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Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: Effects on enzymes of the antioxidant, cholinergic, and adenosinergic systems
Accepted Manuscript Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: Effects on enzymes of the antioxidant, cholinergic, and adenosinergic systems Rovaina L. Doyle, Alexandro Fritzen, Nathieli B. Bottari, Mariana S. Alves, Aniélen D. da Silva, Vera M. Morsch, Maria Rosa C. Schetinger, João R. Martins, Julsan S. Santos, Gustavo Machado, Aleksandro S. da Silva PII:
S0882-4010(16)30231-5
DOI:
10.1016/j.micpath.2016.06.001
Reference:
YMPAT 1849
To appear in:
Microbial Pathogenesis
Received Date: 1 May 2016 Revised Date:
22 May 2016
Accepted Date: 1 June 2016
Please cite this article as: Doyle RL, Fritzen A, Bottari NB, Alves MS, da Silva AD, Morsch VM, Schetinger MRC, Martins JR, Santos JS, Machado G, da Silva AS, Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: Effects on enzymes of the antioxidant, cholinergic, and adenosinergic systems, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: effects on
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enzymes of the antioxidant, cholinergic, and adenosinergic systems
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Rovaina L. Doylea*, Alexandro Fritzenb, Nathieli B. Bottaric, Mariana S. Alvesc, Aniélen D. da
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Silvac, Vera M. Morschc, Maria Rosa C. Schetingerc, João R. Martinsa, Julsan S. Santosa,
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Gustavo Machadod, Aleksandro S. da Silvab,c*
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a
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Brazil.
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b
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de Santa Catarina (UDESC), Chapecó, SC, Brazil.
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c
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Molecular Biology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil.
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d
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Medicine, University of Minnesota, St. Paul, MN, USA.
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Graduate Program in Animal Science, Department of Animal Science, Universidade do Estado
Graduate Program in Toxicology and Biochemistry, Department of Biochemistry and
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STEMMA laboratory, Department of Veterinary Population Medicine, College of Veterinary
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Instituto de Pesquisas Veterinárias Desidério Finamor (FEPAGRO), Eldorado do Sul, RS,
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*Author for correspondence: Department of Animal Science, University of Santa Catarina State
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(UDESC). 680 D, Beloni Trombeta Zanin Street, Chapecó/SC, Brazil Zip: 89815-630, Phone: 55
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49 3322-4202. Fax: 55 49 3311-9316. (E-mail: [email protected];
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[email protected]).
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ABSTRACT Anaplasmosis is a worldwide hemolytic disease in cattle caused by a gram-negative
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obligatory intracellular bacterium, characterized by anemia and jaundice. Among the treatments
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used for anaplasmosis is a drug called imidocarb dipropionate, also indicated as an
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immunomodulator agent. However, it causes side effects associated with increased levels of
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acetylcholine. In view of this, the effects of imidocarb dipropionate on the purinergic system,
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and antioxidant enzymes in animals naturally infected by Anaplasma marginale were evaluated.
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Young cattle (n=22) infected by A. marginale were divided into two groups: the Group A
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consisted of 11 animals used as controls; and the Group B composed of 11 animals. Imidocarb
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dipropionate (5 mg/kg) was used subcutaneously to treat both groups (the Group A on day 6 and
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the Group B on day 0). The treatment reduced acetylcholinesterase (AChE), and adenosine
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deaminase (ADA) activities, and increased the dismutase superoxide and catalase activities. No
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changes on lipid peroxidation (TBARS levels) and BChE activities were noticed. These results
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suggest that imidocarb dipropionate used to treat A. marginale infection in cattle has effect on
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antioxidant enzymes, and significantly inhibits the enzymatic activities of ADA and AChE.
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Keywords: Anaplasma marginale, AChE, ADA, CAT, immunomodulation, SOD.
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1. INTRODUCTION
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Anaplasmosis is a hemolytic disease that affects mainly cattle, and it is caused by a gram-
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negative obligate intracellular bacterium named Anaplasma marginale [1]. This disease occurs in
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tropical and subtropical regions worldwide and is endemic in Mexico, Central and South
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America, resulting in huge economic losses to the cattle industry [2]. Disease transmission
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occurs through mechanical vectors comprising sucking flies and fomites contaminated with
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blood, in addition to biological vectors such as ticks [3, 4]. Prepatent period depends on the
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infectious dose and host immune status, ranging from 7 to 60 days (28 days on average), causing
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mild to severe clinical signs [5]. Infected erythrocytes are phagocytosed by reticuloendothelial
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cells, leading to varying degrees of anemia, jaundice, and hemoglobinuria [6]. During infection
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progress, cattle may show fever, weight loss, lethargy, abortion, jaundice, and sudden death
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specially in animals older than 2 years [7].
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Treatment with imidocarb dipropionate has been proved to be successful against
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hemolytic diseases such as anaplasmosis, requiring only one dose for clinical signs remission.
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However, this drug causes many side effects such as salivation, lacrimation, tachypnea,
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tachycardia, and pain at the injection site, as a result of massive acetylcholine accumulation [8].
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Acetylcholine (Ach) is the main vagal neurotransmitter and has important anti-inflammatory
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effect. It is hydrolyzed by the enzyme acetylcholinesterase, which is an intrinsic regulator of
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inflammation [9]. These clinical changes may cause oxidative stress, characterized by excess of
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production or inadequate removal of reactive oxygen species (ROS), and reactive nitrogen
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molecules [10]. These ROS molecules are involved in lipid peroxidation processes, protein
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oxidation, and damage to DNA, which is one of the mechanisms used by cells of the immune
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system to eradicate infections. ROS reactions are enhanced by the presence of iron, which makes
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red blood cells easy targets for ROS reactions, providing higher levels of lipid peroxidation in
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these cells, exacerbating hemolysis [11]. The persistence presence of intracellular bacteria is
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possible due to ROS production, able to prevent immune system responses. An example of this
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process is shown in the avoidance mechanism by Anaplasma phagocytophilum, which blocks the
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incorporation of p22 and gp91 into the phagolysosome, reducing the oxidative activity [12].
