http://dx.doi.org/10.1016/j.micpath.2016.08.009 0882-4010/© 2016 Elsevier Ltd. All rights reserved.
(M.D.
Baldissera),
erosion of the fins in farmed and wild fish [6]. This bacterium is a serious cause of mortality and economic loss in species such as common carp (Cyprinus carpio), goldfish (Carassius auratus auratus), striped fish (Plotosus lineatus), rohu carp (Labeo rohita) and silver catfish (Rhamdia quelen) [7e10]. Recently, studies have demonstrated the effects of A. hydrophila infection on immune response in head kidney and spleen tissues [11], but the effects on enzymes of cholinergic and purinergic system, that are considered novel markers of inflammatory response [12,13], remain unknown. The cholinergic system is notably involved in anti-inflammatory reactions and cholinesterases enzymes, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), develop an important role during immune and inflammatory response [12,13]. AChE hydrolyses acetylcholine (ACh), a neurotransmitter with antiinflammatory effects, into choline and acetate, while the BChE hydrolyses the butyrylcholine (BCh) and other variety of esters, including ACh [14,15]. Recently, study conducted by Tonin et al. [13] demonstrated that BChE activity is recognized as a novel marker of tissue inflammation and can be used with a marker of acute
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
inflammatory response. Alterations on AChE and BChE activities in liver and serum of laying hens naturally infected with Salmonella gallinarum contribute directly to inflammatory response and the pathogenesis of disease [16]. The adenosine deaminase (ADA) is an important enzyme of the purinergic system that catalyzes the deamination of 2deoxyadenosine to inosine. ADA activity plays an important role on immune and inflammatory response due the regulation of adenosine, a molecule with anti-inflammatory properties, protecting the host tissue from damage and having an important role in the differentiation and proliferation of lymphocytes [17]. In this context, a study has demonstrated that ADA activity is involved in the cell-mediated immunity and could be an important biomarker to determine the severity of inflammatory and immune response during the infection caused by Salmonella gallinarum, another important bacterial pathogen [18]. Based on that head kidney and spleen are considered important lymphatic immune organs in fish [19], and the role of cholinergic and purinergic system during the immune and inflammatory response, the aim of this study was to evaluate the activities of AChE, BChE and ADA in head kidney and spleen tissues, AChE in total blood and BChE and ADA in serum of R. quelen experimentally infected with A. hydrophila. 2. Materials and methods 2.1. Aeromonas hydrophila isolate The isolate of A. hydrophila used in this study was obtained from a naturally infected silver catfish (R. quelen), identified by phenotypic testing and 16S rDNA sequencing, prepared in saline solution from cultures grown in Mueller-Hinton agar (Himedia Laboratories). 2.2. Animal model and water quality Fourteen silver catfish juveniles (50.5 ± 2 g; 19.5 ± 1.3 cm) were used as the experimental model for determination of the enzymes of the cholinergic and purinergic systems in head kidney and spleen. All fish were transferred from a local fish culture to the laboratory, where they were maintained in continuously aerated tanks with controlled water parameters (22.0e24 C, pH 7.2e7.6, dissolved oxygen levels: 5.5e7.5 mg L1). Fish were acclimated for 14 days, and dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512, Ohio, USA). The pH was ~o Paulo, SP, measured using a DMPH-2 pH meter (Digimed, Sa Brazil). Total ammonia levels were determined according to Verdouw et al. [20] and un-ionized ammonia (NH3) levels were calculated according to Colt [21]. 2.3. Experimental design The silver catfish were assigned into two groups with seven animals each: uninfected animals (negative control) and experimentally infected animals (positive control) inoculated intramuscularly with 100 mL of A. hydrophila solution (2.1 109 colony forming unit: OD600 ¼ 1.7e1.9) on the right latero-dorsal side of each fish. The negative control received the same dose of sterile saline by the same route. Fish were fed once a day to satiation with commercial feed, and uneaten food, other residues and feces were removed 30 min after feeding. The methodology used in this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria under protocol number 074/2014.
79
2.4. Sample collection and tissue preparation On day 2 post-infection (PI), blood samples were collected from the caudal vein using tubes containing EDTA and tubes without anticoagulant to obtain serum. Thereafter, the head kidney and spleen were removed and dissected on a glass dish over ice for the measurement of AChE, BChE and ADA activities. All animals were euthanized by spinal cord section according the Ethics Committee recommendations. The blood samples were centrifuged at 3000 rpm for 15 min to obtain serum for measurement the BChE and ADA activities. The total blood collected in tubes containing EDTA was used to determine the AChE in total blood. Head kidney and spleen were homogenized (1:10 w/v) in a glass potter with Tris-HCl buffer (50 mM, pH 7.4), centrifuged at 10000 g for 10 min. Aliquots of the supernatant were stored at e 20 C until utilization. 