- Email: [email protected]
Oxidative stress in dairy cows seropositives for Neospora caninum
Accepted Manuscript Title: Oxidative stress in dairy cows seropositives for Neospora caninum Authors: Patr´ıcia Glombowsky, Nathieli B. Bottari, Vanderlei Klauck, Juscivete F. F´avero, Natan M. Sold´a, Matheus D. Baldissera, Gessica Perin, Vera M. Morsch, Maria Rosa C. Schetinger, Lenita M. Stefani, Aleksandro S. Da Silva PII: DOI: Reference:
S0147-9571(17)30069-3 http://dx.doi.org/doi:10.1016/j.cimid.2017.07.007 CIMID 1156
To appear in: Received date: Revised date: Accepted date:
17-12-2016 17-7-2017 27-7-2017
Please cite this article as: Glombowsky Patr´ıcia, Bottari Nathieli B, Klauck Vanderlei, F´avero Juscivete F, Sold´a Natan M, Baldissera Matheus D, Perin Gessica, Morsch Vera M, Schetinger Maria Rosa C, Stefani Lenita M, Da Silva Aleksandro S.Oxidative stress in dairy cows seropositives for Neospora caninum.Comparative Immunology, Microbiology and Infectious Diseases http://dx.doi.org/10.1016/j.cimid.2017.07.007 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.
Oxidative stress in dairy cows seropositives for Neospora caninum
Patrícia Glombowskya, Nathieli B. Bottarib, Vanderlei Klaucka, Juscivete F. Fáveroa, Natan M. Soldáa, Matheus D. Baldisserac, Gessica Perina, Vera M. Morschb, Maria Rosa C. Schetingerb, Lenita M. Stefania, Aleksandro S. Da Silvaa,b*
a
Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó,
Santa Catarina (SC), Brazil. b
Department of Biochemistry and Molecular Biology, Universidade Federal de Santa Maria
(UFSM), Santa Maria, RS, Brazil. c
Departament of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM),
Santa Maria, RS, Brazil.
*Author for correspondence: Department of Animal Science, University of Santa Catarina State. 680 D, Beloni Trombeta Zanin Street, Chapecó/SC, Brazil Zip: 89815-630, Phone: 55 49 33224202. Fax: 55 49 3311-9316. (E-mail: [email protected]).
Abstract: Bovine neosporosis is caused by the protozoan Neospora caninum and is one of the major causes of abortion in cows. Cattle are intermediate hosts of this parasite and may have asymptomatic or symptomatic infections. Therefore, the aim of this study was to evaluate oxidative stress marker (reactive oxygen species (ROS), thiobarbituric reactive acid substances (TBARS) levels, glutathione S-transferase (GST), adenosine deaminase (ADA), and butyrylcholinesterase (BChE) activities in dairy cows seropositives for N. caninum (asymptomatic or symptomatic). Dairy cows (n=90) were tested by immunofluorescent antibody assay (IFA) for N. caninum and divided accordingly into
three groups: the group A (seronegatives, n=30), the group B (seropositives and asymptomatic, n=30), and the group C (seropositives and symptomatic, n=30). It was observed increased levels of TBARS and reduced (P<0.05) BChE activity in seropositives either asymptomatic or symptomatic animals. ROS levels and ADA activity increased, and GST activity decreased (P<0.05) only in seropositives symptomatic dairy cows (the group C) compared to seronegatives dairy cows (the group A). Based on these results, it was observed that seropositive animals showed cell damage associated with oxidative stress and inflammation, mainly in those with symptomatic infections. Increased seric ROS levels and BChE activity may have influenced N. caninum pathogenesis in symptomatic animals due to increased cell damage and exacerbated inflammatory response, leading to the development of clinical signs.
Keywords: neosporosis; cattle; inflammation; abortion; oxidative damage.
1. Introduction Bovine neosporosis is an important cause of abortion in cows [1]. This disease is caused by Neospora caninum, a protozoan transmitted horizontally by the consumption of infective oocysts or vertically by transplacental transmission of tachyzoites during pregnancy [2], which is considered the major mode of transmission in cattle [3]. Canids are definitive hosts and cattle, along with several mammals, are intermediate hosts of N. caninum [4]. This disease is characterized by embryo mortality, abortion, fetal mummification, stillbirth or birth of weak calves, or healthy animals but with persistent infections, which causes major losses for the cattle industry, mainly in dairy herds [5]. The main clinical sign observed in cows is abortion, but often the animals show no clinical signs, which favor the spread of disease due to transplacental transmission of the parasite [6].
Currently, the consequences of symptomatic and asymptomatic infections to cattle are not well known, however, some biochemical alterations have been reported in N. caninum seropositive asymptomatic goats, which contribute to the pathogenesis of the disease [7]. According to this author, seropositive asymptomatic goats showed elevated levels of nitric oxide (NO) and protein oxidation. Recently, study demonstrated a relationship between nitrate/nitrite (NOx) and oxidative stress [8]. Therefore, studies relating antioxidant/oxidant status and inflammatory markers should be performed to better understand their role in N. caninum infections in dairy cows. Oxidative stress is defined as an imbalance between free radicals and antioxidants in the organism due to increased production of reactive oxygen species (ROS), causing a reduction on antioxidants [9]. In physiological and metabolic processes, free radicals act as mediators of biochemical reactions, however, in pathological conditions, the excessive production of free radicals can cause damage to the organism, because these molecules can damage cells and tissues, contributing to the occurrence of clinical signs [10-12], as abortion in neosporosis. Adenosine deaminase (ADA) is an important enzyme that participates in neuromodulation, apoptosis, necrosis, and proliferation of lymphocytes during cellular response [13]. According to Franco et al. [14], ADA acts during inflammation in injured tissue, i.e., regulates the concentration of extracellular adenosine, an important molecule with anti-inflammatory properties, since this enzyme converts adenosine to inosine. Already, the butyrylcholinesterase (BChE) is an enzyme that participates in the cholinergic system, found mainly in the plasma, liver, spleen, and central nervous system [15]. According to Da Silva et al. [12], increased BChE activity is related to oxidative stress and chronic and acute inflammation, with important role in regulation of acetylcholine neurotransmitter. Alterations in these enzymes have been found during parasitic infections, including neosporosis [16]. According to Tonin et al. [7], ADA and BChE are considered important inflammatory markers during N. caninum infection.
Therefore, the aim of this study was to evaluate
the levels of free radicals, lipid
peroxidation, and glutathione-S-transferase, adenosine deaminase, and butyrylcholinesterase activities in dairy cows seropositives for N. caninum, using asymptomatic and symptomatic animals.