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The adenosinergic system plays an important role on immune and inflammatory
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responses due to the regulation of adenine nucleoside, adenosine. Adenosine deaminase (ADA)
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is considered a key enzyme in the adenosinergic system, being responsible for the irreversible
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deamination of adenosine, controlling its extracellular levels. The anti-inflammatory effects of
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extracellular adenosine are modulated by ADA activity [13], and can be affected by
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chemotherapy. The metabolic waste of the drug are deposited in the liver and kidney for a long
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period, which may result in necrosis of these organs, compromising physiological functioning of
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other systems [14]. Therefore, the aim of this study was to evaluate the effects of treatment with
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imidocarb dipropionate on the cholinergic systems, antioxidant enzymes, and adenosine
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deaminase in cattle naturally infected by A. marginale.
79 2. MATERIALS AND METHODS
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2.1. Animals
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Young cattle (n=22, average of 1.5 years, and 276 kg of body weight) part of a herd from
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the Veterinary Research Institute Desidério Finamor, Eldorado do Sul, South of Brazil, were
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used in this study. The animals were kept in an extensive system with access to native pasture
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and water ad libitum. A. marginale was found in blood smears of all 22 animals at day 0 of the
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experiment, i.e. this study used cattle naturally infected by A. marginale. These animals were
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divided into two groups: the Group A consisting of 11 animals used as control animals for all
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analyzes performed on day 5 of the experiment, and treated subcutaneously on day 6 with
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imidocarb dipropionate as recommendated by the manufacturer (5 mg/kg). The Group B was
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formed by 11 bovines, treated subcutaneously on day 0 with imidocarb dipropionate (same
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dose). Therefore, animals from the Group A were used as control for the analysis performed on
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day 5 post-treatment of the Group B in order to evaluate treatment response. Blood smears were
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performed at the end of the experiment (day 10) for post- treatment parasitemia evaluation.
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For oxidative and antioxidant profile analyses, blood samples were collected at days 0, 5
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and 10 post-treatment, and allocated on two sterile tubes with anticoagulant (EDTA 10%, and
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sodium citrate) and one tube without anticoagulant. The tubes without anticoagulant were
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centrifugated at 3500 rpm for 10 min to obtain serum that was collected, and stored in eppendorf
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tubes at -20 °C up to analysis of TBARS, ADA, and BChE. Tubes with sodium citrate was
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homogenized and frozen at -20ºC for analysis of CAT and SOD. Blood samples with EDTA
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were diluted 1:50 (v/v) in lysis solution (0.1 mmol/L potassium/sodium phosphate buffer
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containing 0.03% Triton X-100) to determine AChE activity. Hematocrit [15] was also
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performed on day 0 in order to assess the clinical status of the animals related to anemia.
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2.3. Biochemical analysis
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2.3.1. Seric TBARS, BChE, and ADA
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Lipid peroxidation was determined based on the levels of thiobarbituric acid reactive
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substances (TBARS) in sera samples according to the method described by Jentzsch et al. [16].
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The results were obtained by spectrophotometry (535 nm) and expressed as nmol of
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malondialdehyde (MAD) per mL.
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To verify butyrylcholinesterase (BChE) activity in the sera the method described by
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Ellman et al. [17] was used, using butyrylthiocholine iodide (BcSCh) instead of acetylcholine,
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and the results were expressed in µmoles BcSCh/h/mg of protein.
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Adenosine deaminase (ADA) activity was measured spectrophotometrically in serum by
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the method of Giusti and Gakis and the reaction was started by the addition of adenosine
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substrate [18]. Ammonia concentration is directly proportional to the absorption of indophenol at
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650 nm, and the specific activity is reported as U/L in serum.
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2.3.2. AChE activity in total blood
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The AChE enzymatic assay in whole blood was determined by Ellman et al. [17] method,
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modified by Worek et al. [19]. The specific activity of whole blood AChE was calculated from
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the quotient between AChE activity and hemoglobin content, and the results were expressed as
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mU/µmol Hb.
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2.3.3. Catalase and superoxide dismutase activities
Determination of CAT activity was carried out in accordance to a modified method of
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Nelson and Kiesow [20]. This assay involved the change in absorbance at 240 nm due to CAT
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dependent decomposition of hydrogen peroxide. CAT activity was calculated using the molar
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extinction coefficient, and the results were expressed as nmol CAT per milligram protein.
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SOD activity measurement was based on the inhibition of radical superoxide reaction
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with adrenalin as described by McCord and Fridovich [21]. SOD activity is determined by
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measuring the speed of adrenochrome formation, observed at 480 nm, in a reaction medium
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containing glycine–NaOH and adrenaline. The results were expressed as UI SOD per milligram
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of protein.
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2.4. Statistical analysis
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The tests were performed in triplicate, and the average used for statistical analysis. All data were
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analyzed first descriptively. Further all variables were submitted to Shapiro Wilk’s test for
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normally distribution verification, since most of the variables did not met assumption of
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parametric testing, it was used a nonparametric test for two independence groups test Friedman
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test to evaluate the influence of time of the experiment on the measured parameters (AChE,
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BChE, ADA, TBARS, CAT, and SOD), and when the differenceover time was present was
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used Mann-Whitney U. Also analyzed the changes in parameters over the study period
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considering the groups (A and B) and in addition was observed difference between two
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independent groups by Mann-Whitney U for every moment of collection (day 0; day 5 and day
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10) as analysis. Critical P value of <0.05 was used. All analyzes were performed using the R
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software v.2.15.2.
