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Assay of alterations in oxidative stress markers in pigs naturally infested with Sarcoptes scabiei var. suis Umesh Dimri ∗ , S. Bandyopadhyay, Shanker Kumar Singh 1 , Rakesh Ranjan 2 , R. Mukherjee, M.I. Yatoo, P.H. Patra 3 , U.K. De, A.A. Dar Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India

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Article history: Received 31 March 2014 Received in revised form 27 May 2014 Accepted 6 June 2014 Keywords: Mange Oxidative-stress Pigs Sarcoptic

a b s t r a c t The present study aimed to examine the status of antioxidant systems of the pigs naturally suffering from sarcoptic mange. Fifty nine pigs were divided into three groups, healthy control (group I, n = 15), subclinical sarcoptic mange (group II, n = 22) and clinical sarcoptic mange (group III, n = 22). To assess the status of antioxidant systems; lipid peroxides (LPO), reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), ascorbic acid, zinc and copper concentrations in the blood samples as well as LPO, SOD, CAT and glutathione-s-transferase (GST) activities in the skin were measured. The GSH, SOD, GPx, ascorbic acid, zinc, copper concentrations in blood were significantly lower in the pigs suffering from clinical and subclinical sarcoptic mange, when compared with the healthy control. However, LPO content of these infested pigs was significantly higher. The CAT, SOD and GST activities in the skin of the diseased pigs were significantly lower, whereas LPO was significantly higher as compared to the healthy control. From the present study, it may be concluded that sarcoptic mange bestows remarkable alterations in the oxidative stress markers and imposes compromisation of the antioxidant status of the infested pigs. © 2014 Published by Elsevier B.V.

1. Introduction Parasitic mites of the genus Sarcoptes infest up to 40 different mammalian hosts across 17 families (Camkerten et al., 2009). Commonly described hosts include dogs, pigs, foxes and wombats. Sarcoptes scabiei var. suis, is one of the

∗ Corresponding author at: Division of Medicine, Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India. E-mail addresses: [email protected], [email protected] (U. Dimri). 1 Address: Department of Veterinary Clinical Medicine, DUVASU, Mathura, Uttar Pradesh, India. 2 Address: Department of Teaching Veterinary Clinical Complex, GADVASU, Ludhiana, Punjab, India. 3 Address: Department of Pharmacology and Toxicology, Veterinary College, Agartala, Tripura, India.

most common causes of mange infestation in swine with significant losses to the primary industries, especially in pig herds, where it leads to decreased growth rate and subsequently reduced feed conversion efficiency (Dimri et al., 2008a). Clinical symptoms of sarcoptic mange include generalized focal erythema, pruritus and later encrustations, especially in the ears progress to the skin of the back, which may become very thickened and crack open leaving deep wounds imparting secondary infections (Das et al., 2010). Previously, S. scabiei var. suis infestation in pigs has been reported globally including India (Lowenstein et al., 2006; Das et al., 2010). Notwithstanding the economic impact and significance of S. scabiei infestation in both the human and animal populations, the pathogenesis to this disease is not well understood. Pro-inflammatory cytokines including interleukin 1␣ (IL-1 ␣) and IL-1␤, tumor necrosis factor␣ (TNF-␣) and interferon-␥ (IFN-␥) appear to be the key

http://dx.doi.org/10.1016/j.vetpar.2014.06.015 0304-4017/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Dimri, U., et al., Assay of alterations in oxidative stress markers in pigs naturally infested with Sarcoptes scabiei var. suis. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.015

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effectors in the pathogenesis of scabies (Arlian et al., 1996; Lalli et al., 2004). Pro-inflammatory cascade can lead to excessive generation of the reactive oxygen species (ROS) and reactive nitrogen species (RNS). Even though, in skin diseases the body possesses an array of potent antioxidants protection such as superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx) and antioxidant vitamins A, E, and C (Bickers and Athar, 2006), changes in the oxidative stress indices has been reported in mites infested buffaloes (Dimri et al., 2008b), dogs (Dimri et al., 2008a; Singh et al., 2011), and sheep (Dimri et al., 2010). Therefore, the present study aimed to investigate the alterations in antioxidant defense parameters of naturally S. scabiei var. suis infested pigs. 2. Materials and methods 2.1. Experimental design The study included pigs of 1–2 years age group naturally infested with S. scabiei var. suis. Pigs revealing dermatological manifestations for instance itching, dermatitis especially in the ears, head, back, neck, shoulders and legs were examined. Skin lesions suggestive of clinical mange were reddening, formation of crusts, hyperkeratosis, alopecia with wrinkled, thickened, rough, raised and thick skin. From each side of the affected pigs and healthy controls two areas of one square centimeter dimension were marked and skin-scraping samples were collected in test tubes containing 3 mL of 10% potassium hydroxide solution. After gentle heating for 2–3 min, a drop of this mixture was put on a glass slide and examined under microscope for presence or absence of the mite S. scabiei. The mite was identified by the third and fourth pairs of legs, which did not project beyond the margin of the body and by other morphological characteristics of the species. Ten microscopic fields were examined and number of mites per microscopic field was counted. Animal with skin lesions (crusts formation, hyperkeratosis and wrinkled, thickened, rough, raised and thick skin) and a minimum of three sarcoptic mites per microscopic field was classified as clinical sarcoptic mange (group III). Animal showing clinical manifestation of itching but no skin lesions (the pigs which had not developed the skin lesions of crusts formation, hyperkeratosis and wrinkled, thickened, rough, raised and thick skin) and 1–2 mites per microscopic field was classified as subclinical sarcoptic mange (group II). Twenty two pigs each from sub-clinical (group II) and clinical mange (group III) were selected randomly for further sampling. Another 15 healthy pigs showing no itching and skin lesions and negative for mites were selected to serve as healthy control (group I). 2.2. Collection of samples Blood samples from selected animals were collected from the ear vein in heparinized vials. Small pieces of skin weighting around 10 g were collected and placed in individual wide mouth glass vial containing 100 mL chilled phosphate buffer saline (pH 7.4). To avoid further oxidative damage, skin samples were placed in ice-boxes at temperature below 4 ◦ C without adding any chemical antioxidant