2. Material and Methods Frozen serum (- 20ºC) from adult’s dairy cows (Holstein) were used in this study. The animals are in semi-extensive farming (diet based on concentrate and free grazing), and present between 3 and 5 years of age, as well as all cows were in lactation (average of production 18.5 liters). All cows had a parasitological examination of feces negative for endoparasites, as well as being seronegative for Leptospira spp. Despite the history of abortion described in 30 dairy cows, all were apparently and clinically healthy at the time of sampling of these animals. It is important to emphasize that the blood samples collected from cows that aborted occurred within 15 days after the event. A total of 90 sera samples helped to classify the animals as seronegatives and seropositives animals using the indirect immunofluorescence assay (IFA). The samples were previously used on a study by Klauck et al. [17]. All sera samples positives for N. caninum at dilutions of 1:100 were subjected to maximum titration. Thus, three groups were formed: the group A consisted of seronegative animals (n=30 animals); the group B was formed by seropositive cows (titration 1:100 (n=30 animals)), but asymptomatic (neither neurological signs of the disease nor history of reproductive problems); and the group C was formed by cows seropositives for N. caninum (titrations: 1:200 (n=8 animals), 1:400 (n=12 animals), 1:800 (n=7 animals), 1:1600 (n=1 animals), and 1:3200 (n=2 animals)), and symptomatic (recent history of abortion - less than 15 days). According to the literature, there is a strong relationship between animals with a titers equal or
greater than 1:200 for N. caninum and occurred of abortion, that is, abortion was caused by the parasite [17]. Lipid peroxidation was determined based on the levels of Thiobarbituric Acid Reactive Substances (TBARS) in sera samples according to the method described by Jentzsch et al. [18]. The results were obtained by spectrophotometry (535 nm) and expressed as nmol of malondialdehyde per mL. Sera samples were prepared as described by Ali et al. [19] and Bass et al. [20] for the determination of ROS levels, and recently described briefly by Radavelli et al. [21]. ROS formation was quantified from a DCF standard curve in methanol (0.05-1.0 µM). GST activity was measured based on methods described by Habig et al. [22]. Freshly prepared cell lysate (25–50 μL) was mixed in a cuvette with 1 mL of GST assay buffer containing either 1 mM -chloro-2,4-dinitrobenzene (CDNB) and 1 mM reduced glutathione [GSH] or 1 mM 4nitrobenzylchloride and 5 mM GSH in 0.11 M sodium phosphate, pH 6.5, and incubated at room temperature. GST was calculated as nmol GS-DNB per min per mg of protein. Seric ADA activity was measured spectrophotometrically by the method of Giusti and Gakis [23]. The reaction was started by addition of the substrate (adenosine) to a final concentration of 21 mmol/L and incubated for 1 h at 37 °C. The specific activity was reported as U/L. The BChE enzymatic assay for sera samples was performed as described by Ellman et al. [24] using butyrylthiocholine as substrate. Sample analyses were carried out in duplicate, and the enzymatic activity was expressed in µmoles BcSCh/h/mg of protein. Data were analyzed initially by descriptive statistics. All variables were subjected to Shapiro-Wilk test for normality verification. Since most variables were normally distributed, the data was used for parametric tests (t test) for three independent groups for each sampling. The P value considered statistically different was <0.05.
3. Results and discussion Results for ROS and TBARS levels, and GST activity is shown in Table 1. Seropositive and symptomatic dairy cows (the group C) showed higher levels of seric ROS (p<0.05) compared to seronegative animals (the group A). TBARS levels were increased on seropositive asymptomatic animals (the group B) and seropositive symptomatic animals (the group C) compared to seronegatives (the group A) (p<0.05). Seropositive symptomatic dairy cows (the group C) showed lower seric GST activity (p<0.05) compared to seronegative animals (the group A). Results of ADA and BChE activities are shown in Table 2. ADA activity increased (p<0.05) on seropositive symptomatic dairy cows (the group C) compared to seronegative dairy cows (the group A). BChE activity decreased (p<0.05) on seropositive asymptomatic and symptomatic dairy cows (groups B and C, respectively) compared to seronegative dairy cows (the group A). In this study, changes in the oxidative stress markers and in the enzymes involved in the regulation of acetylcholine (BChE) and adenosine (ADA) levels were found in animals seropositives for N. caninum; alterations that may contribute directly to the pathogenesis of neosporosis. Oxidative stress was evident as indicated by higher levels of lipid peroxidation biomarker (MDA) and inhibition of antioxidant enzyme GST due to high levels of ROS, such as observed in seropositive symptomatic dairy cows. The accumulation of MDA is an indicative of the excessive formation of free radicals, oxidative stress and damage, which contribute directly to the pathogenesis of disease [25]. In a number of studies, it has been demonstrated that host cells infected with different species of parasites show increased amounts of ROS, which causes increased lipoperoxidation, and consequently cell and tissue damage [26, 27]. Recent studies have demonstrated the oxidative effects caused by N. caninum in cows and rodents, but they did not related to clinical signs of neosporosis. For example, studies conducted by Fidan et al. [28] and
Tonin et al. [16] demonstrated that animals infected by N. caninum showed elevated levels of TBARS, nitric oxide and protein oxidation, similarly to those observed in this present study. In this study, seropositive symptomatic dairy cows showed decreased GST activity concomitantly with increased ROS levels. Antioxidant enzymes, such as GST, protect the animals against oxidative stress and is able to scavenge the excess ROS generated, and it can be used as an indicator of the ROS production [29,30]. Also, GST inactivates lipoperoxidation products, such as MDA content, and inactivates ROS via SH groups [31]. Based on these evidences, we concluded that increased ROS levels observed in seropositive symptomatic animals occur to due an impairment of GST activity caused by the infection. The decreased GST activity in this group could be explained since glutathione (GSH) was largely consumed as a substrate, and many intermediate metabolites were produced in the process of detoxification, which could reduce GST activity or competitively inhibit GST substrates [32]. It is important to emphasize that alterations on the enzymes of the glutathione family, such as glutathione reductase (GR) have been observed during N. caninum infections [16]. Also, decreased GST activity may be considered an indicator of increased ROS production in animals N. caninum seropositives symptomatic. BChE activity was reduced in N. caninum seropositive asymptomatic and symptomatic dairy cows. This result possibly occurs due to hepatic damage observed during neosporosis, since BChE is synthetized by hepatocytes [33], as well as observed by Tonin et al. [16]. Recently, a study demonstrated that mice infected by T. gondii, other important protozoan, showed decreased seric BChE activity associated with liver injury [12]. Some studies have demonstrated the relation between alterations on cholinergic system, including BChE activity, and fetal mummification and abortion [34,35]. Thus, we believe that alteration on BChE activity contributes to pathophysiology of neosporosis, and it may contribute to abortion on seropositive asymptomatic or symptomatic dairy cows.