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3. RESULTS
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Hematocrits performed on day 0 did not show differences between groups (Figure 1).
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Anemia was not detected despite A. marginale infection, since hematocrit results were normal.
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When sampling day was considered, the only significant difference observed was between
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groups A and B regarding ADA activity on day 5 of the experiment (Table 1). All other
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parameters and sampling time analyzed did not show any significant differences between groups
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(Table 1).
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There was influence of time on several measured parameters throughout the experiment,
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for example on AChE (P<0.001), and this was observed between day 0 and 10 for the group B
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(P=0.01) with reduced AChE activity (Figure 2). On the other hand, BChE was not influnced
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over time for both groups (P=0.86). For ADA, there was a significant influence of time
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(P<0.001), and this difference (reduced activity of ADA) was between day 0 and 10 (P=0.004)
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and day 5 and 10 (P=0.01) for the group A; and, a marked decrease was observed on ADA
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activity from day 0 to 10 (P=0.003) and from day 5 to 10 (P=0.01) for the group B (Figure 2).
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For TBARS, no influence of time was observed on both groups (P=0.75). However, for SOD
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there was influence of time (P<0.001), and this difference (increased SOD activity) was between
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day 0 and 5 (P=0.0004) and day 0 and 10 (P<0.001) for the group A; and a high increase on SOD
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activity in the group B from day 0 to 5 (P<0.001) and from day 0 to 10 (P=0.004) (Figure 2).
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Finally, for CAT there was influence of time (P<0.001), and this difference (i.e. increased
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activity) was between day 0 and 5 (P<0.001) and day 0 and 10 (P<0.001) for the group A; and,
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with a high increase on CAT activity in the group B from day 0 to 5 (P<0.001) and from day 0 to
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10 (P<0.001) (Figure 2). On day 10 of the experiment, all treated animals were negative for A.
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marginale by blood smear.
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4. DISCUSSION
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After imidocarb dipropionate treatment, there was a reduction on seric levels of ADA in
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animals from the Group B compared to those from the Group A, and this reduction was also
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observed in the Group A after imidocarb injection on day 5 of experiment. The reduction on
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ADA seric activity lead to increased adenosine, which in turn has antinflammatory activities and
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acts regulating the growth, differentiation, and proliferation of lymphocytes and erythrocytes
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[13]. In studies conducted by Katayama et al. [22], it was observed that treatment with imidocarb
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dipropionate in the presence of LPS (lipopolysaccharide) led to increase dose dependent IL-10
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production by activated macrophages, and a reduction on TNF-α, IL-12, and nitric oxide,
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providing anti-inflammatory and immunomodulatory effects. Therefore, the stimulation of
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adenosine A2A receptors may lead to similar response in the event of increased IL-10 levels, and
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reduced pro-inflammatory cytokines, which demonstrates a response mechanism promoted by
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imidocarb, since its use reduces ADA activity, an enzyme with regulatory effect on extracellular
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levels of adenosine. A recent study evaluated NTPDase and 5'-nucleotidase activities in platelets
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of bovines infected by A. marginale with and without spleens, and observed increased levels of
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degradation of the nucleotides ATP and ADP by NTPDase, in addition to AMP by 5'nucleotidase
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to form the nucleoside adenosine in platelets of splenectomized animals compared to non-
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splenectomized [23], therefore during A. marginale infection, there are alterations in the
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enzymatic cascade of the purinergic system, as in the adenosinergic system mentioned in this
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study.
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Imidocarb dipropionate may cause side effects, which are related to increased levels of
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acetylcholine, a parasympathetic neurotransmitter [8]. It was observed that the use of imidocarb
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dipropionate in cattle led to reduced AChE activity, but had no effect on BChE enzymes which
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associated with acetylcholine degradation. The cholinergic anti-inflammatory pathway is
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composed of an efferent vague nerve with acetylcholine as the main neurotransmitter and the α7
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nicotinic acetylcholine as its receptor, when activated reduces the release of proinflammatory
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cytokines [24]. The control of extracellular levels of acetylcholine is carried out by AChE and by
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BChE in cases of high levels of ACh [9]. Reduction in AChE activity, as observed by the use of
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imidocarb dipropionate, possibly induces higher seric levels of ACh, which contributes to the
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modulation of cholinergic inflammatory response, as a possible route of immunomodulation
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effect of imidocarb dipropionate.
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Formation of reactive oxygen species (ROS) are an important mechanism against
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infectious agents, however their actions may cause damage to the host. Control of ROS levels is
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performed by enzymes, such as CAT and SOD, able to prevent cell damage and thus, they are
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considered important markers of oxidative stress [12, 25]. One of the actions of ROS may cause
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lipid peroxidation, which is a reaction measured by TBARS levels [26], but this variable showed
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no changes in this study current. The use of imidocarb dipropionate led to increased SOD and
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CAT activities, possibly in response to the increase of ROS metabolism. However, this current
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study was unable to determine whether this increase was related to the infection and/or
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treatment. Drug administration, as aceturate diminazene (a diamidine, as imidocarb dipropionate)
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can increase ROS production, however high levels of it may induce tissue damage and host cell
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death [27], and low levels may induce the production of free radicals which is a form of evasion
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found by pathogens that produce persistent infections [12]. Lipid peroxidation can occurr by A.
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marginale infection [5] and/or hemolysis [28], however after application of imidocarb
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dipropionate we did not observe changes in TBARS results, demonstrating that the degree of
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lipid peroxidation was not affected. Thus, the use of imidocarb dipropionate possibly leads to a
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controlled ROS response in order to avoid host damage.