2.5. AChE activity in head kidney, spleen and total blood AChE enzymatic activity in the head kidney and spleen homogenates were determined by a modified spectrophotometric method of Ellman et al. [22] as previously described by Rocha et al. [23]. The reaction mixture was composed by of 1.0 M of potassium phosphate buffer (pH 8.0) and 10 mM of DTNB. The method is based on increased yellow color of the nitrobenzoate (TNB), ion produced from thiocoline after reaction with 5,5-dithio-bis-acid-nitrobenzoic (DTNB) ion, measured by absorbance at 412 nm for 2 min of incubation at 30 C. The reaction was initiated by adding 0.78 mM acetylcholine iodide as substrate. All samples were run in duplicate and the enzymatic activity was expressed in mmol AcSCh/min mg of protein. AChE activity in total blood was determined by Ellman et al. [22] method, modified by Worek et al. [24]. 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/mmol Hb. 2.6. BChE activity in head kidney, spleen and serum Butyrylcholinesterase activity in homogenates and serum was determined by the method of Ellman et al. [22] as previously described by Rocha et al. [23], using the substrate butyrylcholine. Pre-incubation of samples was done at 37 C for 2 min, and reading performed for 2 min, at intervals of 20-20 s on a spectrophotometer (412 nm). The samples analysis was carried out in duplicate, and the enzymatic activity was expressed in mmol BcSCh/min mg of protein. 2.7. ADA activity in head kidney, spleen and serum ADA activity in head kidney and spleen were measured according to Giusti [25], which is based on the direct measurement of the formation of ammonia produced when the enzyme acts on adenosine. A volume of 100 mL of head kidney or spleen was used. The enzymatic reaction was started by addition of 500 mL of 21 mM adenosine as substrate and stopped by adding 1.5 mL of 106/ 0.16 mM phenol-sodium nitroprusside to the reaction mixture, which was immediately mixed with 1.5 mL of 125/11 mM alkalinehypochlorite (sodium hypochlorite). Released ammonia reacted with alkaline-hypochlorite and phenol in the presence of the catalyst-sodium nitroprusside to produce indophenol (a blue color). The concentration of ammonia was directly proportional to the absorbance of indophenol, read at 620 nm. Ammonium sulfate 75 mM was used as standard. All measures were carried out in duplicate and ADA activity in head kidney and spleen homogenates
80
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
were expressed as U/mg of protein. The activity of ADA in serum was determined spectrophotometrically, according to the method described by Giusti and Gakis [26]. The reaction started by addition of adenosine (substrate) to a final concentration of 21 mMol/L; incubations were carried out for 1 h at 37 C. The reaction stopped by adding 106 mMol/L 0.16 mMol/ L of phenolnitroprusside/mL solution. The reaction mixtures were mixed immediately to 125 mMol/L; 11mMol/L of alkaline hypochlorite (sodium hypochlorite) and vortexed. Ammonium sulfate, at concentration of 75 mmol/L served as ammonium standard. The ammonium concentration is directly proportional to the absorption of indophenol at 650 nm. The specificity activity was reported as U/ L. 2.8. Protein determination Protein content in head kidney, spleen and serum was determined by the Bradford method using bovine albumin serum as the standard [27]. 2.9. Statistical analyses The data were tested for normality using the Shapiro-Wilk test. Then, the data were subjected to Student's t-test for independent samples to verify the statistical difference. Differences between groups were considered significant when p < 0.05. The effect size (r2) was described and scored as follows: 0.1 (small), 0.1 to 0.3 (medium) and 0.5 (large). Data were expressed as mean ± standard deviation.
Fig. 1. Acetylcholinesterase (AChE) activity in head kidney and spleen [A], and in total blood [B] of silver catfish experimentally infected by Aeromonas hydrophila compared to uninfected group (*p < 0.05 and ***p < 0.001 using Student's t-test; n ¼ 7 fish per group).
3. Results 3.1. Water quality parameters The mean water quality parameters were: temperature 23 C, dissolved oxygen level 6.50 ± 0.2 mg/L, pH 7.25 ± 0.05, total ammonia 0.85 ± 0.03 mg/L and non-ionized ammonia of 0.007 ± 0.0004 mg/L. 3.2. AChE and BChE activities AChE activity decreased 37% in head kidney [t(12) ¼ 2.93; p < 0.05; r2 ¼ 0.63], 62% in spleen [t(12) ¼ 2.71; p < 0.05; r2 ¼ 0.55] and 45% [t(12) ¼ 4.11; p < 0.001; r2 ¼ 0.63] in total blood of animals infected by A. hydrophila compared with the uninfected control group (Fig. 1). BChE activity in head kidney, spleen and serum were not expressed in the silver catfish. 3.3. ADA activity ADA activity decreased 52% in head kidney [t(12) ¼ 5.73; p < 0.001; r2 ¼ 0.84], 61% in spleen [t(12) ¼ 4.97; p < 0.001; r2 ¼ 0.72] and 40% [t(12) ¼ 3.13; p < 0.001; r2 ¼ 0.52] in serum of animals infected by A. hydrophila compared with the uninfected control group (Fig. 2). 4. Discussion Several studies have reported that both purinergic [28,29] and cholinergic systems [30,31] are important in the modulation of the inflammatory and immune responses. In this study, alterations in the activities of enzymes involved in the regulation of acetylcholine and adenosine levels were found in fish infected with A. hydrophila. The analysis of our data clearly shows the inhibition of the AChE and ADA activities in head kidney and spleen, AChE in total blood
Fig. 2. Adenosine deaminase (ADA) activity in head kidney and spleen [A], and in serum [B] of silver catfish experimentally infected by Aeromonas hydrophila compared to uninfected group (***p < 0.001 using Student's t-test; n ¼ 7 fish per group).