Increased ADA activity in dairy cows seropositives symptomatic for neosporosis was observed. A study conducted by Abbrachio and Ceruti [36] demonstrated that reduction on ADA activity contributes to limit the inflammatory response and subsequent cellular damage, since it would increase adenosine tissue levels, an anti-inflammatory molecule, conferring protection to the host tissue. Thus, we believe that increased ADA activity leads to decreased adenosine levels, which contributes to the inflammatory process and tissue damage, because adenosine protects host cells from excessive tissue injury associated with severe inflammation. Also, there are evidences that elevated levels of adenosine potently down-regulates the activation of lymphocytes during inflammation, playing a regulatory role on neutrophils in immune responses [37,38]. These results clarify the need to check all animals, regardless of the presence of clinical signs, since these asymptomatic animals may have lower performance due to imperceptible cell damage. Therefore, based on the results obtained and discussed here, it is possible to assure that seropositive dairy cows showed tissue damage associated with oxidative stress and inflammation, mainly those that were symptomatic for neosporosis. The increase of ROS levels and BChE activity in sera samples may have influenced the pathogenesis of N. caninum infection in symptomatic animals due to increased cell damage and exacerbated inflammatory response, respectively, leading to the development of clinical signs.
Ethics Committee The experiment was approved by the Ethics Committee in Animal Research at the University of Santa Catarina State (UDESC).
Conflict of Interest: The authors declare that they have no conflict of interest.
References [1] McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473-1478. [2] Anderson, M.L., Andrianarivo, A.G., Conrad, P.A., 2000. Neosporosis in cattle. Anim. Reprod. Sci. 60, 417-431. [3] Schares G., Peters M., Wurm R., Barwald A., Conraths F.J., 1998. The efficiency of vertical transmission of Neospora caninum in dairy cattle analyzed by serological techniques. Vet. Parasitol. 80, 87-98. [4] Dubey J.P., Schares G., Ortega-Mora L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbial. Rev. 20, 323-367. [5] Innes A.E., Andrianarivo G., Bjorkman C., Williams D.J.L., Conrad P.A., 2002. Immune responses to Neospora caninum and prospects for vaccination. Trends Parasitol. 18, 497-504. [6] Dubey J.P., Buxton D., Wouda W., 2006. Pathogenesis of Bovine Neosporosis. J. Comp. Path. 134, 267-289. [7] Tonin A.A., Weber A., Ribeiro A., Camilo G., Vogel F.F., Moura A.B., Bochi G.V., Moresco R.N., Da Silva A.S., 2015. Serum levels of nitric oxide and protein oxidation in goats seropositive for Toxoplasma gondii and Neospora caninum. Comp. Immunol. Microbiol. Infect. Dis. 41, 55-58. [8] Da Silva A.S., Paim F.C., Santos R.C., Sangoi M.B., Moresco R.N., Lopes S.T., Jaques J.A., Baldissarelli J., Morsch V., Monteiro S.G., 2012. Nitric oxide level, protein oxidation and antioxidant enzymes in rats infected by Trypanosoma evansi. Exp. Parasitol. 132, 166-170. [9] Winterboun C.C., Hampton M.B., 2015. Redox biology: signaling via a peroxiredoxin sensor. Nat. Chem. Biol. 11, 5-6. [10] Barbosa K.B.F, Costa N.M.B., Alfenas R.C.G., De Paula S.O., Minim V.P.R., Bressan J., 2010. Estresse oxidativo: conceito, implicações e fatores modulatórios. Rev. Nutr. 23, 629-643.
[11] Bouwstra R.J., Nielen M., Newbold J.R., Jansen E.H., Jelinek H.F., Van Werven T., 2010. Vitamin e supplementation during the dry period in dairy cattle. Oxidative stress following vitamin e supplementation may increase clinical mastitis incidence postpartum. J. Dairy. Sci. 93, 5696-5706. [12] Da Silva A.S., Munhoz T.D., Faria J.L., Vargas-Hérnandez G., Machado R.Z., Almeida T.C., Moresco R.N., Stefani L.M., Tinucci-Costa M., 2013. Increase nitric oxide and oxidative stress in dogs experimentally infected by Ehrlichia canis: Effect on the pathogenesis of the disease. Vet. Microbiol. 4, 366-369. [13] Da Silva, A.S., Bellé, L.P., Bitencourt, P.E., Souza, V.C., Costa, M.M., Oliveira, C.B., Jaques, J.A., Leal, D.B., Moretto, M.B., Maz zanti, C.M., Lopes, S.T., Monteiro, S.G., 2011. Activity of the enzyme adenosine deaminase in serum, erythrocytes and lymphocytes of rats infected with Trypanosoma evansi. Parasitol. 138, 201 – 208. [14] Franco, R., Casadó, V., Ciruela, F., Saura C., Malloi, J., Canela, El., Lluis, C., 1997. Cell surface adenosine deaminase: much more than an ectoenzyme. Prog. Neurobiol. 52 (4), 283–294. [15] Kaplay, S.S., 1976 Acetylcholinesterase and butyrylcholinesterase of developing human brain. Biol. Neonate. 28, 65-73. [16] Tonin, A.A., Da Silva, A.S., Thomé, G.R., Oliveira, L.S., Schetinger, M.R., Morsch, V.M., Flores, M.M., Fighera, R.A., Toscan, G., Vogel, F.F., Lopes, S.T.A., 2013. Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils. Exp. Parasitol. 135 (4), 669-674. [17] Klauck, V., Machado, G., Pazinato, R., Radavelli, W.M., Santos, D.S., Berwaguer, J.C., Braunig, P., Volgel F.F., Da Silva, A.S., 2016. Relation between Neospora caninum and abortion in dairy cows: Risk factors and pathogenesis of disease. Microb. Pathog. 92, 46-49. [18] Jentzsch, A.M., Bachmann, H., Furst, P., Biesalski, H., 1996. Improved analysis of malondialdehyde in human body fluids. Free Rad. Biol. Med. 20 (2), 251–256.
