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antioxidant balance in dogs with sarcoptic mange
Veterinary Parasitology 161 (2009) 106–109
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Evaluation of blood oxidant/antioxidant balance in dogs with sarcoptic mange Ilker Camkerten a,*, T. Sahin a, G. Borazan b, A. Gokcen c, O. Erel d, A. Das a a
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Harran, 63300 Sanliurfa, Turkey Department of Biochemistry, Faculty of Veterinary Medicine, University of Harran, 63300 Sanliurfa, Turkey c Department of Parasitology, Faculty of Veterinary Medicine, University of Harran, 63300 Sanliurfa, Turkey d Department of Biochemistry, Faculty of Medicine, University of Harran, 63300 Sanliurfa, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 June 2008 Received in revised form 15 December 2008 Accepted 23 December 2008
The aim of this study was to investigate of oxidant/antioxidant balance in dogs with sarcoptic mange. The study materials consisted of totally 30 cross-breed male dogs; 15 with sarcoptic mange (study group) and 15 healthy as control. Blood samples for analyses were taken from control and study group. In study group, microscopic examination of dermal scrapings of 15 dogs revealed S. scabies. Lipid hydroperoxide level, total oxidant status and oxidative stress index in dogs with sarcoptic mange were higher (P < 0.01, P < 0.01 and P < 0.05, respectively) than the control. Otherwise; sulphydril levels in dogs with sarcoptic mange were lower (P < 0.05) than that of control. No significant differences were observed in total antioxidant capacity between groups. Our results suggest a possible relationship between oxidant/antioxidant imbalance and sarcoptic mange infestation in dogs. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Sarcoptic mange Oxidant Antioxidant Dog
1. Introduction Sarcoptic mange is a contagious parasitic skin disease. Clinical signs result from the development, multiplication and pathogenic action at the skin surface and in the stratum corneum of the mite, Sarcoptes scabiei var canis. This disease is characterized by intense pruritus associated with a vesiculopapular eruption and pinpoint crusts in combination with alopecia (Pin et al., 2006). The mechanism(s) by which cellular defense kills microorganisms has been the subject of intense research. Numerous studies demonstrated that a variety of inflammatory cells are activated which induce or activate various oxidant-generating enzymes to kill intra-cellular and extra-cellular parasites (Kocyigit et al., 2005). Humoral and cellular immune response (IgG, IgM and
* Corresponding author. Tel.: +90 414 312 84 56/2436; fax: +90 414 314 41 58. E-mail address: [email protected] (I. Camkerten). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.12.019
especially IgE) has been reported an important role in the pathogenesis of mange. Total leukocyte, T-lymphocyte, mast cells, neutrophyles, eosinophiles, and a, b, g globulin levels increases in animals with mange. Alterations in the free radicals due to these parameters may be effective in the phsiopathogenesis of mange (Yaralıog˘luGu¨rgo¨ze et al., 2003). Free radicals and other reactive oxygen species (ROS) in the form of the superoxide anion (O2 ), Hydrogen Peroxide (H2O2) and the hydroxyl radical (OH) cause damage to DNA, lipids and proteins (Cemek et al., 2006). ROS are produced in metabolic and physiological processes, and harmful oxidative reactions may occur in organisms which remove them via enzymatic and nonenzymatic antioxidative mechanisms. Under certain conditions such as human and canine leishmaniosis, Behcet’s diseases, pneumonia, etc., the increase in oxidants and decrease in antioxidants cannot be prevented, and the oxidative/ antioxidative balance shifts towards the oxidative status (Bildik et al., 2004; Erel, 2005; Kocyigit et al., 2005; Cemek ¨ stu¨ndag, 2006). et al., 2006; Is¸ık and U
I. Camkerten et al. / Veterinary Parasitology 161 (2009) 106–109
Lipid peroxidation is a well-established mechanism of cellular injury and is used as an indicator of oxidative stress in cells and tissues. Lipid hydroperoxides (LOOH) are by product of lipid peroxidation. Increased levels of lipid peroxidation products have been associated with a variety of diseases including parasitic infections (Yaralıog˘luGu¨rgo¨ze et al., 2003; Arab and Steghens, 2004; Bildik et al., 2004; Kiral et al., 2005). Plasma or sera concentrations of oxidants can be measured separately in the laboratory, since the effects of the oxidant components in plasma are additive, the measurement of the total oxidant status (TOS) accurately reflects the oxidative status of plasma or sera. TOS of sera has been evaluated using a recently developed measurement method by Erel (2005). Blood contains many antioxidant molecules that prevent and/or inhibit harmful free radical reactions. Sulphydril groups (SH) prevent tissue damage