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In this study, animals treated with imidocarb dipropionate showed absence of bacteria in
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the blood on day 10 of the experiment. However, a recent study found that imidocarb-treated
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calves remained PCR positive, i.e. the treatment reliably eliminated persistent A. marginale
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infections in cattle [29]. In the literature, other alternatives for disease control has emerged as the
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development of a cell culture system for A. marginale provides a potential source of antigen for
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the development of improved killed and live vaccines, and the availability of cell culture-derived
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antigen would eliminate the use of cattle in vaccine production [30], and thus the development of
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new strategies for control and prevention of bovine anaplasmosis [31]. In conclusion, the use of imidocarb dipropionate on cattle naturally infected by A.
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marginale reduced the ADA activity by interfering adenosine regulation, as well as decreased the
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AChE activity, demonstrating modulation of the inflammatory system. Also it caused increase in
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SOD and CAT activities, enzymes involved in the antioxidant metabolism against free radicals.
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These results demonstrated a possible mechanism of action with immunomodulation and
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regulatory effect on antioxidant activities of cattle as a result of parasitic infection followed
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imidocarb dipropionate treatment, however, more studies are needed to define the mechanisms of
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action of this drug.
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Ethics Committee
The study protocol was approved by the Ethics and Animal Welfare Committee of
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Veterinary Research Institute Desidério Finamor under rotocol 01/2011 - CEUA/IPVDF.
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[25] Lau, A.T.; Wang, Y.; Chiu, J.F. 2008. Reactive oxygen species: current knowledge and
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applications in cancer research and therapeutic. J. Cell. Biochem. 104, 657–667.
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[26] Esterbauer, H. 1993. Cytotoxicity and genotoxicity of lipid-oxidation products. Am. J. Clin.
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Nutr. 57, 779-785.
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[27] Baldissera, M.D., Gonçalves, R.A., Sagrillo, M.R., Grando, T.H., Ritter, C.S., Grotto, F.S.,
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Brum, G.F., Da Luz, S.C.A., Silveira, S.O., Fausto, V.P., Boligon, Aline A., Vaucher, R.A.,
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Stefani, L.M., Da Silva, A.S., Souza, C.F., Monteiro, S.G. 2016. Effects of treatment with the
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anti-parasitic drug diminazene aceturate on antioxidant enzymes in rat liver and kidney. N-S
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Arch. Pharmacol. 389, 429-438.
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[28] Saleh, M.A. 2009. Erythrocytic oxidative damage in crossbred cattle naturally infected with
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Babesia bigemina. Res. Vet. Sci. 86, 43-48.
339
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[29] Alberton L.R., Orlandini C.F., Zampieri T.M. , Nakamur A.Y., Gonçalves D.D.,
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Piau Júnior R., Zaniolo M.M., Cardim S.T., Vidotto O., Garcia J.L. 2015. Efficacy of imidocarb
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dipropionate, enrofloxacin and oxytetracycline chlorydrate on the treatment of cattle naturally
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infected by Anaplasma marginale. Arq. Bras. Med. Vet. Zootec. 67, 1056-1062.
344 [30] Kocan K.M., de la Fuente J, Guglielmone A.A., Meléndez R.D. 2003. Antigens and
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alternatives for control of Anaplasma marginale infection in cattle. Clin. Microbiol. Rev. 16,
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698-712.
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[31] Kocan K.M., de la Fuente J., Blouin E.F., Coetzee J.F., Ewing S.A. 2010. The natural
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history of Anaplasma marginale. Vet. Parasitol. 167, 95-107.
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Legends for figures
367 Figure 1: Hematocrit of cattle naturally infected by Anaplasma marginale on day 0 of the
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experiment. Groups A (n=11) and B (n=11) treated with imidocarb dipropionate on day 6 and 0
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of the experiment, respectively. There is no significant difference between groups (P>0.05;
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Mann-Whitney U). Reference values for hematocrit were 24 - 46%, according to Thrall et al.
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[15].
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Figure 2: Activities of acetylcholinesterase (AChE), adenosine deaminase (ADA), catalase
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(CAT), and superoxide dismutase (SOD) in cattle naturally infected by Anaplasma marginale.
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Groups A and B treated with imidocarb dipropionate on day 6 and 0 of the experiment,
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respectively. Group analysis over time with statistical difference at P<0.05. (Mann-Whitney U)
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was considered. Results showed as median and minimum-maximum.
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Table 1: Median and minimum-maximum of variables acetylcholinesterase (AChE), butyrylcholinesterase (BChE), adenosine deaminase (ADA), Thiobarbituric Acid Reactive Substances (TBARS), catalase (CAT) and dismutase superoxide (SOD) in
dipropionate. Group A
AChE mU/µmol/Hb
0 5 10
1193 (669.4-1586.85) 1307.63 (809.09-1844.71) 1173.12 (64.81-1881.43)
BChE µmoles/BcSCh/h/mg/protein
0 5 10
ADA U/L
0 5 10
TBARS nmol/MAD/mL
0 5 10
CAT nmol/CAT/mg/protein
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SOD UI/SOD/mg/protein
Group B
P value
1100 (598.82-2973.42) 1341.51 (794.45-1992.53) 902.43 (48.83-1759.44)
0.82 0.66 0.14
48.23 (38.88-52.58) 48.23 (38.88-54.54) 50.92 (45.38-57.39)
49.14 (40.2-60.01) 49.76 (40.20-60.01) 49.04 (40.69-77.09)
0.25 0.21 0.13
34.93 (2.19-79.41) 33.52 (8.39-65.83) 11.75 (2.46-53.08)
31.73 (20.15-93.92) 20.36 (12.06-52.20) 18.23 (3.67-39.20)
0.74 0.02* 0.19
9.51 (7.88-11.13) 8.88 (8.11-11.45) 9.32 (5.88-10.07)
9.18 (7.23-10.68) 8.87 (7.79-11.75) 8.96 (7.75-10.77)
0.28 0.70 0.72
0 5 10
3.05 (2.4-4.98) 4.84 (2.73-5.48) 4.98 (4.66-5.62)
2.90 (1.12-5.75) 4.70 (4.56-5.40) 4.84 (4.73-5.30)
0.25 0.26 0.86
0 5 10
7.07 (3.91-13.51) 13.41 (5.57-16.66) 16.53 (3.25-27.04)
5.21 (1.44-12.08) 11.34 (2.77-35.80) 14.92 (3.15-29.04)
0.23 0.87 0.90
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serum of bovines naturally infected by Anaplasma marginale treated with imidocarb
*Values with P<0.05 were considered statistically different between groups of some
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Highlights - Cattle naturally infected by Anaplasma marginale and treated with imidocarb dipropionate.