[19] Ali, S.F., Lebel, C.P., Bondy, S.C., 1992. Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity. Neurotoxicol. 13 (3), 637-648. [20] Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C., Thomas, M., 1983. Flow cytometric studies of oxidative product formation by neurotrophils: a graded response to membrane stimulation. J. Immunol. 1130, 1910-1917. [21] Radavelli, W. M., Campigotto, G., Machado, G., Bottari, N.B., Bochi, G., Moresco, R.N., Morsch, V.M., Schetinger, M.R.C., Bianchi, A., Baldissera, M.D., Ferreira, R., Da Silva, A.S., 2016. Effect of lactation induction on milk production and composition, oxidative and antioxidant status and biochemical variables. Comp. Clin. Pathol. DOI: 10.1007/s00580-016-2243-z [22] Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249 (22), 7130-7139. [23] Giusti, G., Gakis, C., 1971. Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues. Enzyme. 12 (4), 417-442. [24] Ellman, G.L., Courtney, K.D., Andres V.J., Feather-stone R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biocherm. Phamacol. 7, 88-95. [25] Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41, 1819-1828. [26] Sarin, K., Kumar, A., Prskash, A., Sharma, A., 1993. Oxidative stress and antioxidant defense mechanism in Plasmodium vivax malaria before and after chloroquine treatment. Indian J. Malariol. 30, 127-133. [27] Deger, Y., Ertekin, A., Deger, S., Mert, H., 2008. Lipid peroxidation and antioxidant potential of sheep liver infected naturally with distomatosis. Turkiye Parazitoloji Dergisi 32, 23-36.
[28] Fidan, A.F., Cingi, C.C., Karafakioglu, Y.S., Utuk, A.E., Pekkaya, S., Piskin, F.C., 2010. The levels of antioxidant activity, malondialdehyde and nitric oxide in cows naturally infected with Neospora caninum. J. Animal Vet. Adv. 9, 1707-1711. [29] Holovska, J.K., Sobekova, B., Holovska, K., Lenartova, V., Javorsky, P., Legath, J., Legath, L., Maretta, M., 2005. Antioxidant and detoxifying enzymes in the liver of rats after subchronic inhalation of the mixture of cyclic hydrocarbons. Exp. Toxicol. Pathol. 56, 377-383. [30] Martins, F., Pereira. J.A., Baptista, P., 2014. Oxidative stress response of Beauveria bassiana to Bordeaux mixture and its influence on fungus growth and development. Pestic. Manag. Sci. 70, 1220-1227. [31] Choi, C.Y., An, K.W., An, M.I., 2008. Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus). Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 149, 330-337. [32] Eggas, E., Sandvik, M., Fjeld, E., Kallqvist, T., Goksoyr, A., Svensen, A., 1999. Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta). Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol. 122, 337-344. [33] Wescoe, W.C., Hunt, C.H., Riker, W.F., Litt, I.C., 1947. Regeneration rates of serum cholinesterase in normal individuals and in patients with liver damage. Am. J. Physiol. 149, 549551. [34] Sternfeld, M., Rachmilewitz, J., Lichtenstein-Loewenstein, Y., Andres, C., Timberg, R., BenAri, S., Glick, D., Soreq, H., Zakut, H., 1997. Normal and atypical butyrylcholinesterases in placental development, function and malfunction. Cell. Mol. Neurobiol. 17, 315-332. [35] Mahmoud, F.F., Abul, H.T., Haines, D.D., Omu, A.E., Diejomaoh, M., Wise J.A., Abu, Donia, M.B., 2008. Butyrylcholinesterase activity and lymphocyte subpopulations in peripheral blood of Kuwaiti women experiencing recurrent spontaneous abortion. J. Reprod. Immunol. 77, 186-194.
[36] Abbrachio, M.P., Ceruti, S., 2007. P1 receptors and cytokine secretion. Purinergic Signal. 3, 13-25. [37] Desrosiers, M.D., Cembrola, K.M., Fakir, M.J., Sthepens, L.A., Jama, F.M., Shameli, A., Mehal, W.Z., Santamaria, P., Shi, Y., 2007. Adenosine deamination sustains dendritic cell activation in inflammation. J. Immunol. 179, 1884-1892. [38] Mills, J.H., Kim, D.G., Krenz, A., Chen, J.F., Byone, M.S., 2012. A2S adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis. J. Immunol. 118, 5713-5722.
- Bovine neosporosis is an important cause of abortion in cows. - Seropositive dairy cows showed oxidative stress and increase of inflammation markers which indicate tissue damage. - The increase of ROS levels, ADA and BChE activities in sera samples may have influenced the pathogenesis of N. caninum. - Due to increased cell damage and exacerbated inflammatory response occurred the development of abortion. - ADA and BChE activities participates in immunomodulation.
Table 1: Seric levels of reactive oxygen species (ROS), thiobarbituric acid reactive species (TBARS), and glutathione S-transferase (GST) in seronegative dairy cattle (the group A, control), seropositive/asymptomatic dairy cattle (the group B), and seropositive/symptomatic dairy cattle (the group C) to Neospora caninum.
Group
ROS (U DCFA/μL)
TBARS (nmol MDA/mL)
GST (μmol/Cdnb/min)
A - control
2.26 ± 0.45b
6.58 ± 0.59b
87.8 ± 12.6a
B - seropositive/asymptomatic
3.26 ± 1.06ab
11.02 ± 1.74a
92.4 ± 8.95a
C - seropositive/symptomatic
4.15 ± 0.29a
12.78 ± 1.35a
36.4 ± 15.3b
P<0.05
P<0.05
P<0.05
Note: Means with equal letters, on the same column, do not differ statistically among themselves for 5 % of significance (P>0.05).
Table 2: Seric activities of adenosine deaminase (ADA) and butyrylcholinesterase (BChE) of seronegative dairy cattle (the group A, control), seropositive/asymptomatic dairy cattle (the group B), and seropositive/symptomatic dairy (the group C) to Neospora caninum.
Group
ADA (U/L)
BChE (μmol BuSCh/h/mg of protein)
A - control
14.8 ± 5.3b
66.3 ± 10.6a
B - seropositive/asymptomatic
15.7 ± 4.6b
46.5 ± 13.5b
C - seropositive/symptomatic
41.5 ± 18.9a
41.3 ± 8.3b
P<0.05
P<0.05
Note: Means with equal letters, on the same column, do not differ statistically among themselves for 5 % of significance (P>0.05).