through reacting with free oxygen radicals and lipid peroxides and neutralization of these molecules (Erbay et al., 2003). Plasma or sera concentrations of antioxidants can be measured one by one, but this procedure is timeconsuming, labor-intensive and costly, and requires complicated techniques (Erel, 2004). On the other hand, total antioxidant capacity (TAC) whose measurement method has been recently specified and developed can reflect the total antioxidant status of the plasma (Erel, 2004). In this method, TAC of sera which acts especially against potent free radical reactions strongly leading to oxidative damage of biomolecules such as lipids, proteins and DNA, is measured (Kosecik et al., 2005). A possible roles of the highly ROS in the pathogenesis of parasitic infections has been an active area of research in recent years and we could not determine a study in the literature that examined oxidant and antioxidant status in dogs with sarcoptic mange. Aim of this study was to investigate oxidant/antioxidant balance in dogs with sarcoptic mange. 2. Material and methods 2.1. Selection of animals Study included 15 dogs diagnosed as sarcoptic mange by clinical and parasitological examination (study group) and 15 healthy dogs residing in Dog Shelter of Sanliurfa Municipality (control group) making a total of 30 crossbreed, in the age group between 1 and 2 years, male dogs. Dogs had skin lesions including severe itching dermatitis, excoriation, alopecia, scabs of pinnae, neck, brisket, elbow and around root of tail. Dogs had also pinnal pedal reflex in study group. Fecal samples were negative for eggs of internal parasites, and no detectable sings of any other systemic disease were observed in study group. Healthy dogs had good body condition. Vital signs and feed intake were normal in control group. For parasitological examination, a dulled scalpel blade is held perpendicular to the skin and used with moderate pressure to scrape in the direction of hair growth. Skinscraping samples of 5 cm 5 cm were taken. Scraping samples of the dogs from the study group were transferred into test tube, and 10% KOH was added; samples were
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mildly heated for 1–2 min and then centrifuged on 1500 rpm for 5 min. Obtained precipitates were examined under microscope. The mites were identified according to morphological features. Ten microscopic fields were examined and number of mites per microscopic field was count. Animals with skin lesion and a minimum of three sarcoptic mites per microscopic field were classified as clinical sarcoptic mange. 2.2. Sample collection 2.2.1. Sera Blood samples of 9 mL volume were drawn from both study and control group. Samples were transferred to laboratory in a cold media and sera were extracted by a cooler centrifuge with 1500 rpm in 10 min. Sera were preserved at 80 8C and analyzed within 2 months. 2.3. Estimations 2.3.1. Total antioxidant capacity (TAC) The total antioxidant capacity of the sera was measured using a novel automated colorimetric measurement method for TAC developed by Erel (2004). In this method the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction, and reacts with the colorless substrate O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in color. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the color change and thereby providing an effective measure of the total antioxidant status of the plasma. The assay results are expressed as mmol Trolox equivalent/L (Eq/L), and the precision of this assay is excellent, being lower than 3% (Cao and Prior, 1998). 2.3.2. Sera sulfhydryl (SH) group Free sulfhydryl groups of sera samples were assayed according to the method of Ellman (1959) as modified by Hu et al. (1993). Briefly, 1 mL of buffer containing 0.1 M Tris, 10 mM EDTA, pH 8.2, and 50 mL sera was added to cuvettes followed by 50 mL 10 mM DTNB in methanol. Blanks were run for each sample as a test, but there was no DTNB in the methanol. Following incubation for 15 min at room temperature, sample absorbance was read at 412 nm on a spectrophotometer. Sample and reagent blanks were subtracted. The concentration of sulfhydryl groups was calculated using reduced glutathione as free sulfhydryl group standard and the result was expressed as millimolars (Erel, 2004). 2.3.3. Total oxidant status (TOS) The total oxidant status of the sera was measured using a novel automated colorimetric measurement method for TOS developed by Erel (2005). In this method, oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color
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I. Camkerten et al. / Veterinary Parasitology 161 (2009) 106–109
intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (mmol H2O2 equivalent/L).
significantly lower in DSM as compared with HD. No significant differences were observed in TAC between DSM and HD. Results of analyzed parameters are presented in Table 1.