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- Post-treatment there was a reduction in the ativity of acetilcolinestarase and adenosine deaminase activities.
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- Post-treatment also caused an increase in catalase and superoxide dismutase activities.
S0882-4010(16)30231-5
DOI:
10.1016/j.micpath.2016.06.001
Reference:
YMPAT 1849
To appear in:
Microbial Pathogenesis
Received Date: 1 May 2016 Revised Date:
22 May 2016
Accepted Date: 1 June 2016
Please cite this article as: Doyle RL, Fritzen A, Bottari NB, Alves MS, da Silva AD, Morsch VM, Schetinger MRC, Martins JR, Santos JS, Machado G, da Silva AS, Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: Effects on enzymes of the antioxidant, cholinergic, and adenosinergic systems, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.06.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Imidocarb dipropionate in the treatment of Anaplasma marginale in cattle: effects on
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enzymes of the antioxidant, cholinergic, and adenosinergic systems
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Rovaina L. Doylea*, Alexandro Fritzenb, Nathieli B. Bottaric, Mariana S. Alvesc, Aniélen D. da
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Silvac, Vera M. Morschc, Maria Rosa C. Schetingerc, João R. Martinsa, Julsan S. Santosa,
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Gustavo Machadod, Aleksandro S. da Silvab,c*
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a
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Brazil.
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b
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de Santa Catarina (UDESC), Chapecó, SC, Brazil.
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c
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Molecular Biology, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brazil.
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d
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Medicine, University of Minnesota, St. Paul, MN, USA.
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Graduate Program in Animal Science, Department of Animal Science, Universidade do Estado
Graduate Program in Toxicology and Biochemistry, Department of Biochemistry and
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STEMMA laboratory, Department of Veterinary Population Medicine, College of Veterinary
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Instituto de Pesquisas Veterinárias Desidério Finamor (FEPAGRO), Eldorado do Sul, RS,
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*Author for correspondence: Department of Animal Science, University of Santa Catarina State
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(UDESC). 680 D, Beloni Trombeta Zanin Street, Chapecó/SC, Brazil Zip: 89815-630, Phone: 55
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49 3322-4202. Fax: 55 49 3311-9316. (E-mail: [email protected];
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[email protected]).
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ABSTRACT Anaplasmosis is a worldwide hemolytic disease in cattle caused by a gram-negative
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obligatory intracellular bacterium, characterized by anemia and jaundice. Among the treatments
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used for anaplasmosis is a drug called imidocarb dipropionate, also indicated as an
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immunomodulator agent. However, it causes side effects associated with increased levels of
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acetylcholine. In view of this, the effects of imidocarb dipropionate on the purinergic system,
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and antioxidant enzymes in animals naturally infected by Anaplasma marginale were evaluated.
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Young cattle (n=22) infected by A. marginale were divided into two groups: the Group A
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consisted of 11 animals used as controls; and the Group B composed of 11 animals. Imidocarb
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dipropionate (5 mg/kg) was used subcutaneously to treat both groups (the Group A on day 6 and
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the Group B on day 0). The treatment reduced acetylcholinesterase (AChE), and adenosine
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deaminase (ADA) activities, and increased the dismutase superoxide and catalase activities. No
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changes on lipid peroxidation (TBARS levels) and BChE activities were noticed. These results
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suggest that imidocarb dipropionate used to treat A. marginale infection in cattle has effect on
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antioxidant enzymes, and significantly inhibits the enzymatic activities of ADA and AChE.
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Keywords: Anaplasma marginale, AChE, ADA, CAT, immunomodulation, SOD.
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1. INTRODUCTION
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Anaplasmosis is a hemolytic disease that affects mainly cattle, and it is caused by a gram-
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negative obligate intracellular bacterium named Anaplasma marginale [1]. This disease occurs in
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tropical and subtropical regions worldwide and is endemic in Mexico, Central and South
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America, resulting in huge economic losses to the cattle industry [2]. Disease transmission
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occurs through mechanical vectors comprising sucking flies and fomites contaminated with
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blood, in addition to biological vectors such as ticks [3, 4]. Prepatent period depends on the
48
infectious dose and host immune status, ranging from 7 to 60 days (28 days on average), causing
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mild to severe clinical signs [5]. Infected erythrocytes are phagocytosed by reticuloendothelial
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cells, leading to varying degrees of anemia, jaundice, and hemoglobinuria [6]. During infection
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progress, cattle may show fever, weight loss, lethargy, abortion, jaundice, and sudden death
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specially in animals older than 2 years [7].
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Treatment with imidocarb dipropionate has been proved to be successful against
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hemolytic diseases such as anaplasmosis, requiring only one dose for clinical signs remission.
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However, this drug causes many side effects such as salivation, lacrimation, tachypnea,
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tachycardia, and pain at the injection site, as a result of massive acetylcholine accumulation [8].