S0147-9571(17)30069-3 http://dx.doi.org/doi:10.1016/j.cimid.2017.07.007 CIMID 1156
To appear in: Received date: Revised date: Accepted date:
17-12-2016 17-7-2017 27-7-2017
Please cite this article as: Glombowsky Patr´ıcia, Bottari Nathieli B, Klauck Vanderlei, F´avero Juscivete F, Sold´a Natan M, Baldissera Matheus D, Perin Gessica, Morsch Vera M, Schetinger Maria Rosa C, Stefani Lenita M, Da Silva Aleksandro S.Oxidative stress in dairy cows seropositives for Neospora caninum.Comparative Immunology, Microbiology and Infectious Diseases http://dx.doi.org/10.1016/j.cimid.2017.07.007 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.
Oxidative stress in dairy cows seropositives for Neospora caninum
Patrícia Glombowskya, Nathieli B. Bottarib, Vanderlei Klaucka, Juscivete F. Fáveroa, Natan M. Soldáa, Matheus D. Baldisserac, Gessica Perina, Vera M. Morschb, Maria Rosa C. Schetingerb, Lenita M. Stefania, Aleksandro S. Da Silvaa,b*
a
Department of Animal Science, Universidade do Estado de Santa Catarina (UDESC), Chapecó,
Santa Catarina (SC), Brazil. b
Department of Biochemistry and Molecular Biology, Universidade Federal de Santa Maria
(UFSM), Santa Maria, RS, Brazil. c
Departament of Microbiology and Parasitology, Universidade Federal de Santa Maria (UFSM),
Santa Maria, RS, Brazil.
*Author for correspondence: Department of Animal Science, University of Santa Catarina State. 680 D, Beloni Trombeta Zanin Street, Chapecó/SC, Brazil Zip: 89815-630, Phone: 55 49 33224202. Fax: 55 49 3311-9316. (E-mail: [email protected]).
Abstract: Bovine neosporosis is caused by the protozoan Neospora caninum and is one of the major causes of abortion in cows. Cattle are intermediate hosts of this parasite and may have asymptomatic or symptomatic infections. Therefore, the aim of this study was to evaluate oxidative stress marker (reactive oxygen species (ROS), thiobarbituric reactive acid substances (TBARS) levels, glutathione S-transferase (GST), adenosine deaminase (ADA), and butyrylcholinesterase (BChE) activities in dairy cows seropositives for N. caninum (asymptomatic or symptomatic). Dairy cows (n=90) were tested by immunofluorescent antibody assay (IFA) for N. caninum and divided accordingly into
three groups: the group A (seronegatives, n=30), the group B (seropositives and asymptomatic, n=30), and the group C (seropositives and symptomatic, n=30). It was observed increased levels of TBARS and reduced (P<0.05) BChE activity in seropositives either asymptomatic or symptomatic animals. ROS levels and ADA activity increased, and GST activity decreased (P<0.05) only in seropositives symptomatic dairy cows (the group C) compared to seronegatives dairy cows (the group A). Based on these results, it was observed that seropositive animals showed cell damage associated with oxidative stress and inflammation, mainly in those with symptomatic infections. Increased seric ROS levels and BChE activity may have influenced N. caninum pathogenesis in symptomatic animals due to increased cell damage and exacerbated inflammatory response, leading to the development of clinical signs.
Keywords: neosporosis; cattle; inflammation; abortion; oxidative damage.
1. Introduction Bovine neosporosis is an important cause of abortion in cows [1]. This disease is caused by Neospora caninum, a protozoan transmitted horizontally by the consumption of infective oocysts or vertically by transplacental transmission of tachyzoites during pregnancy [2], which is considered the major mode of transmission in cattle [3]. Canids are definitive hosts and cattle, along with several mammals, are intermediate hosts of N. caninum [4]. This disease is characterized by embryo mortality, abortion, fetal mummification, stillbirth or birth of weak calves, or healthy animals but with persistent infections, which causes major losses for the cattle industry, mainly in dairy herds [5]. The main clinical sign observed in cows is abortion, but often the animals show no clinical signs, which favor the spread of disease due to transplacental transmission of the parasite [6].
Currently, the consequences of symptomatic and asymptomatic infections to cattle are not well known, however, some biochemical alterations have been reported in N. caninum seropositive asymptomatic goats, which contribute to the pathogenesis of the disease [7]. According to this author, seropositive asymptomatic goats showed elevated levels of nitric oxide (NO) and protein oxidation. Recently, study demonstrated a relationship between nitrate/nitrite (NOx) and oxidative stress [8]. Therefore, studies relating antioxidant/oxidant status and inflammatory markers should be performed to better understand their role in N. caninum infections in dairy cows. Oxidative stress is defined as an imbalance between free radicals and antioxidants in the organism due to increased production of reactive oxygen species (ROS), causing a reduction on antioxidants [9]. In physiological and metabolic processes, free radicals act as mediators of biochemical reactions, however, in pathological conditions, the excessive production of free radicals can cause damage to the organism, because these molecules can damage cells and tissues, contributing to the occurrence of clinical signs [10-12], as abortion in neosporosis. Adenosine deaminase (ADA) is an important enzyme that participates in neuromodulation, apoptosis, necrosis, and proliferation of lymphocytes during cellular response [13]. According to Franco et al. [14], ADA acts during inflammation in injured tissue, i.e., regulates the concentration of extracellular adenosine, an important molecule with anti-inflammatory properties, since this enzyme converts adenosine to inosine. Already, the butyrylcholinesterase (BChE) is an enzyme that participates in the cholinergic system, found mainly in the plasma, liver, spleen, and central nervous system [15]. According to Da Silva et al. [12], increased BChE activity is related to oxidative stress and chronic and acute inflammation, with important role in regulation of acetylcholine neurotransmitter. Alterations in these enzymes have been found during parasitic infections, including neosporosis [16]. According to Tonin et al. [7], ADA and BChE are considered important inflammatory markers during N. caninum infection.
Therefore, the aim of this study was to evaluate
the levels of free radicals, lipid
peroxidation, and glutathione-S-transferase, adenosine deaminase, and butyrylcholinesterase activities in dairy cows seropositives for N. caninum, using asymptomatic and symptomatic animals.