2.3.4. Lipid hydroperoxide (LOOH) Sera LOOH levels were measured with the ferrous ion oxidation–xylenol orange assay. The principle of the assay depends on the oxidation of ferrous ion to ferric ion via various oxidants and the produced ferric ion is measured with xylenol orange. LOOHs are reduced by triphenyl phosphine (TPP), which is a specific reductant for lipids. The difference between with and without TPP pretreatment gives LOOH levels (Arab and Steghens, 2004).
In the present study, we found that the oxidative/ antioxidative balance shifted towards oxidative in DSM. TOS, LOOH levels, and OSI were significantly higher and SH levels were significantly lower in DSM compared with HD. To the best of our knowledge, this is the first report showing that an association between high TOS, LOOH, OSI and low SH in DSM. It is known that inflammatory cells are increased as a result of inflammation in animals with mange; recruited neutrophiles and macrophages produce reactive oxidants such as hydrogen peroxide (H2O2), hypochlorite and oxygen radicals, and these reactive oxygen substances produced by cells of the immune system show potent cytotoxic effects on parasites as well as other pathogenic organisms (Yaralıog˘lu-Gu¨rgo¨ze et al., 2003). Free radicals induce or contribute to adverse effects on the skin, including erythema, edema, wrinkling, inflammation, autoimmune reactions, hypersensitivity, and keratinization abnormalities (Bickers and Athar, 2006). Lipid peroxidation can be harmful for skin due to alternations in the membrane structure and permeability (Portugal et al., 2007). Some authors showed lipid peroxidation levels increased in sheep with sarcoptic mange (Yaralıog˘lu-Gu¨rgo¨ze et al., 2003), in dog with demodicosis (Dimri et al., 2008) and in patient with dermatitis (Tsukahara et al., 2003). Our study has also shown that LOOHs levels and TOS were significantly higher in DSM than that of HD which revealed that DSM is associated with oxidative stress. Sera SH groups act as important cellular scavengers of peroxides and so help to protect cells from damage by these molecules. Decrease in SH level not only impairs cells’ response to oxidants, but also changes the functions of inflammatory cells (McKeown et al., 1984). It has been reported that SH levels decrease in some skin diseases including Behcet’s Disease and measles (Erbay et al., 2003; ¨ stu¨ndag, 2006). Individual antioxidants level or Is¸ık and U activity indicates the antioxidant characteristics of only one antioxidant, whereas TAC may represent the total antioxidant characteristics of all antioxidants found in the sera (Erel, 2004). Our study has also confirmed a significant decrease in SH levels in DSM compared to HD. No significant difference was found between TAC levels of both groups. This amazing condition can be originated from increase in enzymatic antioxidant response. However, TAC levels were not reduced, and the oxidant/antioxidant balance shifted significantly to the oxidant side, because other indicators of oxidative status, i.e., LOOH, TOS and OSI levels, were significantly increased in DSM. Our search of the literature showed no other study investigating the relation between the sarcoptic mange in animals and oxidative stress, except the study by Yaralıog˘lu-Gu¨rgo¨ze et al. (2003) and Dimri et al. (2008a).