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Acetylcholine (Ach) is the main vagal neurotransmitter and has important anti-inflammatory
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effect. It is hydrolyzed by the enzyme acetylcholinesterase, which is an intrinsic regulator of
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inflammation [9]. These clinical changes may cause oxidative stress, characterized by excess of
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production or inadequate removal of reactive oxygen species (ROS), and reactive nitrogen
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molecules [10]. These ROS molecules are involved in lipid peroxidation processes, protein
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oxidation, and damage to DNA, which is one of the mechanisms used by cells of the immune
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system to eradicate infections. ROS reactions are enhanced by the presence of iron, which makes
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red blood cells easy targets for ROS reactions, providing higher levels of lipid peroxidation in
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these cells, exacerbating hemolysis [11]. The persistence presence of intracellular bacteria is
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possible due to ROS production, able to prevent immune system responses. An example of this
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process is shown in the avoidance mechanism by Anaplasma phagocytophilum, which blocks the
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incorporation of p22 and gp91 into the phagolysosome, reducing the oxidative activity [12].
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The adenosinergic system plays an important role on immune and inflammatory
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responses due to the regulation of adenine nucleoside, adenosine. Adenosine deaminase (ADA)
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is considered a key enzyme in the adenosinergic system, being responsible for the irreversible
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deamination of adenosine, controlling its extracellular levels. The anti-inflammatory effects of
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extracellular adenosine are modulated by ADA activity [13], and can be affected by
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chemotherapy. The metabolic waste of the drug are deposited in the liver and kidney for a long
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period, which may result in necrosis of these organs, compromising physiological functioning of
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other systems [14]. Therefore, the aim of this study was to evaluate the effects of treatment with
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imidocarb dipropionate on the cholinergic systems, antioxidant enzymes, and adenosine
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deaminase in cattle naturally infected by A. marginale.
79 2. MATERIALS AND METHODS
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2.1. Animals
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Young cattle (n=22, average of 1.5 years, and 276 kg of body weight) part of a herd from
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the Veterinary Research Institute Desidério Finamor, Eldorado do Sul, South of Brazil, were
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used in this study. The animals were kept in an extensive system with access to native pasture
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and water ad libitum. A. marginale was found in blood smears of all 22 animals at day 0 of the
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experiment, i.e. this study used cattle naturally infected by A. marginale. These animals were
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divided into two groups: the Group A consisting of 11 animals used as control animals for all
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analyzes performed on day 5 of the experiment, and treated subcutaneously on day 6 with
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imidocarb dipropionate as recommendated by the manufacturer (5 mg/kg). The Group B was
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formed by 11 bovines, treated subcutaneously on day 0 with imidocarb dipropionate (same
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dose). Therefore, animals from the Group A were used as control for the analysis performed on
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day 5 post-treatment of the Group B in order to evaluate treatment response. Blood smears were
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performed at the end of the experiment (day 10) for post- treatment parasitemia evaluation.
94 2.2. Sampling
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For oxidative and antioxidant profile analyses, blood samples were collected at days 0, 5
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and 10 post-treatment, and allocated on two sterile tubes with anticoagulant (EDTA 10%, and
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sodium citrate) and one tube without anticoagulant. The tubes without anticoagulant were
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centrifugated at 3500 rpm for 10 min to obtain serum that was collected, and stored in eppendorf
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tubes at -20 °C up to analysis of TBARS, ADA, and BChE. Tubes with sodium citrate was
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homogenized and frozen at -20ºC for analysis of CAT and SOD. Blood samples with EDTA
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were diluted 1:50 (v/v) in lysis solution (0.1 mmol/L potassium/sodium phosphate buffer
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containing 0.03% Triton X-100) to determine AChE activity. Hematocrit [15] was also
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performed on day 0 in order to assess the clinical status of the animals related to anemia.
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2.3. Biochemical analysis
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2.3.1. Seric TBARS, BChE, and ADA
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Lipid peroxidation was determined based on the levels of thiobarbituric acid reactive
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substances (TBARS) in sera samples according to the method described by Jentzsch et al. [16].
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The results were obtained by spectrophotometry (535 nm) and expressed as nmol of
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malondialdehyde (MAD) per mL.
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To verify butyrylcholinesterase (BChE) activity in the sera the method described by
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Ellman et al. [17] was used, using butyrylthiocholine iodide (BcSCh) instead of acetylcholine,
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and the results were expressed in µmoles BcSCh/h/mg of protein.
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Adenosine deaminase (ADA) activity was measured spectrophotometrically in serum by
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the method of Giusti and Gakis and the reaction was started by the addition of adenosine
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substrate [18]. Ammonia concentration is directly proportional to the absorption of indophenol at
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650 nm, and the specific activity is reported as U/L in serum.
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2.3.2. AChE activity in total blood
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The AChE enzymatic assay in whole blood was determined by Ellman et al. [17] method,
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modified by Worek et al. [19]. The specific activity of whole blood AChE was calculated from
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the quotient between AChE activity and hemoglobin content, and the results were expressed as
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mU/µmol Hb.
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2.3.3. Catalase and superoxide dismutase activities
Determination of CAT activity was carried out in accordance to a modified method of
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Nelson and Kiesow [20]. This assay involved the change in absorbance at 240 nm due to CAT
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dependent decomposition of hydrogen peroxide. CAT activity was calculated using the molar
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extinction coefficient, and the results were expressed as nmol CAT per milligram protein.
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SOD activity measurement was based on the inhibition of radical superoxide reaction
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with adrenalin as described by McCord and Fridovich [21]. SOD activity is determined by
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measuring the speed of adrenochrome formation, observed at 480 nm, in a reaction medium
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containing glycine–NaOH and adrenaline. The results were expressed as UI SOD per milligram
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of protein.