and ADA in serum at 2 days PI. Similar responses were observed in cattle infected with Anasplasma marginale [31] and hens infected with Salmonella gallinarum [18]. Due to this decrease in AChE
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
activity, less ACh is hydrolyzed, possibly in order to reduce the proinflammatory response and tissue damage, since ACh has antiinflammatory effects [32]. In this line, AChE arises as a potential contributor in the cholinergic pathways controlling inflammatory and immune response mediated by muscarinic and nicotinic receptors during A. hydrophila infection. Adenosine exhibits potent anti-inflammatory and immunosuppressive action, inhibiting the secretion of pro-inflammatory cytokines [33], acting as regulator of immune response, protecting the host from excessive tissue damage associated with inflammation [34,35]. The reduction on ADA activity in head kidney, spleen and serum observed in this study could contribute to restrict the inflammatory response and subsequent cellular damage because it would increase adenosine tissue levels. Adenosine, an anti-inflammatory nucleoside, confers to the host tissue protection, a compensatory mechanism against bacterial infections [18]. Also, in other infectious diseases, the enzymes AChE and ADA increase, contributing to inflammatory process, because decrease the levels of ACh and adenosine, considered anti-inflammatory molecules [30]. We observed that BChE activity was not expressed in the evaluated tissues and serum of this species. BChE in serum is not expressed in the serum or plasma of many fish families, such as Percidae, Esocidae and Coregonidae [36,37]. The serum and plasma of some cyprinid species contain both AChE and BChE, but the common carp (Cyprinus carpio) has only AChE [37]. According this author, a difference between species suggests the existence of cross-species variability in these enzymes. Based on these evidences and in the absence of related data in literature (to our knowledge) regarding BChE, we can conclude that it possible that silver catfish does not express the BChE enzyme in the evaluated organs, as well as in the serum. Based on the results, we concluded that infection by A. hydrophila in silver catfish alters the cholinergic and purinergic systems, suggesting the involvement of AChE and ADA activities on pathogenesis of disease, regulating the ACh and adenosine levels, molecules known to participate in physiological and pathological events as inflammatory mediator. In summary, AChE and ADA reduction probably played an effect on inflammatory response, decreasing hydrolysis of ACh and adenosine, important antiinflammatory molecules, in an attempt to reduce or prevent the inflammatory tissue damage. These alterations probably reduce or prevent the inflammatory tissue damage caused by A. hydrophila.
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25] [26]
References [27] [1] M.G. Rivera-Ferre, Can export-oriented aquaculture in developing countries be sustainable and promote sustainable development? the shrimp case, J. Agric. Environ. Ethics 22 (2009) 301e321. [2] M. Reverter, N. Bontemps, D. Lecchini, B. Banaigs, P. Sasal, Use of plant extracts in fish as an alternative to chemotherapy: current status and future perspectives, Aquaculture 433 (2014) 50e61. [3] Z.Z. Qi, Z.H. Zhang, N. Boon, P. Bossier, Probiotics in aquaculture of Chinacurrent state, problems and prospect, Aquaculture 290 (2009) 15e21. [4] F.J. Zagmutt, S.H. Sempier, T.R. Hanson, Disease spread models to estimate highly uncertain emerging diseases losses for animal agriculture insurance policies: an application to the U.S. farm-raised catfish industry, Risk Anal. 33 (2013) 1924e1937. [5] M.Y. Huang, C.I. Chang, C.C. Chang, L.W. Tseng, C.L. Pan, Effects of dietary levan on growth performance, nonspecific immunity, pathogen resistance and body composition of orange-spotted grouper (Epinephelus coioides H.), Aquac. Res. 46 (2015) 2752e2767. [6] J.M. Janda, S.L. Abbott, The genus Aeromonas, taxonomy, pathogenicity, and infection, Clin, Microbiol. Rev. 23 (2010) 35e73. [7] S. Maji, P. Mali, S.N. Joardar, Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.), Fish. Shellfish Immun. 20 (2006) 462e473. [8] W. Sirimanapong, K.D. Thompson, K. Kledmanee, P. Thaijongrak, B. Collet, E.L. Ooi, et al., Optimization and standardisation of functional immune assays for striped catfish (Pangasianodon hypophthalmus) to compare their immune
[28]
[29]
[30]
[31]
[32] [33]
[34]
81
response to live and heat killed Aeromonas hydrophila as models of infection and vaccination, Fish. Shellfish Immun. 40 (2014) 374e383. J.L. Wang, X.L. Meng, R.H. Lu, C. Wu, T.Y. Luo, X. Yan, et al., Effects of Rehmania glutinosa on growth performance, immunological parameters and disease resistance to Aeromonas hydrophila in common carp (Cyprinus carpio L.), Aquaculture 435 (2015) 293e300. C.F. Souza, M.D. Baldissera, R.A. Vaucher, L.Q.S. Lopes, B.S. Vizzotto, R.P. Raffin, et al., In vivo bactericidal effect of Melaleuca alternifolia essential oil against Aeromonas hydrophila: silver catfish (Rhamdia quelen) as an experimental model, Microb. Pathog. 98 (2016) 82e87. P.J. Ni, W.D. Jiang, P. Wu, Y. Liu, S.Y. Kuang, L. Tang, et al., Dietary low or excess of lipids reduced growth performance, and impaired immune function and structure of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella) under the infection of Aeromonas hydrophila, Fish. Shellfish Immun. 55 (2016) 28e47. G.M. Do Carmo, L.Z. Crivellenti, N.B. Bottari, G. Machado, S. Borin-Crivellenti, R.N. Moresco, et al., Butyrylcholinesterase as a marker of inflammation and liver injury in the acute and subclinical phases of canine ehrlichiosis, Comp. Immunol. Microbiol. Infect. Dis. 43 (2015) 16e21. , T. Duarte, et A.A. Tonin, A.M.C. Calado, N.B. Bottari, D. Dalegonare, G.R. Thome al., Novel markers of inflammatory response and hepatic dysfunction in canine leishmaniasis, Comp. Immunol. Microbiol. Infect. Dis. 44 (2016) 61e64. L. Anglister, A. Etlin, E. Finkel, A.R. Durrant, A. Lev-Tov, Cholinesterases in development and disease, Chem. Biol. Interact. 175 (2008) 92e100. J.A.D.S. Jacques, J.F.P. Rezer, J.F. Gonçalves, R.M. Spanevello, J.M. Gutierres, V.C. Pimentel, et al., The effect of curcumin in the ectonucleotidases and acetylcholinesterase activities in synaptosomes from the cerebral cortex of cigarette smoke-exposed rats, Cell biochem. Funct. 29 (2011) 703e707. A.S. Da Silva, M.M. Boiago, N.B. Bottari, G.M. do Carmo, M.S. Alves, C. Boscato, et al., Hepatic cholinesterase of laying hens naturally infected by Salmonella Gallinarum (fowl typhoid), Microb. Pathog. 