2.3.5. Oxidative stress index (OSI) The TOS to TAC ratio was regarded as the OSI. To perform the calculation, the result unit of TAC, mmol Trolox equivalent/L, was changed to mmol Trolox equivalent/L, and the OSI value was calculated as follows: OSI = [(TOS, mmol/L)/(TAC, mmol Trolox equivalent/ L) 100] (Aycicek et al., 2005). 2.4. Apparatus A Cecil 3000 spectrophotometer with a temperature controlled cuvette holder (Cecil) and an Aeroset automated analyzer (Abbott) were used. 2.5. Statistical analysis The statistical analysis of the data was carried out with the SPSS software 11.0 (SPSS Inc., Chicago, IL, USA). Wilcoxon signed rank test (SH, TOS and LOOH) and paired t test (TAK and OSI) were used to compare two groups. Values obtained were expressed as mean S.D. The differences were considered to be significant when P 0.05. 3. Results Dermal scraping for microscopic examination in all dogs of study group revealed S. scabies. The TOS and LOOH sera levels were found to be significantly higher in the dogs with sarcoptic mange (DSM) compared to healthy dogs (HD). The OSI was also significantly higher in the DSM than in the HD. Otherwise, SH sera levels were found to be Table 1 Oxidant/antioxidant parameters in dogs with sarcoptic mange. Parameters
Control group
Study group
TOS (mmol H2O2 equivalent/L) LOOH (mmol H2O2 equivalent/L) TAC (mmol Trolox equivalent/L) SH (mmol/L) OSI (arbitrary unit)
12.22 0.95 6.99 1.12 1.65 0.50 0.20 0.15 0.08 0.21
20.39 4.31** 15.56 5.74** 1.67 0.69a 0.17 0.35* 0.15 0.76**
TOS: total oxidant status; LOOH: lipid hydroperoxide; TAC: total antioxidant capacity; SH: sulphydril; OSI: oxidative stress index. a Insignificant. * P 0.05. ** P 0.01.
4. Discussion
I. Camkerten et al. / Veterinary Parasitology 161 (2009) 106–109
As a conclusion, TOS, LOOH, TAC, SH and OSI levels determined in our study may contribute to the studies in the future as basic data in dogs with sarcoptic mange. Our results suggest a possible relationship between oxidant/ antioxidant imbalance and sarcoptic mange infestation in dogs. This can be explained by higher increase in free radicals, occurred due to inflammatory response against dermal scabby agents, than antioxidant capacity; however, in order to more definitively delineate the pathogenesis of sarcoptic mange, further studies are necessary. The determination of oxidative stress may require that clinicians treating the disease should include antioxidative drugs in their treatment regime. References Arab, K., Steghens, J.P., 2004. Plasma lipid hydroperoxides measurement by an automated xylenol orange method. Anal. Biochem. 325, 158–163. Aycicek, A., Erel, O., Kocyigit, A., 2005. Decreased total antioxidant capacity and increased oxidative stress in passive smoker infants and their mothers. Pediatr. Int. 47, 635–639. Bickers, D.R., Athar, M., 2006. Oxidative stress in the pathogenesis of skin disease. J. Invest. Derm. 126, 2565–2575. doi:10.1038/sj.jid.5700340. Bildik, A., Kargin, F., Seyrek, K., Pasa, S., Ozensoy, S., 2004. Oxidative stress and non-enzymatic antioxidative status in dogs with visceral leishmaniasis. Res. Vet. Sci. 77, 63–66. Cao, G., Prior, R.L., 1998. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin. Chem. 44 (6 Pt. 1), 1309–1315. Cemek, M., Caksen, H., Bayiroglu, F., Cemek, F., Dede, S., 2006. Oxidative stress and enzymic-non-enzymic antioxidant responses in children with acute pneumonia. Cell Biochem. Funct. 24 (3), 269–273. Dimri, U., Sharma, M.C., Swarup, D., Ranjan, R., Kataria, M., 2008a. Alterations in hepatic lipid peroxides and antioxidant profile in Indian water buffaloes suffering from sarcoptic mange. Res. Vet. Sci. 85 (1), 101–105. Dimri, U., Ranjan, R., Kumar, N., Sharma, M.C., Swarup, D., Sharma, B., Kataria, M., 2008. Changes in oxidative stress indices, zinc and copper