2. Material and Methods Frozen serum (- 20ºC) from adult’s dairy cows (Holstein) were used in this study. The animals are in semi-extensive farming (diet based on concentrate and free grazing), and present between 3 and 5 years of age, as well as all cows were in lactation (average of production 18.5 liters). All cows had a parasitological examination of feces negative for endoparasites, as well as being seronegative for Leptospira spp. Despite the history of abortion described in 30 dairy cows, all were apparently and clinically healthy at the time of sampling of these animals. It is important to emphasize that the blood samples collected from cows that aborted occurred within 15 days after the event. A total of 90 sera samples helped to classify the animals as seronegatives and seropositives animals using the indirect immunofluorescence assay (IFA). The samples were previously used on a study by Klauck et al. [17]. All sera samples positives for N. caninum at dilutions of 1:100 were subjected to maximum titration. Thus, three groups were formed: the group A consisted of seronegative animals (n=30 animals); the group B was formed by seropositive cows (titration 1:100 (n=30 animals)), but asymptomatic (neither neurological signs of the disease nor history of reproductive problems); and the group C was formed by cows seropositives for N. caninum (titrations: 1:200 (n=8 animals), 1:400 (n=12 animals), 1:800 (n=7 animals), 1:1600 (n=1 animals), and 1:3200 (n=2 animals)), and symptomatic (recent history of abortion - less than 15 days). According to the literature, there is a strong relationship between animals with a titers equal or
greater than 1:200 for N. caninum and occurred of abortion, that is, abortion was caused by the parasite [17]. Lipid peroxidation was determined based on the levels of Thiobarbituric Acid Reactive Substances (TBARS) in sera samples according to the method described by Jentzsch et al. [18]. The results were obtained by spectrophotometry (535 nm) and expressed as nmol of malondialdehyde per mL. Sera samples were prepared as described by Ali et al. [19] and Bass et al. [20] for the determination of ROS levels, and recently described briefly by Radavelli et al. [21]. ROS formation was quantified from a DCF standard curve in methanol (0.05-1.0 µM). GST activity was measured based on methods described by Habig et al. [22]. Freshly prepared cell lysate (25–50 μL) was mixed in a cuvette with 1 mL of GST assay buffer containing either 1 mM -chloro-2,4-dinitrobenzene (CDNB) and 1 mM reduced glutathione [GSH] or 1 mM 4nitrobenzylchloride and 5 mM GSH in 0.11 M sodium phosphate, pH 6.5, and incubated at room temperature. GST was calculated as nmol GS-DNB per min per mg of protein. Seric ADA activity was measured spectrophotometrically by the method of Giusti and Gakis [23]. The reaction was started by addition of the substrate (adenosine) to a final concentration of 21 mmol/L and incubated for 1 h at 37 °C. The specific activity was reported as U/L. The BChE enzymatic assay for sera samples was performed as described by Ellman et al. [24] using butyrylthiocholine as substrate. Sample analyses were carried out in duplicate, and the enzymatic activity was expressed in µmoles BcSCh/h/mg of protein. Data were analyzed initially by descriptive statistics. All variables were subjected to Shapiro-Wilk test for normality verification. Since most variables were normally distributed, the data was used for parametric tests (t test) for three independent groups for each sampling. The P value considered statistically different was <0.05.
3. Results and discussion Results for ROS and TBARS levels, and GST activity is shown in Table 1. Seropositive and symptomatic dairy cows (the group C) showed higher levels of seric ROS (p<0.05) compared to seronegative animals (the group A). TBARS levels were increased on seropositive asymptomatic animals (the group B) and seropositive symptomatic animals (the group C) compared to seronegatives (the group A) (p<0.05). Seropositive symptomatic dairy cows (the group C) showed lower seric GST activity (p<0.05) compared to seronegative animals (the group A). Results of ADA and BChE activities are shown in Table 2. ADA activity increased (p<0.05) on seropositive symptomatic dairy cows (the group C) compared to seronegative dairy cows (the group A). BChE activity decreased (p<0.05) on seropositive asymptomatic and symptomatic dairy cows (groups B and C, respectively) compared to seronegative dairy cows (the group A). In this study, changes in the oxidative stress markers and in the enzymes involved in the regulation of acetylcholine (BChE) and adenosine (ADA) levels were found in animals seropositives for N. caninum; alterations that may contribute directly to the pathogenesis of neosporosis. Oxidative stress was evident as indicated by higher levels of lipid peroxidation biomarker (MDA) and inhibition of antioxidant enzyme GST due to high levels of ROS, such as observed in seropositive symptomatic dairy cows. The accumulation of MDA is an indicative of the excessive formation of free radicals, oxidative stress and damage, which contribute directly to the pathogenesis of disease [25]. In a number of studies, it has been demonstrated that host cells infected with different species of parasites show increased amounts of ROS, which causes increased lipoperoxidation, and consequently cell and tissue damage [26, 27]. Recent studies have demonstrated the oxidative effects caused by N. caninum in cows and rodents, but they did not related to clinical signs of neosporosis. For example, studies conducted by Fidan et al. [28] and
Tonin et al. [16] demonstrated that animals infected by N. caninum showed elevated levels of TBARS, nitric oxide and protein oxidation, similarly to those observed in this present study. In this study, seropositive symptomatic dairy cows showed decreased GST activity concomitantly with increased ROS levels. Antioxidant enzymes, such as GST, protect the animals against oxidative stress and is able to scavenge the excess ROS generated, and it can be used as an indicator of the ROS production [29,30]. Also, GST inactivates lipoperoxidation products, such as MDA content, and inactivates ROS via SH groups [31]. Based on these evidences, we concluded that increased ROS levels observed in seropositive symptomatic animals occur to due an impairment of GST activity caused by the infection. The decreased GST activity in this group could be explained since glutathione (GSH) was largely consumed as a substrate, and many intermediate metabolites were produced in the process of detoxification, which could reduce GST activity or competitively inhibit GST substrates [32]. It is important to emphasize that alterations on the enzymes of the glutathione family, such as glutathione reductase (GR) have been observed during N. caninum infections [16]. Also, decreased GST activity may be considered an indicator of increased ROS production in animals N. caninum seropositives symptomatic. BChE activity was reduced in N. caninum seropositive asymptomatic and symptomatic dairy cows. This result possibly occurs due to hepatic damage observed during neosporosis, since BChE is synthetized by hepatocytes [33], as well as observed by Tonin et al. [16]. Recently, a study demonstrated that mice infected by T. gondii, other important protozoan, showed decreased seric BChE activity associated with liver injury [12]. Some studies have demonstrated the relation between alterations on cholinergic system, including BChE activity, and fetal mummification and abortion [34,35]. Thus, we believe that alteration on BChE activity contributes to pathophysiology of neosporosis, and it may contribute to abortion on seropositive asymptomatic or symptomatic dairy cows.