98 (2016) 93e97. T.S. Frode, Y.S. Medeiros, Myeloperoxidase and adenosine-deaminase levels in the pleural fluid leakage induced by carrageenan in the mouse model of pleurisy, Mediat. Inflamm. 10 (2011) 223e227. M.M. Boiago, M.D. Baldissera, P.H. Doleski, N.B. Bottari, G.M. do Carmo, D.N. Araujo, et al., Ectonucleotidases and adenosine deaminase activity in laying hens naturally infected by Salmonella gallinarum ad their effects on the pathogenesis of the disease, Microb. Pathog. 93 (2016) 180e184. E. Seker, U. Ispir, M. Dorucu, Immunostimulating effect of levamisole on spleen and head-kidney leucocytes of rainbow trout (Oncorhynchus mykiss, walbaum 1792), Kafkas Univ. Vet. Fal 17 (2011) 239e242. H. Verdouw, C.J.A. Vanechteld, E.M.J. Deckkers, Ammonia determinations based on indophenol formation with sodium salicylate, Water Res. 12 (1978) 399e402. J. Colt, List of spreadsheets prepared as a complement. In: Wedemeyer GA (Ed.), Fish Hatchery Management, second ed., American Fisheries Society. G.L. Ellman, K.D. Courtney, V. Andres, R.M. Feather-Stone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88e95. J.B.T. Rocha, T. Emanuelli, M.E. Pereira, Effects of early undernutrition on kinetic parameters of brain acetylcholinesterase from adult rats, Acta Neurobiol. Exp. 53 (1993) 431e437. F. Worek, U. Mast, D. Kiderlen, Improved determination of acetylcholinesterase activity in human whole blood, Clin. Chim. Acta 90 (1999) 28873e28890. G. Giusti, Adenosine deaminase, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, second ed., Academic Press, New York, 1974, pp. 1092e1099. G. Giusti, C. Gakis, Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues, Enzyme 12 (1971) 417e424. M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248e254. M.R. Schetinger, V.M. Morsch, C.D. Bonan, A.T. Wyse, NTPDase and 5’-nucleotidase activities in physiological and disease conditions: new perspectives for human health, Biofactors 31 (2007) 77e98. , L.S. Oliveira, M.R. Schetinger, V.M. Morsch, R.M. Spanevello, G.R. Thome A.M. Fiorenza, et al., Nicotine alters the ectonucleotidases activities in lymphocytes: in vitro and in vivo studies, Biomed. Pharmacother. 66 (2012) 206e212. M.D. Baldissera, N.B. Bottari, R.E. Mendes, C.I. Schwertz, N.J. Lucca, D. Dalenogare, et al., Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: influence of these enzymes on inflammatory response and pathological findings, Pathol. Res. Pract. 211 (2015) 871e876. R.L. Doyle, A. Fritzen, N.B. Bottari, M.S. Alves, A.D. Da Silva, V.M. Morsch, et al., Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: effects on enzymes of the antioxidant, cholinergic and adenosinergic systems, Microb. Pathog. 97 (2016) 226e230. U.N. Das, Acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation, Med. Sci. Monit. 13 (2007) 214e221. S. Deaglio, K.M. Dwyer, W. Gao, D. Friedman, A. Usheva, A. Erat, et al., Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression, J. Exp. Med. 204 (2007) 1257e1265. G. Hasko, B.N. Cronstein, Adenosine: an endogenous regulator of innate
82
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
immunity, Trends Immunol. 25 (2004) 33e39. [35] M.D. Desrosiers, M.C. Katherine, M.J. Fakir, L.A. Stephens, F.M. Jama, A. Shameli, Adenosine deamination sustains dendritic cell activation in inflammation, J. Immunol. 179 (2007) 1884e1892. [36] Y.Y. Zheinin, G.M. Chuiko, V.A. Pod’gornaya, Blood plasma cholinesterase
activity in various freshwater teleosts, Biol. Inland Waters 1 (1998) 74e79. [37] G.M. Chuiko, Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities in vitro inhibition by DDVP, an organophosphorus pesticide, Comp. Biochem. Physiology Part C 127 (2000) 233e242.
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Involvement of cholinergic and purinergic systems during the inflammatory response caused by Aeromonas hydrophila in Rhamdia quelen Matheus D. Baldissera a, *, Carine F. Souza b, Pedro Henrique Doleski a, Guerino B. Júnior b, Agueda C. de Vargas c, Bernardo Baldisserotto b, ** a b c
Department of Microbiology and Parasitology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Department of Physiology and Pharmacology, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil Department of Preventive Veterinary Medicine, Universidade Federal de Santa Maria, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 July 2016 Received in revised form 4 August 2016 Accepted 8 August 2016 Available online 9 August 2016
The aim of this study was to evaluate the cholinergic (acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)) and purinergic (adenosine deaminase (ADA)) systems in head kidney, spleen, total blood and serum samples in experimentally infected fish with A. hydrophila, and the involvement of these systems during the inflammatory process. Silver catfish (Rhamdia quelen) juveniles were divided into two groups with seven fish each: uninfected (negative control) and infected (positive control). On day 2 post-infection, animals were euthanized and the head kidney, spleen, total blood and serum were collected. AChE and ADA activities in head kidney and spleen decreased in infected animals compared to uninfected animals, as well as AChE in total blood and seric ADA activities. BChE activity was not expressed in the evaluated tissues. Therefore, our results lead to the hypothesis that cholinergic and purinergic systems play an important role on the immune response against A. hydrophila with an antiinflammatory effect. In summary, AChE and ADA activities reduced probably in order to protect against tissue inflammatory damage caused by infection. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Adenosine deaminase Acetylcholine Fish Infectious diseases
1. Introduction Aquaculture is one of the fastest growing food and food producing sectors due to increased demand for fish and seafood, providing one-third of seafood consumed worldwide in the last decade [1,2]. However, the intensification of aquaculture has increased the risk of infectious diseases, such as those caused by Aeromonas hydrophila, that are recognized as one the most serious challenges in the fish production, limiting the development of aquaculture and causing important economic losses [3,4]. Aeromonas hydrophila is an important aquatic bacterium pathogen of freshwater aquaculture, the causative agent of epizootic ulcerative syndrome [5]. The main symptoms of this pathogen are ulcerations, hemorrhagic focus, infectious abdominal dropsy and
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] [email protected] (B. Baldisserotto). http://dx.doi.org/10.1016/j.micpath.2016.08.009 0882-4010/© 2016 Elsevier Ltd. All rights reserved.