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concentrations in blood in canine demodicosis. Vet. Parasitol. 154, 98–102. Ellman, G.L., 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77. Erbay, A., Erbay, A.R., Baykam, N., Eren, S¸., Dokuzouz, B., 2003. Oxidative stress in measles. Turkish J. Infect. 17, 381–383. Erel, O., 2004. A novel automated method to measure total antioxidant response against potent free radical reactions. Clin. Biochem. 37, 112– 119. Erel, O., 2005. A new automated colorimetric method for measuring total oxidant status. Clin. Biochem. 38, 1103–1111. Hu, M.L., Louie, S., Cross, C.E., Motchnik, P., Halliwell, B., 1993. Antioxidant protection against hydrochlorous acid in human plasma. J. Lab. Clin. Med. 121, 257–262. ¨ stu¨ndag, B., 2006. Paraoxonase and arylesterase levels in Is¸ık, A., U Behc¸et’s disease. J. Health Sci. Univ. Firat 20 (4), 307–315. Kiral, F., Karagenc, T., Pasa, S., Yenisey, C., Seyrek, K., 2005. Dogs with Hepatozoon canis respond to the oxidative stress by increased production of glutathione and nitric oxide. Vet. Parasitol. 131, 15–21. Kocyigit, A., Keles, H., Selek, S., Guzel, S., Celik, H., Erel, O., 2005. Increased DNA damage and oxidative stress in patients with cutaneous leishmaniasis. Mutat. Res. 585, 71–78. Kosecik, M., Erel, O., Sevinc, E., Selek, S., 2005. Increased oxidative stress in children exposed to passive smoking. Int. J. Cardiol. 100, 61–64. McKeown, M.J., Hall, N.D., Corvalan, J.R., 1984. Defective monocyte accessory function due to surface sulphydryl (SH) oxidation in rheumatoid arthritis. Clin. Exp. Immunol. 56, 607–613. Pin, D., Bensignor, E., Carlotti, D.N. and Cadiergues, M.C., 2006. Localised sarcoptic mange in dogs: a retrospective study of 10 cases. J. Small Anim. Pract. 47, 611–614. doi:10.1111/j.1748-5827.2006.00111.x. Portugal, M., Barak, V., Ginsburg, I., Kohen, R., 2007. Interplay among oxidants, antioxidants, and cytokines in skin disorders: present status and future considerations. Biomed. Pharmacother. 61 (7), 412–422. Tsukahara, H., Shibata, R., Ohshima, Y., Todoroki, Y., Sato, S., Ohta, N., Hiraoka, M., Yoshida, A., Nishima, S., Mayumi, M., 2003. Oxidative stress and altered antioxidant defences in children with acute exacerbation of atopic dermatitis. Life Sci. 72, 2509–2516. ¨ zkutlu, Z., Temizer-Ozan, S., Yaralıog˘lu-Gu¨rgo¨ze, S., S¸ahin, T., Sevgili, M., O 2003. The effects of ivermectin or doramectin treatment on some antioxidant enzymes and the level of lipid peroxidation in sheep with natural sarcoptic scap. J. Fac. Vet. Med. YYU 14, 30–34.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Evaluation of blood oxidant/antioxidant balance in dogs with sarcoptic mange Ilker Camkerten a,*, T. Sahin a, G. Borazan b, A. Gokcen c, O. Erel d, A. Das a a
Department of Internal Medicine, Faculty of Veterinary Medicine, University of Harran, 63300 Sanliurfa, Turkey Department of Biochemistry, Faculty of Veterinary Medicine, University of Harran, 63300 Sanliurfa, Turkey c Department of Parasitology, Faculty of Veterinary Medicine, University of Harran, 63300 Sanliurfa, Turkey d Department of Biochemistry, Faculty of Medicine, University of Harran, 63300 Sanliurfa, Turkey b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 June 2008 Received in revised form 15 December 2008 Accepted 23 December 2008
The aim of this study was to investigate of oxidant/antioxidant balance in dogs with sarcoptic mange. The study materials consisted of totally 30 cross-breed male dogs; 15 with sarcoptic mange (study group) and 15 healthy as control. Blood samples for analyses were taken from control and study group. In study group, microscopic examination of dermal scrapings of 15 dogs revealed S. scabies. Lipid hydroperoxide level, total oxidant status and oxidative stress index in dogs with sarcoptic mange were higher (P < 0.01, P < 0.01 and P < 0.05, respectively) than the control. Otherwise; sulphydril levels in dogs with sarcoptic mange were lower (P < 0.05) than that of control. No significant differences were observed in total antioxidant capacity between groups. Our results suggest a possible relationship between oxidant/antioxidant imbalance and sarcoptic mange infestation in dogs. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Sarcoptic mange Oxidant Antioxidant Dog