and transported to the laboratory within an hour. The tissue samples were washed with chilled phosphate buffer saline (pH 7.4) and blot dried before being homogenized in 1.15% Tris–potassium chloride buffer. 2.3. Laboratory examinations 2.3.1. Oxidative stress indices The concentration of malonaldialdehyde (MDA), a reliable marker of lipid peroxidation, was estimated in haemolysate following the method suggested by Placer et al. (1966). The nmole of MDA per mL of haemolysate was calculated using 1.56 × 105 as extinction coefficient (Utley et al., 1967). Lipid peroxide level in erythrocytes was expressed as nmole MDA/mg of haemoglobin. Endogenous lipid peroxides in tissue homogenates were measured after suitable dilution following the method of Okhawa et al. (1979). Total protein in tissue homogenate was determined spectrophotometrically by the modified Biuret and Dumas method (Varley, 1980). Values were expressed as nmole MDA/mg of protein. Superoxide dismutase (SOD) activity in haemolysate and tissue homogenate was measured after suitable dilution according to the method of Marklund and Markund (1974) with certain modifications suggested by Minami and Yoshikawa (1979). Each unit of SOD activity is defined as the quantity of enzyme that inhibited auto-oxidation of pyrogallol by 50% under suitable experimental conditions. Activity was expressed as units/mg of protein. Catalase activity in tissue homogenate was estimated spectrophotometrically at wavelength of 240 nm after appropriate dilution, according to the method of Cohen et al. (1970). The value was expressed as units/mg of protein. The concentration of GSH in RBC suspension was estimated by dithio-bis-2-nitro benzoic acid (DTNB) method as per the procedure of Prins and Loos (1969). The GSH concentration in the test sample was calculated by employing the molar extinction coefficient of DTNB–GSH conjugate 13600/M/X cm and expressed as ␮m/g of haemoglobin. GPx activity in haemolysate was determined by the method of Paglie and Valentie (1967). The change in the absorbance was recorded at 340 nm for 4 min GPx activity and expressed as NADPH nmol oxidized per minute per mg of haemoglobin (U/mg Hb). Glutathione-s-transferase was estimated following the increase in absorbance at 340 nm using 1-chloro-2,4-116 dinitrobenzene (CDNB) as substrate (Habig et al., 1974). Phosphate buffer (2.8 mL) was taken in a test tube and 0.1 mL of GSH and 20 mL of tissue homogenates were added to it. At last, 0.1 mL of CDNB was added and increase in absorbance was noted at 340 nm after every 30 s for 3 min. 2.3.2. Zinc and copper concentrations Zinc and copper concentrations in serum were estimated by atomic absorption spectrophotometer (AAS 4141, Electronic Corporation of India Limited, Hyderabad) after acid digestion. Briefly, 5 mL of blood sample was mixed with equal volume of concentrated nitric acid and kept overnight at room temperature. Next day, this mixture was wet digested using micro-digestion bench after mixing with double acid mixture (four parts conc. nitric acid

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Table 1 Blood levels of lipid peroxides, reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx), ascorbic acid, zinc and copper in pigs with and without sarcoptic mange. Parameters

Healthy

LPO (␮mol/L) GSH (␮M GSH/mg Hb) SOD (U/mg Hb) GPx (mU/mg Hb) Ascorbic acid (mg/100 mL) Zinc (mg/mL) Copper (mg/mL)

3.58 ± 0.25 0.52 ± 0.01c 1.22 ± 0.03c 578 ± 9.51c 0.18 ± 0.00c 1.59 ± 0.00c 2.12 ± 0.05c

a

Sub-clinical mange

Clinical mange

5.68 ± 0.11 0.32 ± 0.00b 0.93 ± 0.01b 408 ± 3.01b 0.06 ± 0.00b 1.41 ± 0.00b 1.92 ± 0.03b