(M.D.
Baldissera),
erosion of the fins in farmed and wild fish [6]. This bacterium is a serious cause of mortality and economic loss in species such as common carp (Cyprinus carpio), goldfish (Carassius auratus auratus), striped fish (Plotosus lineatus), rohu carp (Labeo rohita) and silver catfish (Rhamdia quelen) [7e10]. Recently, studies have demonstrated the effects of A. hydrophila infection on immune response in head kidney and spleen tissues [11], but the effects on enzymes of cholinergic and purinergic system, that are considered novel markers of inflammatory response [12,13], remain unknown. The cholinergic system is notably involved in anti-inflammatory reactions and cholinesterases enzymes, such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), develop an important role during immune and inflammatory response [12,13]. AChE hydrolyses acetylcholine (ACh), a neurotransmitter with antiinflammatory effects, into choline and acetate, while the BChE hydrolyses the butyrylcholine (BCh) and other variety of esters, including ACh [14,15]. Recently, study conducted by Tonin et al. [13] demonstrated that BChE activity is recognized as a novel marker of tissue inflammation and can be used with a marker of acute
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
inflammatory response. Alterations on AChE and BChE activities in liver and serum of laying hens naturally infected with Salmonella gallinarum contribute directly to inflammatory response and the pathogenesis of disease [16]. The adenosine deaminase (ADA) is an important enzyme of the purinergic system that catalyzes the deamination of 2deoxyadenosine to inosine. ADA activity plays an important role on immune and inflammatory response due the regulation of adenosine, a molecule with anti-inflammatory properties, protecting the host tissue from damage and having an important role in the differentiation and proliferation of lymphocytes [17]. In this context, a study has demonstrated that ADA activity is involved in the cell-mediated immunity and could be an important biomarker to determine the severity of inflammatory and immune response during the infection caused by Salmonella gallinarum, another important bacterial pathogen [18]. Based on that head kidney and spleen are considered important lymphatic immune organs in fish [19], and the role of cholinergic and purinergic system during the immune and inflammatory response, the aim of this study was to evaluate the activities of AChE, BChE and ADA in head kidney and spleen tissues, AChE in total blood and BChE and ADA in serum of R. quelen experimentally infected with A. hydrophila. 2. Materials and methods 2.1. Aeromonas hydrophila isolate The isolate of A. hydrophila used in this study was obtained from a naturally infected silver catfish (R. quelen), identified by phenotypic testing and 16S rDNA sequencing, prepared in saline solution from cultures grown in Mueller-Hinton agar (Himedia Laboratories). 2.2. Animal model and water quality Fourteen silver catfish juveniles (50.5 ± 2 g; 19.5 ± 1.3 cm) were used as the experimental model for determination of the enzymes of the cholinergic and purinergic systems in head kidney and spleen. All fish were transferred from a local fish culture to the laboratory, where they were maintained in continuously aerated tanks with controlled water parameters (22.0e24 C, pH 7.2e7.6, dissolved oxygen levels: 5.5e7.5 mg L1). Fish were acclimated for 14 days, and dissolved oxygen and temperature were measured with a YSI oxygen meter (Model Y5512, Ohio, USA). The pH was ~o Paulo, SP, measured using a DMPH-2 pH meter (Digimed, Sa Brazil). Total ammonia levels were determined according to Verdouw et al. [20] and un-ionized ammonia (NH3) levels were calculated according to Colt [21]. 2.3. Experimental design The silver catfish were assigned into two groups with seven animals each: uninfected animals (negative control) and experimentally infected animals (positive control) inoculated intramuscularly with 100 mL of A. hydrophila solution (2.1 109 colony forming unit: OD600 ¼ 1.7e1.9) on the right latero-dorsal side of each fish. The negative control received the same dose of sterile saline by the same route. Fish were fed once a day to satiation with commercial feed, and uneaten food, other residues and feces were removed 30 min after feeding. The methodology used in this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria under protocol number 074/2014.