1. Introduction Sarcoptic mange is a contagious parasitic skin disease. Clinical signs result from the development, multiplication and pathogenic action at the skin surface and in the stratum corneum of the mite, Sarcoptes scabiei var canis. This disease is characterized by intense pruritus associated with a vesiculopapular eruption and pinpoint crusts in combination with alopecia (Pin et al., 2006). The mechanism(s) by which cellular defense kills microorganisms has been the subject of intense research. Numerous studies demonstrated that a variety of inflammatory cells are activated which induce or activate various oxidant-generating enzymes to kill intra-cellular and extra-cellular parasites (Kocyigit et al., 2005). Humoral and cellular immune response (IgG, IgM and
* Corresponding author. Tel.: +90 414 312 84 56/2436; fax: +90 414 314 41 58. E-mail address: [email protected] (I. Camkerten). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.12.019
especially IgE) has been reported an important role in the pathogenesis of mange. Total leukocyte, T-lymphocyte, mast cells, neutrophyles, eosinophiles, and a, b, g globulin levels increases in animals with mange. Alterations in the free radicals due to these parameters may be effective in the phsiopathogenesis of mange (Yaralıog˘luGu¨rgo¨ze et al., 2003). Free radicals and other reactive oxygen species (ROS) in the form of the superoxide anion (O2 ), Hydrogen Peroxide (H2O2) and the hydroxyl radical (OH) cause damage to DNA, lipids and proteins (Cemek et al., 2006). ROS are produced in metabolic and physiological processes, and harmful oxidative reactions may occur in organisms which remove them via enzymatic and nonenzymatic antioxidative mechanisms. Under certain conditions such as human and canine leishmaniosis, Behcet’s diseases, pneumonia, etc., the increase in oxidants and decrease in antioxidants cannot be prevented, and the oxidative/ antioxidative balance shifts towards the oxidative status (Bildik et al., 2004; Erel, 2005; Kocyigit et al., 2005; Cemek ¨ stu¨ndag, 2006). et al., 2006; Is¸ık and U
I. Camkerten et al. / Veterinary Parasitology 161 (2009) 106–109
Lipid peroxidation is a well-established mechanism of cellular injury and is used as an indicator of oxidative stress in cells and tissues. Lipid hydroperoxides (LOOH) are by product of lipid peroxidation. Increased levels of lipid peroxidation products have been associated with a variety of diseases including parasitic infections (Yaralıog˘luGu¨rgo¨ze et al., 2003; Arab and Steghens, 2004; Bildik et al., 2004; Kiral et al., 2005). Plasma or sera concentrations of oxidants can be measured separately in the laboratory, since the effects of the oxidant components in plasma are additive, the measurement of the total oxidant status (TOS) accurately reflects the oxidative status of plasma or sera. TOS of sera has been evaluated using a recently developed measurement method by Erel (2005). Blood contains many antioxidant molecules that prevent and/or inhibit harmful free radical reactions. Sulphydril groups (SH) prevent tissue damage through reacting with free oxygen radicals and lipid peroxides and neutralization of these molecules (Erbay et al., 2003). Plasma or sera concentrations of antioxidants can be measured one by one, but this procedure is timeconsuming, labor-intensive and costly, and requires complicated techniques (Erel, 2004). On the other hand, total antioxidant capacity (TAC) whose measurement method has been recently specified and developed can reflect the total antioxidant status of the plasma (Erel, 2004). In this method, TAC of sera which acts especially against potent free radical reactions strongly leading to oxidative damage of biomolecules such as lipids, proteins and DNA, is measured (Kosecik et al., 2005). A possible roles of the highly ROS in the pathogenesis of parasitic infections has been an active area of research in recent years and we could not determine a study in the literature that examined oxidant and antioxidant status in dogs with sarcoptic mange. Aim of this study was to investigate oxidant/antioxidant balance in dogs with sarcoptic mange. 