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2.4. Statistical analysis
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The tests were performed in triplicate, and the average used for statistical analysis. All data were
139
analyzed first descriptively. Further all variables were submitted to Shapiro Wilk’s test for
140
normally distribution verification, since most of the variables did not met assumption of
141
parametric testing, it was used a nonparametric test for two independence groups test Friedman
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test to evaluate the influence of time of the experiment on the measured parameters (AChE,
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BChE, ADA, TBARS, CAT, and SOD), and when the differenceover time was present was
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used Mann-Whitney U. Also analyzed the changes in parameters over the study period
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considering the groups (A and B) and in addition was observed difference between two
146
independent groups by Mann-Whitney U for every moment of collection (day 0; day 5 and day
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10) as analysis. Critical P value of <0.05 was used. All analyzes were performed using the R
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software v.2.15.2.
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3. RESULTS
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Hematocrits performed on day 0 did not show differences between groups (Figure 1).
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Anemia was not detected despite A. marginale infection, since hematocrit results were normal.
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When sampling day was considered, the only significant difference observed was between
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groups A and B regarding ADA activity on day 5 of the experiment (Table 1). All other
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parameters and sampling time analyzed did not show any significant differences between groups
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(Table 1).
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There was influence of time on several measured parameters throughout the experiment,
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for example on AChE (P<0.001), and this was observed between day 0 and 10 for the group B
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(P=0.01) with reduced AChE activity (Figure 2). On the other hand, BChE was not influnced
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over time for both groups (P=0.86). For ADA, there was a significant influence of time
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(P<0.001), and this difference (reduced activity of ADA) was between day 0 and 10 (P=0.004)
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and day 5 and 10 (P=0.01) for the group A; and, a marked decrease was observed on ADA
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activity from day 0 to 10 (P=0.003) and from day 5 to 10 (P=0.01) for the group B (Figure 2).
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For TBARS, no influence of time was observed on both groups (P=0.75). However, for SOD
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there was influence of time (P<0.001), and this difference (increased SOD activity) was between
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day 0 and 5 (P=0.0004) and day 0 and 10 (P<0.001) for the group A; and a high increase on SOD
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activity in the group B from day 0 to 5 (P<0.001) and from day 0 to 10 (P=0.004) (Figure 2).
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Finally, for CAT there was influence of time (P<0.001), and this difference (i.e. increased
170
activity) was between day 0 and 5 (P<0.001) and day 0 and 10 (P<0.001) for the group A; and,
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with a high increase on CAT activity in the group B from day 0 to 5 (P<0.001) and from day 0 to
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10 (P<0.001) (Figure 2). On day 10 of the experiment, all treated animals were negative for A.
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marginale by blood smear.
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4. DISCUSSION
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After imidocarb dipropionate treatment, there was a reduction on seric levels of ADA in
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animals from the Group B compared to those from the Group A, and this reduction was also
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observed in the Group A after imidocarb injection on day 5 of experiment. The reduction on
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ADA seric activity lead to increased adenosine, which in turn has antinflammatory activities and
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acts regulating the growth, differentiation, and proliferation of lymphocytes and erythrocytes
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[13]. In studies conducted by Katayama et al. [22], it was observed that treatment with imidocarb
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dipropionate in the presence of LPS (lipopolysaccharide) led to increase dose dependent IL-10
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production by activated macrophages, and a reduction on TNF-α, IL-12, and nitric oxide,
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providing anti-inflammatory and immunomodulatory effects. Therefore, the stimulation of
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adenosine A2A receptors may lead to similar response in the event of increased IL-10 levels, and
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reduced pro-inflammatory cytokines, which demonstrates a response mechanism promoted by
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imidocarb, since its use reduces ADA activity, an enzyme with regulatory effect on extracellular
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levels of adenosine. A recent study evaluated NTPDase and 5'-nucleotidase activities in platelets
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of bovines infected by A. marginale with and without spleens, and observed increased levels of
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degradation of the nucleotides ATP and ADP by NTPDase, in addition to AMP by 5'nucleotidase
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to form the nucleoside adenosine in platelets of splenectomized animals compared to non-
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splenectomized [23], therefore during A. marginale infection, there are alterations in the
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enzymatic cascade of the purinergic system, as in the adenosinergic system mentioned in this
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study.
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Imidocarb dipropionate may cause side effects, which are related to increased levels of
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acetylcholine, a parasympathetic neurotransmitter [8]. It was observed that the use of imidocarb
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dipropionate in cattle led to reduced AChE activity, but had no effect on BChE enzymes which
198
associated with acetylcholine degradation. The cholinergic anti-inflammatory pathway is
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composed of an efferent vague nerve with acetylcholine as the main neurotransmitter and the α7
200
nicotinic acetylcholine as its receptor, when activated reduces the release of proinflammatory
201
cytokines [24]. The control of extracellular levels of acetylcholine is carried out by AChE and by
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BChE in cases of high levels of ACh [9]. Reduction in AChE activity, as observed by the use of
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imidocarb dipropionate, possibly induces higher seric levels of ACh, which contributes to the
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modulation of cholinergic inflammatory response, as a possible route of immunomodulation
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effect of imidocarb dipropionate.
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Formation of reactive oxygen species (ROS) are an important mechanism against
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infectious agents, however their actions may cause damage to the host. Control of ROS levels is
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performed by enzymes, such as CAT and SOD, able to prevent cell damage and thus, they are
209
considered important markers of oxidative stress [12, 25]. One of the actions of ROS may cause
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lipid peroxidation, which is a reaction measured by TBARS levels [26], but this variable showed
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no changes in this study current. The use of imidocarb dipropionate led to increased SOD and
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CAT activities, possibly in response to the increase of ROS metabolism. However, this current
213
study was unable to determine whether this increase was related to the infection and/or
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treatment. Drug administration, as aceturate diminazene (a diamidine, as imidocarb dipropionate)
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can increase ROS production, however high levels of it may induce tissue damage and host cell
216
death [27], and low levels may induce the production of free radicals which is a form of evasion
217
found by pathogens that produce persistent infections [12]. Lipid peroxidation can occurr by A.