79
2.4. Sample collection and tissue preparation On day 2 post-infection (PI), blood samples were collected from the caudal vein using tubes containing EDTA and tubes without anticoagulant to obtain serum. Thereafter, the head kidney and spleen were removed and dissected on a glass dish over ice for the measurement of AChE, BChE and ADA activities. All animals were euthanized by spinal cord section according the Ethics Committee recommendations. The blood samples were centrifuged at 3000 rpm for 15 min to obtain serum for measurement the BChE and ADA activities. The total blood collected in tubes containing EDTA was used to determine the AChE in total blood. Head kidney and spleen were homogenized (1:10 w/v) in a glass potter with Tris-HCl buffer (50 mM, pH 7.4), centrifuged at 10000 g for 10 min. Aliquots of the supernatant were stored at e 20 C until utilization. 2.5. AChE activity in head kidney, spleen and total blood AChE enzymatic activity in the head kidney and spleen homogenates were determined by a modified spectrophotometric method of Ellman et al. [22] as previously described by Rocha et al. [23]. The reaction mixture was composed by of 1.0 M of potassium phosphate buffer (pH 8.0) and 10 mM of DTNB. The method is based on increased yellow color of the nitrobenzoate (TNB), ion produced from thiocoline after reaction with 5,5-dithio-bis-acid-nitrobenzoic (DTNB) ion, measured by absorbance at 412 nm for 2 min of incubation at 30 C. The reaction was initiated by adding 0.78 mM acetylcholine iodide as substrate. All samples were run in duplicate and the enzymatic activity was expressed in mmol AcSCh/min mg of protein. AChE activity in total blood was determined by Ellman et al. [22] method, modified by Worek et al. [24]. 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/mmol Hb. 2.6. BChE activity in head kidney, spleen and serum Butyrylcholinesterase activity in homogenates and serum was determined by the method of Ellman et al. [22] as previously described by Rocha et al. [23], using the substrate butyrylcholine. Pre-incubation of samples was done at 37 C for 2 min, and reading performed for 2 min, at intervals of 20-20 s on a spectrophotometer (412 nm). The samples analysis was carried out in duplicate, and the enzymatic activity was expressed in mmol BcSCh/min mg of protein. 2.7. ADA activity in head kidney, spleen and serum ADA activity in head kidney and spleen were measured according to Giusti [25], which is based on the direct measurement of the formation of ammonia produced when the enzyme acts on adenosine. A volume of 100 mL of head kidney or spleen was used. The enzymatic reaction was started by addition of 500 mL of 21 mM adenosine as substrate and stopped by adding 1.5 mL of 106/ 0.16 mM phenol-sodium nitroprusside to the reaction mixture, which was immediately mixed with 1.5 mL of 125/11 mM alkalinehypochlorite (sodium hypochlorite). Released ammonia reacted with alkaline-hypochlorite and phenol in the presence of the catalyst-sodium nitroprusside to produce indophenol (a blue color). The concentration of ammonia was directly proportional to the absorbance of indophenol, read at 620 nm. Ammonium sulfate 75 mM was used as standard. All measures were carried out in duplicate and ADA activity in head kidney and spleen homogenates
80
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
were expressed as U/mg of protein. The activity of ADA in serum was determined spectrophotometrically, according to the method described by Giusti and Gakis [26]. The reaction started by addition of adenosine (substrate) to a final concentration of 21 mMol/L; incubations were carried out for 1 h at 37 C. The reaction stopped by adding 106 mMol/L 0.16 mMol/ L of phenolnitroprusside/mL solution. The reaction mixtures were mixed immediately to 125 mMol/L; 11mMol/L of alkaline hypochlorite (sodium hypochlorite) and vortexed. Ammonium sulfate, at concentration of 75 mmol/L served as ammonium standard. The ammonium concentration is directly proportional to the absorption of indophenol at 650 nm. The specificity activity was reported as U/ L. 2.8. Protein determination Protein content in head kidney, spleen and serum was determined by the Bradford method using bovine albumin serum as the standard [27]. 2.9. Statistical analyses The data were tested for normality using the Shapiro-Wilk test. Then, the data were subjected to Student's t-test for independent samples to verify the statistical difference. Differences between groups were considered significant when p < 0.05. The effect size (r2) was described and scored as follows: 0.1 (small), 0.1 to 0.3 (medium) and 0.5 (large). Data were expressed as mean ± standard deviation.
Fig. 1. Acetylcholinesterase (AChE) activity in head kidney and spleen [A], and in total blood [B] of silver catfish experimentally infected by Aeromonas hydrophila compared to uninfected group (*p < 0.05 and ***p < 0.001 using Student's t-test; n ¼ 7 fish per group).
3. Results 3.1. Water quality parameters The mean water quality parameters were: temperature 23 C, dissolved oxygen level 6.50 ± 0.2 mg/L, pH 7.25 ± 0.05, total ammonia 0.85 ± 0.03 mg/L and non-ionized ammonia of 0.007 ± 0.0004 mg/L. 3.2. AChE and BChE activities AChE activity decreased 37% in head kidney [t(12) ¼ 2.93; p < 0.05; r2 ¼ 0.63], 62% in spleen [t(12) ¼ 2.71; p < 0.05; r2 ¼ 0.55] and 45% [t(12) ¼ 4.11; p < 0.001; r2 ¼ 0.63] in total blood of animals infected by A. hydrophila compared with the uninfected control group (Fig. 1). BChE activity in head kidney, spleen and serum were not expressed in the silver catfish. 3.3. ADA activity ADA activity decreased 52% in head kidney [t(12) ¼ 5.73; p < 0.001; r2 ¼ 0.84], 61% in spleen [t(12) ¼ 4.97; p < 0.001; r2 ¼ 0.72] and 40% [t(12) ¼ 3.13; p < 0.001; r2 ¼ 0.52] in serum of animals infected by A. hydrophila compared with the uninfected control group (Fig. 2). 4. Discussion Several studies have reported that both purinergic [28,29] and cholinergic systems [30,31] are important in the modulation of the inflammatory and immune responses. In this study, alterations in the activities of enzymes involved in the regulation of acetylcholine and adenosine levels were found in fish infected with A. hydrophila. The analysis of our data clearly shows the inhibition of the AChE and ADA activities in head kidney and spleen, AChE in total blood
Fig. 2. Adenosine deaminase (ADA) activity in head kidney and spleen [A], and in serum [B] of silver catfish experimentally infected by Aeromonas hydrophila compared to uninfected group (***p < 0.001 using Student's t-test; n ¼ 7 fish per group).