Increased ADA activity in dairy cows seropositives symptomatic for neosporosis was observed. A study conducted by Abbrachio and Ceruti [36] demonstrated that reduction on ADA activity contributes to limit the inflammatory response and subsequent cellular damage, since it would increase adenosine tissue levels, an anti-inflammatory molecule, conferring protection to the host tissue. Thus, we believe that increased ADA activity leads to decreased adenosine levels, which contributes to the inflammatory process and tissue damage, because adenosine protects host cells from excessive tissue injury associated with severe inflammation. Also, there are evidences that elevated levels of adenosine potently down-regulates the activation of lymphocytes during inflammation, playing a regulatory role on neutrophils in immune responses [37,38]. These results clarify the need to check all animals, regardless of the presence of clinical signs, since these asymptomatic animals may have lower performance due to imperceptible cell damage. Therefore, based on the results obtained and discussed here, it is possible to assure that seropositive dairy cows showed tissue damage associated with oxidative stress and inflammation, mainly those that were symptomatic for neosporosis. The increase of ROS levels and BChE activity in sera samples may have influenced the pathogenesis of N. caninum infection in symptomatic animals due to increased cell damage and exacerbated inflammatory response, respectively, leading to the development of clinical signs.
Ethics Committee The experiment was approved by the Ethics Committee in Animal Research at the University of Santa Catarina State (UDESC).
Conflict of Interest: The authors declare that they have no conflict of interest.
References [1] McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473-1478. [2] Anderson, M.L., Andrianarivo, A.G., Conrad, P.A., 2000. Neosporosis in cattle. Anim. Reprod. Sci. 60, 417-431. [3] Schares G., Peters M., Wurm R., Barwald A., Conraths F.J., 1998. The efficiency of vertical transmission of Neospora caninum in dairy cattle analyzed by serological techniques. Vet. Parasitol. 80, 87-98. [4] Dubey J.P., Schares G., Ortega-Mora L.M., 2007. Epidemiology and control of neosporosis and Neospora caninum. Clin. Microbial. Rev. 20, 323-367. [5] Innes A.E., Andrianarivo G., Bjorkman C., Williams D.J.L., Conrad P.A., 2002. Immune responses to Neospora caninum and prospects for vaccination. Trends Parasitol. 18, 497-504. [6] Dubey J.P., Buxton D., Wouda W., 2006. Pathogenesis of Bovine Neosporosis. J. Comp. Path. 134, 267-289. [7] Tonin A.A., Weber A., Ribeiro A., Camilo G., Vogel F.F., Moura A.B., Bochi G.V., Moresco R.N., Da Silva A.S., 2015. Serum levels of nitric oxide and protein oxidation in goats seropositive for Toxoplasma gondii and Neospora caninum. Comp. Immunol. Microbiol. Infect. Dis. 41, 55-58. [8] Da Silva A.S., Paim F.C., Santos R.C., Sangoi M.B., Moresco R.N., Lopes S.T., Jaques J.A., Baldissarelli J., Morsch V., Monteiro S.G., 2012. Nitric oxide level, protein oxidation and antioxidant enzymes in rats infected by Trypanosoma evansi. Exp. Parasitol. 132, 166-170. [9] Winterboun C.C., Hampton M.B., 2015. Redox biology: signaling via a peroxiredoxin sensor. Nat. Chem. Biol. 11, 5-6. [10] Barbosa K.B.F, Costa N.M.B., Alfenas R.C.G., De Paula S.O., Minim V.P.R., Bressan J., 2010. Estresse oxidativo: conceito, implicações e fatores modulatórios. Rev. Nutr. 23, 629-643.
[11] Bouwstra R.J., Nielen M., Newbold J.R., Jansen E.H., Jelinek H.F., Van Werven T., 2010. Vitamin e supplementation during the dry period in dairy cattle. Oxidative stress following vitamin e supplementation may increase clinical mastitis incidence postpartum. J. Dairy. Sci. 93, 5696-5706. [12] Da Silva A.S., Munhoz T.D., Faria J.L., Vargas-Hérnandez G., Machado R.Z., Almeida T.C., Moresco R.N., Stefani L.M., Tinucci-Costa M., 2013. Increase nitric oxide and oxidative stress in dogs experimentally infected by Ehrlichia canis: Effect on the pathogenesis of the disease. Vet. Microbiol. 4, 366-369. [13] Da Silva, A.S., Bellé, L.P., Bitencourt, P.E., Souza, V.C., Costa, M.M., Oliveira, C.B., Jaques, J.A., Leal, D.B., Moretto, M.B., Maz zanti, C.M., Lopes, S.T., Monteiro, S.G., 2011. Activity of the enzyme adenosine deaminase in serum, erythrocytes and lymphocytes of rats infected with Trypanosoma evansi. Parasitol. 138, 201 – 208. [14] Franco, R., Casadó, V., Ciruela, F., Saura C., Malloi, J., Canela, El., Lluis, C., 1997. Cell surface adenosine deaminase: much more than an ectoenzyme. Prog. Neurobiol. 52 (4), 283–294. [15] Kaplay, S.S., 1976 Acetylcholinesterase and butyrylcholinesterase of developing human brain. Biol. Neonate. 28, 65-73. [16] Tonin, A.A., Da Silva, A.S., Thomé, G.R., Oliveira, L.S., Schetinger, M.R., Morsch, V.M., Flores, M.M., Fighera, R.A., Toscan, G., Vogel, F.F., Lopes, S.T.A., 2013. Neospora caninum: Activity of cholinesterases during the acute and chronic phases of an experimental infection in gerbils. Exp. Parasitol. 135 (4), 669-674. [17] Klauck, V., Machado, G., Pazinato, R., Radavelli, W.M., Santos, D.S., Berwaguer, J.C., Braunig, P., Volgel F.F., Da Silva, A.S., 2016. Relation between Neospora caninum and abortion in dairy cows: Risk factors and pathogenesis of disease. Microb. Pathog. 92, 46-49. [18] Jentzsch, A.M., Bachmann, H., Furst, P., Biesalski, H., 1996. Improved analysis of malondialdehyde in human body fluids. Free Rad. Biol. Med. 20 (2), 251–256.