2. Material and methods 2.1. Selection of animals Study included 15 dogs diagnosed as sarcoptic mange by clinical and parasitological examination (study group) and 15 healthy dogs residing in Dog Shelter of Sanliurfa Municipality (control group) making a total of 30 crossbreed, in the age group between 1 and 2 years, male dogs. Dogs had skin lesions including severe itching dermatitis, excoriation, alopecia, scabs of pinnae, neck, brisket, elbow and around root of tail. Dogs had also pinnal pedal reflex in study group. Fecal samples were negative for eggs of internal parasites, and no detectable sings of any other systemic disease were observed in study group. Healthy dogs had good body condition. Vital signs and feed intake were normal in control group. For parasitological examination, a dulled scalpel blade is held perpendicular to the skin and used with moderate pressure to scrape in the direction of hair growth. Skinscraping samples of 5 cm 5 cm were taken. Scraping samples of the dogs from the study group were transferred into test tube, and 10% KOH was added; samples were
107
mildly heated for 1–2 min and then centrifuged on 1500 rpm for 5 min. Obtained precipitates were examined under microscope. The mites were identified according to morphological features. Ten microscopic fields were examined and number of mites per microscopic field was count. Animals with skin lesion and a minimum of three sarcoptic mites per microscopic field were classified as clinical sarcoptic mange. 2.2. Sample collection 2.2.1. Sera Blood samples of 9 mL volume were drawn from both study and control group. Samples were transferred to laboratory in a cold media and sera were extracted by a cooler centrifuge with 1500 rpm in 10 min. Sera were preserved at 80 8C and analyzed within 2 months. 2.3. Estimations 2.3.1. Total antioxidant capacity (TAC) The total antioxidant capacity of the sera was measured using a novel automated colorimetric measurement method for TAC developed by Erel (2004). In this method the hydroxyl radical, the most potent biological radical, is produced by the Fenton reaction, and reacts with the colorless substrate O-dianisidine to produce the dianisyl radical, which is bright yellowish-brown in color. Upon the addition of a plasma sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the plasma, preventing the color change and thereby providing an effective measure of the total antioxidant status of the plasma. The assay results are expressed as mmol Trolox equivalent/L (Eq/L), and the precision of this assay is excellent, being lower than 3% (Cao and Prior, 1998). 2.3.2. Sera sulfhydryl (SH) group Free sulfhydryl groups of sera samples were assayed according to the method of Ellman (1959) as modified by Hu et al. (1993). Briefly, 1 mL of buffer containing 0.1 M Tris, 10 mM EDTA, pH 8.2, and 50 mL sera was added to cuvettes followed by 50 mL 10 mM DTNB in methanol. Blanks were run for each sample as a test, but there was no DTNB in the methanol. Following incubation for 15 min at room temperature, sample absorbance was read at 412 nm on a spectrophotometer. Sample and reagent blanks were subtracted. The concentration of sulfhydryl groups was calculated using reduced glutathione as free sulfhydryl group standard and the result was expressed as millimolars (Erel, 2004). 2.3.3. Total oxidant status (TOS) The total oxidant status of the sera was measured using a novel automated colorimetric measurement method for TOS developed by Erel (2005). In this method, oxidants present in the sample oxidize the ferrous ion-o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color
108
I. Camkerten et al. / Veterinary Parasitology 161 (2009) 106–109
intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (mmol H2O2 equivalent/L).
significantly lower in DSM as compared with HD. No significant differences were observed in TAC between DSM and HD. Results of analyzed parameters are presented in Table 1.
2.3.4. Lipid hydroperoxide (LOOH) Sera LOOH levels were measured with the ferrous ion oxidation–xylenol orange assay. The principle of the assay depends on the oxidation of ferrous ion to ferric ion via various oxidants and the produced ferric ion is measured with xylenol orange. LOOHs are reduced by triphenyl phosphine (TPP), which is a specific reductant for lipids. The difference between with and without TPP pretreatment gives LOOH levels (Arab and Steghens, 2004).