and ADA in serum at 2 days PI. Similar responses were observed in cattle infected with Anasplasma marginale [31] and hens infected with Salmonella gallinarum [18]. Due to this decrease in AChE
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
activity, less ACh is hydrolyzed, possibly in order to reduce the proinflammatory response and tissue damage, since ACh has antiinflammatory effects [32]. In this line, AChE arises as a potential contributor in the cholinergic pathways controlling inflammatory and immune response mediated by muscarinic and nicotinic receptors during A. hydrophila infection. Adenosine exhibits potent anti-inflammatory and immunosuppressive action, inhibiting the secretion of pro-inflammatory cytokines [33], acting as regulator of immune response, protecting the host from excessive tissue damage associated with inflammation [34,35]. The reduction on ADA activity in head kidney, spleen and serum observed in this study could contribute to restrict the inflammatory response and subsequent cellular damage because it would increase adenosine tissue levels. Adenosine, an anti-inflammatory nucleoside, confers to the host tissue protection, a compensatory mechanism against bacterial infections [18]. Also, in other infectious diseases, the enzymes AChE and ADA increase, contributing to inflammatory process, because decrease the levels of ACh and adenosine, considered anti-inflammatory molecules [30]. We observed that BChE activity was not expressed in the evaluated tissues and serum of this species. BChE in serum is not expressed in the serum or plasma of many fish families, such as Percidae, Esocidae and Coregonidae [36,37]. The serum and plasma of some cyprinid species contain both AChE and BChE, but the common carp (Cyprinus carpio) has only AChE [37]. According this author, a difference between species suggests the existence of cross-species variability in these enzymes. Based on these evidences and in the absence of related data in literature (to our knowledge) regarding BChE, we can conclude that it possible that silver catfish does not express the BChE enzyme in the evaluated organs, as well as in the serum. Based on the results, we concluded that infection by A. hydrophila in silver catfish alters the cholinergic and purinergic systems, suggesting the involvement of AChE and ADA activities on pathogenesis of disease, regulating the ACh and adenosine levels, molecules known to participate in physiological and pathological events as inflammatory mediator. In summary, AChE and ADA reduction probably played an effect on inflammatory response, decreasing hydrolysis of ACh and adenosine, important antiinflammatory molecules, in an attempt to reduce or prevent the inflammatory tissue damage. These alterations probably reduce or prevent the inflammatory tissue damage caused by A. hydrophila.
[9]
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
[25] [26]
References [27] [1] M.G. Rivera-Ferre, Can export-oriented aquaculture in developing countries be sustainable and promote sustainable development? the shrimp case, J. Agric. Environ. Ethics 22 (2009) 301e321. [2] M. Reverter, N. Bontemps, D. Lecchini, B. Banaigs, P. Sasal, Use of plant extracts in fish as an alternative to chemotherapy: current status and future perspectives, Aquaculture 433 (2014) 50e61. [3] Z.Z. Qi, Z.H. Zhang, N. Boon, P. Bossier, Probiotics in aquaculture of Chinacurrent state, problems and prospect, Aquaculture 290 (2009) 15e21. [4] F.J. Zagmutt, S.H. Sempier, T.R. Hanson, Disease spread models to estimate highly uncertain emerging diseases losses for animal agriculture insurance policies: an application to the U.S. farm-raised catfish industry, Risk Anal. 33 (2013) 1924e1937. [5] M.Y. Huang, C.I. Chang, C.C. Chang, L.W. Tseng, C.L. Pan, Effects of dietary levan on growth performance, nonspecific immunity, pathogen resistance and body composition of orange-spotted grouper (Epinephelus coioides H.), Aquac. Res. 46 (2015) 2752e2767. [6] J.M. Janda, S.L. Abbott, The genus Aeromonas, taxonomy, pathogenicity, and infection, Clin, Microbiol. Rev. 23 (2010) 35e73. [7] S. Maji, P. Mali, S.N. Joardar, Immunoreactive antigens of the outer membrane protein of Aeromonas hydrophila, isolated from goldfish, Carassius auratus (Linn.), Fish. Shellfish Immun. 20 (2006) 462e473. [8] W. Sirimanapong, K.D. Thompson, K. Kledmanee, P. Thaijongrak, B. Collet, E.L. Ooi, et al., Optimization and standardisation of functional immune assays for striped catfish (Pangasianodon hypophthalmus) to compare their immune
[28]
[29]
[30]
[31]
[32] [33]
[34]
81