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marginale infection [5] and/or hemolysis [28], however after application of imidocarb
219
dipropionate we did not observe changes in TBARS results, demonstrating that the degree of
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lipid peroxidation was not affected. Thus, the use of imidocarb dipropionate possibly leads to a
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controlled ROS response in order to avoid host damage.
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In this study, animals treated with imidocarb dipropionate showed absence of bacteria in
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the blood on day 10 of the experiment. However, a recent study found that imidocarb-treated
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calves remained PCR positive, i.e. the treatment reliably eliminated persistent A. marginale
225
infections in cattle [29]. In the literature, other alternatives for disease control has emerged as the
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development of a cell culture system for A. marginale provides a potential source of antigen for
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the development of improved killed and live vaccines, and the availability of cell culture-derived
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antigen would eliminate the use of cattle in vaccine production [30], and thus the development of
229
new strategies for control and prevention of bovine anaplasmosis [31]. In conclusion, the use of imidocarb dipropionate on cattle naturally infected by A.
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marginale reduced the ADA activity by interfering adenosine regulation, as well as decreased the
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AChE activity, demonstrating modulation of the inflammatory system. Also it caused increase in
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SOD and CAT activities, enzymes involved in the antioxidant metabolism against free radicals.
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These results demonstrated a possible mechanism of action with immunomodulation and
235
regulatory effect on antioxidant activities of cattle as a result of parasitic infection followed
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imidocarb dipropionate treatment, however, more studies are needed to define the mechanisms of
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action of this drug.
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Ethics Committee
The study protocol was approved by the Ethics and Animal Welfare Committee of
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Veterinary Research Institute Desidério Finamor under rotocol 01/2011 - CEUA/IPVDF.
242 REFERENCES
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[1] Theiler, A., 1910. Anaplasma marginale (gen. and spec. nov). The marginal points in the
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blood of cattle suffering from a specific disease. In: Theiler, A. (Ed.), Report of the Government
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on Veterinary Bacteriology in Transvaal. 1908–1909, pp.7–64.
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Legends for figures
367 Figure 1: Hematocrit of cattle naturally infected by Anaplasma marginale on day 0 of the
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experiment. Groups A (n=11) and B (n=11) treated with imidocarb dipropionate on day 6 and 0
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of the experiment, respectively. There is no significant difference between groups (P>0.05;
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Mann-Whitney U). Reference values for hematocrit were 24 - 46%, according to Thrall et al.
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[15].
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Figure 2: Activities of acetylcholinesterase (AChE), adenosine deaminase (ADA), catalase
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(CAT), and superoxide dismutase (SOD) in cattle naturally infected by Anaplasma marginale.
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Groups A and B treated with imidocarb dipropionate on day 6 and 0 of the experiment,
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respectively. Group analysis over time with statistical difference at P<0.05. (Mann-Whitney U)
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was considered. Results showed as median and minimum-maximum.
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Table 1: Median and minimum-maximum of variables acetylcholinesterase (AChE), butyrylcholinesterase (BChE), adenosine deaminase (ADA), Thiobarbituric Acid Reactive Substances (TBARS), catalase (CAT) and dismutase superoxide (SOD) in
dipropionate. Group A
AChE mU/µmol/Hb
0 5 10
1193 (669.4-1586.85) 1307.63 (809.09-1844.71) 1173.12 (64.81-1881.43)
BChE µmoles/BcSCh/h/mg/protein
0 5 10
ADA U/L
0 5 10
TBARS nmol/MAD/mL
0 5 10
CAT nmol/CAT/mg/protein
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SOD UI/SOD/mg/protein
Group B
P value
1100 (598.82-2973.42) 1341.51 (794.45-1992.53) 902.43 (48.83-1759.44)
0.82 0.66 0.14
48.23 (38.88-52.58) 48.23 (38.88-54.54) 50.92 (45.38-57.39)
49.14 (40.2-60.01) 49.76 (40.20-60.01) 49.04 (40.69-77.09)
0.25 0.21 0.13
34.93 (2.19-79.41) 33.52 (8.39-65.83) 11.75 (2.46-53.08)
31.73 (20.15-93.92) 20.36 (12.06-52.20) 18.23 (3.67-39.20)
0.74 0.02* 0.19
9.51 (7.88-11.13) 8.88 (8.11-11.45) 9.32 (5.88-10.07)
9.18 (7.23-10.68) 8.87 (7.79-11.75) 8.96 (7.75-10.77)
0.28 0.70 0.72
0 5 10
3.05 (2.4-4.98) 4.84 (2.73-5.48) 4.98 (4.66-5.62)
2.90 (1.12-5.75) 4.70 (4.56-5.40) 4.84 (4.73-5.30)
0.25 0.26 0.86
0 5 10
7.07 (3.91-13.51) 13.41 (5.57-16.66) 16.53 (3.25-27.04)
5.21 (1.44-12.08) 11.34 (2.77-35.80) 14.92 (3.15-29.04)
0.23 0.87 0.90
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*Values with P<0.05 were considered statistically different between groups of some
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Highlights - Cattle naturally infected by Anaplasma marginale and treated with imidocarb dipropionate.
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- Post-treatment there was a reduction in the ativity of acetilcolinestarase and adenosine deaminase activities.
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- Post-treatment also caused an increase in catalase and superoxide dismutase activities.