In the present study, we found that the oxidative/ antioxidative balance shifted towards oxidative in DSM. TOS, LOOH levels, and OSI were significantly higher and SH levels were significantly lower in DSM compared with HD. To the best of our knowledge, this is the first report showing that an association between high TOS, LOOH, OSI and low SH in DSM. It is known that inflammatory cells are increased as a result of inflammation in animals with mange; recruited neutrophiles and macrophages produce reactive oxidants such as hydrogen peroxide (H2O2), hypochlorite and oxygen radicals, and these reactive oxygen substances produced by cells of the immune system show potent cytotoxic effects on parasites as well as other pathogenic organisms (Yaralıog˘lu-Gu¨rgo¨ze et al., 2003). Free radicals induce or contribute to adverse effects on the skin, including erythema, edema, wrinkling, inflammation, autoimmune reactions, hypersensitivity, and keratinization abnormalities (Bickers and Athar, 2006). Lipid peroxidation can be harmful for skin due to alternations in the membrane structure and permeability (Portugal et al., 2007). Some authors showed lipid peroxidation levels increased in sheep with sarcoptic mange (Yaralıog˘lu-Gu¨rgo¨ze et al., 2003), in dog with demodicosis (Dimri et al., 2008) and in patient with dermatitis (Tsukahara et al., 2003). Our study has also shown that LOOHs levels and TOS were significantly higher in DSM than that of HD which revealed that DSM is associated with oxidative stress. Sera SH groups act as important cellular scavengers of peroxides and so help to protect cells from damage by these molecules. Decrease in SH level not only impairs cells’ response to oxidants, but also changes the functions of inflammatory cells (McKeown et al., 1984). It has been reported that SH levels decrease in some skin diseases including Behcet’s Disease and measles (Erbay et al., 2003; ¨ stu¨ndag, 2006). Individual antioxidants level or Is¸ık and U activity indicates the antioxidant characteristics of only one antioxidant, whereas TAC may represent the total antioxidant characteristics of all antioxidants found in the sera (Erel, 2004). Our study has also confirmed a significant decrease in SH levels in DSM compared to HD. No significant difference was found between TAC levels of both groups. This amazing condition can be originated from increase in enzymatic antioxidant response. However, TAC levels were not reduced, and the oxidant/antioxidant balance shifted significantly to the oxidant side, because other indicators of oxidative status, i.e., LOOH, TOS and OSI levels, were significantly increased in DSM. Our search of the literature showed no other study investigating the relation between the sarcoptic mange in animals and oxidative stress, except the study by Yaralıog˘lu-Gu¨rgo¨ze et al. (2003) and Dimri et al. (2008a).
2.3.5. Oxidative stress index (OSI) The TOS to TAC ratio was regarded as the OSI. To perform the calculation, the result unit of TAC, mmol Trolox equivalent/L, was changed to mmol Trolox equivalent/L, and the OSI value was calculated as follows: OSI = [(TOS, mmol/L)/(TAC, mmol Trolox equivalent/ L) 100] (Aycicek et al., 2005). 2.4. Apparatus A Cecil 3000 spectrophotometer with a temperature controlled cuvette holder (Cecil) and an Aeroset automated analyzer (Abbott) were used. 2.5. Statistical analysis The statistical analysis of the data was carried out with the SPSS software 11.0 (SPSS Inc., Chicago, IL, USA). Wilcoxon signed rank test (SH, TOS and LOOH) and paired t test (TAK and OSI) were used to compare two groups. Values obtained were expressed as mean S.D. The differences were considered to be significant when P 0.05. 3. Results Dermal scraping for microscopic examination in all dogs of study group revealed S. scabies. The TOS and LOOH sera levels were found to be significantly higher in the dogs with sarcoptic mange (DSM) compared to healthy dogs (HD). The OSI was also significantly higher in the DSM than in the HD. Otherwise, SH sera levels were found to be Table 1 Oxidant/antioxidant parameters in dogs with sarcoptic mange. Parameters
Control group
Study group
TOS (mmol H2O2 equivalent/L) LOOH (mmol H2O2 equivalent/L) TAC (mmol Trolox equivalent/L) SH (mmol/L) OSI (arbitrary unit)
12.22 0.95 6.99 1.12 1.65 0.50 0.20 0.15 0.08 0.21
20.39 4.31** 15.56 5.74** 1.67 0.69a 0.17 0.35* 0.15 0.76**
TOS: total oxidant status; LOOH: lipid hydroperoxide; TAC: total antioxidant capacity; SH: sulphydril; OSI: oxidative stress index. a Insignificant. * P 0.05. ** P 0.01.
4. Discussion
I. Camkerten et al. / Veterinary Parasitology 161 (2009) 106–109
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