response to live and heat killed Aeromonas hydrophila as models of infection and vaccination, Fish. Shellfish Immun. 40 (2014) 374e383. J.L. Wang, X.L. Meng, R.H. Lu, C. Wu, T.Y. Luo, X. Yan, et al., Effects of Rehmania glutinosa on growth performance, immunological parameters and disease resistance to Aeromonas hydrophila in common carp (Cyprinus carpio L.), Aquaculture 435 (2015) 293e300. C.F. Souza, M.D. Baldissera, R.A. Vaucher, L.Q.S. Lopes, B.S. Vizzotto, R.P. Raffin, et al., In vivo bactericidal effect of Melaleuca alternifolia essential oil against Aeromonas hydrophila: silver catfish (Rhamdia quelen) as an experimental model, Microb. Pathog. 98 (2016) 82e87. P.J. Ni, W.D. Jiang, P. Wu, Y. Liu, S.Y. Kuang, L. Tang, et al., Dietary low or excess of lipids reduced growth performance, and impaired immune function and structure of head kidney, spleen and skin in young grass carp (Ctenopharyngodon idella) under the infection of Aeromonas hydrophila, Fish. Shellfish Immun. 55 (2016) 28e47. G.M. Do Carmo, L.Z. Crivellenti, N.B. Bottari, G. Machado, S. Borin-Crivellenti, R.N. Moresco, et al., Butyrylcholinesterase as a marker of inflammation and liver injury in the acute and subclinical phases of canine ehrlichiosis, Comp. Immunol. Microbiol. Infect. Dis. 43 (2015) 16e21. , T. Duarte, et A.A. Tonin, A.M.C. Calado, N.B. Bottari, D. Dalegonare, G.R. Thome al., Novel markers of inflammatory response and hepatic dysfunction in canine leishmaniasis, Comp. Immunol. Microbiol. Infect. Dis. 44 (2016) 61e64. L. Anglister, A. Etlin, E. Finkel, A.R. Durrant, A. Lev-Tov, Cholinesterases in development and disease, Chem. Biol. Interact. 175 (2008) 92e100. J.A.D.S. Jacques, J.F.P. Rezer, J.F. Gonçalves, R.M. Spanevello, J.M. Gutierres, V.C. Pimentel, et al., The effect of curcumin in the ectonucleotidases and acetylcholinesterase activities in synaptosomes from the cerebral cortex of cigarette smoke-exposed rats, Cell biochem. Funct. 29 (2011) 703e707. A.S. Da Silva, M.M. Boiago, N.B. Bottari, G.M. do Carmo, M.S. Alves, C. Boscato, et al., Hepatic cholinesterase of laying hens naturally infected by Salmonella Gallinarum (fowl typhoid), Microb. Pathog. 98 (2016) 93e97. T.S. Frode, Y.S. Medeiros, Myeloperoxidase and adenosine-deaminase levels in the pleural fluid leakage induced by carrageenan in the mouse model of pleurisy, Mediat. Inflamm. 10 (2011) 223e227. M.M. Boiago, M.D. Baldissera, P.H. Doleski, N.B. Bottari, G.M. do Carmo, D.N. Araujo, et al., Ectonucleotidases and adenosine deaminase activity in laying hens naturally infected by Salmonella gallinarum ad their effects on the pathogenesis of the disease, Microb. Pathog. 93 (2016) 180e184. E. Seker, U. Ispir, M. Dorucu, Immunostimulating effect of levamisole on spleen and head-kidney leucocytes of rainbow trout (Oncorhynchus mykiss, walbaum 1792), Kafkas Univ. Vet. Fal 17 (2011) 239e242. H. Verdouw, C.J.A. Vanechteld, E.M.J. Deckkers, Ammonia determinations based on indophenol formation with sodium salicylate, Water Res. 12 (1978) 399e402. J. Colt, List of spreadsheets prepared as a complement. In: Wedemeyer GA (Ed.), Fish Hatchery Management, second ed., American Fisheries Society. G.L. Ellman, K.D. Courtney, V. Andres, R.M. Feather-Stone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88e95. J.B.T. Rocha, T. Emanuelli, M.E. Pereira, Effects of early undernutrition on kinetic parameters of brain acetylcholinesterase from adult rats, Acta Neurobiol. Exp. 53 (1993) 431e437. F. Worek, U. Mast, D. Kiderlen, Improved determination of acetylcholinesterase activity in human whole blood, Clin. Chim. Acta 90 (1999) 28873e28890. G. Giusti, Adenosine deaminase, in: H.U. Bergmeyer (Ed.), Methods of Enzymatic Analysis, second ed., Academic Press, New York, 1974, pp. 1092e1099. G. Giusti, C. Gakis, Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues, Enzyme 12 (1971) 417e424. M.M. Bradford, A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248e254. M.R. Schetinger, V.M. Morsch, C.D. Bonan, A.T. Wyse, NTPDase and 5’-nucleotidase activities in physiological and disease conditions: new perspectives for human health, Biofactors 31 (2007) 77e98. , L.S. Oliveira, M.R. Schetinger, V.M. Morsch, R.M. Spanevello, G.R. Thome A.M. Fiorenza, et al., Nicotine alters the ectonucleotidases activities in lymphocytes: in vitro and in vivo studies, Biomed. Pharmacother. 66 (2012) 206e212. M.D. Baldissera, N.B. Bottari, R.E. Mendes, C.I. Schwertz, N.J. Lucca, D. Dalenogare, et al., Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: influence of these enzymes on inflammatory response and pathological findings, Pathol. Res. Pract. 211 (2015) 871e876. R.L. Doyle, A. Fritzen, N.B. Bottari, M.S. Alves, A.D. Da Silva, V.M. Morsch, et al., Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: effects on enzymes of the antioxidant, cholinergic and adenosinergic systems, Microb. Pathog. 97 (2016) 226e230. U.N. Das, Acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation, Med. Sci. Monit. 13 (2007) 214e221. S. Deaglio, K.M. Dwyer, W. Gao, D. Friedman, A. Usheva, A. Erat, et al., Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression, J. Exp. Med. 204 (2007) 1257e1265. G. Hasko, B.N. Cronstein, Adenosine: an endogenous regulator of innate
82
M.D. Baldissera et al. / Microbial Pathogenesis 99 (2016) 78e82
immunity, Trends Immunol. 25 (2004) 33e39. [35] M.D. Desrosiers, M.C. Katherine, M.J. Fakir, L.A. Stephens, F.M. Jama, A. Shameli, Adenosine deamination sustains dendritic cell activation in inflammation, J. Immunol. 179 (2007) 1884e1892. [36] Y.Y. Zheinin, G.M. Chuiko, V.A. Pod’gornaya, Blood plasma cholinesterase
activity in various freshwater teleosts, Biol. Inland Waters 1 (1998) 74e79. [37] G.M. Chuiko, Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities in vitro inhibition by DDVP, an organophosphorus pesticide, Comp. Biochem. Physiology Part C 127 (2000) 233e242.