[19] Ali, S.F., Lebel, C.P., Bondy, S.C., 1992. Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity. Neurotoxicol. 13 (3), 637-648. [20] Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C., Thomas, M., 1983. Flow cytometric studies of oxidative product formation by neurotrophils: a graded response to membrane stimulation. J. Immunol. 1130, 1910-1917. [21] Radavelli, W. M., Campigotto, G., Machado, G., Bottari, N.B., Bochi, G., Moresco, R.N., Morsch, V.M., Schetinger, M.R.C., Bianchi, A., Baldissera, M.D., Ferreira, R., Da Silva, A.S., 2016. Effect of lactation induction on milk production and composition, oxidative and antioxidant status and biochemical variables. Comp. Clin. Pathol. DOI: 10.1007/s00580-016-2243-z [22] Habig, W.H., Pabst, M.J., Jakoby, W.B., 1974. Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249 (22), 7130-7139. [23] Giusti, G., Gakis, C., 1971. Temperature conversion factors, activation energy, relative substrate specificity and optimum pH of adenosine deaminase from human serum and tissues. Enzyme. 12 (4), 417-442. [24] Ellman, G.L., Courtney, K.D., Andres V.J., Feather-stone R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biocherm. Phamacol. 7, 88-95. [25] Gutteridge, J.M., 1995. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 41, 1819-1828. [26] Sarin, K., Kumar, A., Prskash, A., Sharma, A., 1993. Oxidative stress and antioxidant defense mechanism in Plasmodium vivax malaria before and after chloroquine treatment. Indian J. Malariol. 30, 127-133. [27] Deger, Y., Ertekin, A., Deger, S., Mert, H., 2008. Lipid peroxidation and antioxidant potential of sheep liver infected naturally with distomatosis. Turkiye Parazitoloji Dergisi 32, 23-36.
[28] Fidan, A.F., Cingi, C.C., Karafakioglu, Y.S., Utuk, A.E., Pekkaya, S., Piskin, F.C., 2010. The levels of antioxidant activity, malondialdehyde and nitric oxide in cows naturally infected with Neospora caninum. J. Animal Vet. Adv. 9, 1707-1711. [29] Holovska, J.K., Sobekova, B., Holovska, K., Lenartova, V., Javorsky, P., Legath, J., Legath, L., Maretta, M., 2005. Antioxidant and detoxifying enzymes in the liver of rats after subchronic inhalation of the mixture of cyclic hydrocarbons. Exp. Toxicol. Pathol. 56, 377-383. [30] Martins, F., Pereira. J.A., Baptista, P., 2014. Oxidative stress response of Beauveria bassiana to Bordeaux mixture and its influence on fungus growth and development. Pestic. Manag. Sci. 70, 1220-1227. [31] Choi, C.Y., An, K.W., An, M.I., 2008. Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus). Comp. Biochem. Physiol. A: Mol. Integr. Physiol. 149, 330-337. [32] Eggas, E., Sandvik, M., Fjeld, E., Kallqvist, T., Goksoyr, A., Svensen, A., 1999. Some effects of the fungicide propiconazole on cytochrome P450 and glutathione S-transferase in brown trout (Salmo trutta). Comp. Biochem. Physiol. Part C: Pharmacol. Toxicol. Endocrinol. 122, 337-344. [33] Wescoe, W.C., Hunt, C.H., Riker, W.F., Litt, I.C., 1947. Regeneration rates of serum cholinesterase in normal individuals and in patients with liver damage. Am. J. Physiol. 149, 549551. [34] Sternfeld, M., Rachmilewitz, J., Lichtenstein-Loewenstein, Y., Andres, C., Timberg, R., BenAri, S., Glick, D., Soreq, H., Zakut, H., 1997. Normal and atypical butyrylcholinesterases in placental development, function and malfunction. Cell. Mol. Neurobiol. 17, 315-332. [35] Mahmoud, F.F., Abul, H.T., Haines, D.D., Omu, A.E., Diejomaoh, M., Wise J.A., Abu, Donia, M.B., 2008. Butyrylcholinesterase activity and lymphocyte subpopulations in peripheral blood of Kuwaiti women experiencing recurrent spontaneous abortion. J. Reprod. Immunol. 77, 186-194.
[36] Abbrachio, M.P., Ceruti, S., 2007. P1 receptors and cytokine secretion. Purinergic Signal. 3, 13-25. [37] Desrosiers, M.D., Cembrola, K.M., Fakir, M.J., Sthepens, L.A., Jama, F.M., Shameli, A., Mehal, W.Z., Santamaria, P., Shi, Y., 2007. Adenosine deamination sustains dendritic cell activation in inflammation. J. Immunol. 179, 1884-1892. [38] Mills, J.H., Kim, D.G., Krenz, A., Chen, J.F., Byone, M.S., 2012. A2S adenosine receptor signaling in lymphocytes and the central nervous system regulates inflammation during experimental autoimmune encephalomyelitis. J. Immunol. 118, 5713-5722.
- Bovine neosporosis is an important cause of abortion in cows. - Seropositive dairy cows showed oxidative stress and increase of inflammation markers which indicate tissue damage. - The increase of ROS levels, ADA and BChE activities in sera samples may have influenced the pathogenesis of N. caninum. - Due to increased cell damage and exacerbated inflammatory response occurred the development of abortion. - ADA and BChE activities participates in immunomodulation.
Table 1: Seric levels of reactive oxygen species (ROS), thiobarbituric acid reactive species (TBARS), and glutathione S-transferase (GST) in seronegative dairy cattle (the group A, control), seropositive/asymptomatic dairy cattle (the group B), and seropositive/symptomatic dairy cattle (the group C) to Neospora caninum.
Group
ROS (U DCFA/μL)
TBARS (nmol MDA/mL)
GST (μmol/Cdnb/min)
A - control
2.26 ± 0.45b
6.58 ± 0.59b
87.8 ± 12.6a
B - seropositive/asymptomatic
3.26 ± 1.06ab
11.02 ± 1.74a
92.4 ± 8.95a
C - seropositive/symptomatic
4.15 ± 0.29a
12.78 ± 1.35a
36.4 ± 15.3b
P<0.05
P<0.05
P<0.05
Note: Means with equal letters, on the same column, do not differ statistically among themselves for 5 % of significance (P>0.05).
Table 2: Seric activities of adenosine deaminase (ADA) and butyrylcholinesterase (BChE) of seronegative dairy cattle (the group A, control), seropositive/asymptomatic dairy cattle (the group B), and seropositive/symptomatic dairy (the group C) to Neospora caninum.
Group
ADA (U/L)
BChE (μmol BuSCh/h/mg of protein)
A - control
14.8 ± 5.3b
66.3 ± 10.6a
B - seropositive/asymptomatic
15.7 ± 4.6b
46.5 ± 13.5b
C - seropositive/symptomatic
41.5 ± 18.9a
41.3 ± 8.3b
P<0.05
P<0.05
Note: Means with equal letters, on the same column, do not differ statistically among themselves for 5 % of significance (P>0.05).