8.31 ± 0.09c 0.14 ± 0.00a 0.54 ± 0.00a 178 ± 1.33a 0.05 ± 0.00a 0.66 ± 0.01a 0.82 ± 0.00a

b

Values bearing different superscript (a, b and c) in a row differ significantly (P < 0.05). Table 2 Status of different oxidative indices in skin of pigs. Parameters

Healthy

Sub-clinical mange

Clinical mange

LPO (nmole MDA/mg protein) SOD (U/mg protein) Catalase (U/mg protein) GST (␮mol/mg protein)

3.05 ± 0.00a 8.53 ± 0.02c 428 ± 6.05c 11.59 ± 0.03c

4.27 ± 0.00b 7.14 ± 0.01b 329 ± 3.80b 9.92 ± 0.01b

5.79 ± 0.00c 5.27 ± 0.01a 311 ± 2.47a 7.14 ± 0.02a

Values bearing different superscript (a, b and c) in a row differ significantly (P < 0.05).

and one part 70% perchloric acid) (Kolmer et al., 1951). The concentrations of zinc and copper in digested samples were estimated by recording absorbance of digested samples at specific wavelengths of 239.5 and 324.7 nm, respectively and at an operating current of 5 mA using acetylene and air mixture as fuel and oxidant. The analytical quality was maintained by running a reagent blank and standard with each batch. 2.3.3. Serum ascorbic acid Clear serum, separated by centrifugation and stored at −20 ◦ C was used for ascorbic acid assay. Ascorbic acid level in the serum was determined by 2,6dichlorophenolindophenol (DCPIP) titration method (Varley, 1980). This blue compound, on titration with a solution of ascorbic acid, was reduced to a colourless leucobase, the vitamin C being oxidized to dehydroascorbic acid.

healthy control. Interestingly, the values of GSH, SOD, GPx, and ascorbic acid of subclinical group were significantly higher than the clinical mange group (P ≤ 0.05), whereas the LPO level of subclinical group was significantly lower (P ≤ 0.05) than the clinical group. Blood zinc and copper concentrations in pigs with clinical and subclinical mange were significantly (P ≤ 0.05) lower than the healthy group, whereas both the concentrations were significantly (P ≤ 0.05) lower in clinical mange group as compared with the subclinical mange group. Skin LPO level in pigs with clinical mange was approximately twice to the level observed in the healthy pigs (Table 2). Activities of all other oxidative stress marker enzymes (SOD, CAT and GST) in the skin samples were remarkably lower (P ≤ 0.05) in the both subclinical and clinical mange groups in comparison to the healthy control. Activities of SOD, CAT and GST in pigs with clinical mange were also significantly lower (P ≤ 0.05) than the corresponding values observed in the subclinical group (Table 2).

2.4. Statistical analysis The values were expressed as mean ± SE and data were analyzed by one way Analysis of Variance followed by the post Hoc Duncan test using statistical software package, SPSS 16.0 (2007). The level of statistical significance for all comparison was established at P ≤ 0.05. 3. Results Blood LPO level in pigs with subclinical and clinical mange was significantly higher (P ≤ 0.05) than that in healthy pigs (Table 1). While, GSH level was significantly lower (P ≤ 0.05) in the both subclinical and clinical groups as compared with the healthy pigs. Similarly, activities of blood antioxidant enzymes GPx and SOD were also significantly lower (P ≤ 0.05) in these groups as compared with the healthy ones (Table 1). Serum ascorbic acid level was also significantly reduced (P ≤ 0.05) in the both subclinical and clinical mange groups in comparison to the

4. Discussion and conclusion During the normal metabolic processes free radicals are generated continuously inside the living system, but their rate of production increases when there are certain inflammatory or other disease conditions. Oxidative stress has been implicated to play important roles in aetiopathogenesis of various human and animal parasitic diseases (Chandramathi et al., 2009). It supervenes when generated free radicals exceedthe capacity of antioxidant defense of the body. Increased levels of lipid peroxides may be implicated in the pathology of skin lesions induced by Sarcoptes mites. Lipid peroxidation is a wellestablished mechanism of cellular injury and is used as an indicator of oxidative stress in cells and tissues. Lipid hydroperoxides are by-products of lipid peroxidation and increased levels of lipid peroxidation products are associated with parasitic infestations (Dimri et al., 2010; Singh et al., 2011; Dimri et al., 2008a,b). LPO is known to cause

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cellular injury by inactivation of membrane enzymes and receptors, depolymerizaton of polysaccharide, as well as protein cross linking and fragmentation (Luqman and Rizvi, 2006). LPO can be harmful for skin due to alteration in the structure and permeability (Portugal et al., 2008). Increased levels of MDA may be implicated in Sarcoptes-induced skin lesions, conferring clinical manifestations in the pigs with sarcoptic mange. Results of the present study are in agreement with the previous scientific reports demonstrating increased LPO levels to be associated with ecto-parasitic infestations in various species of animals including sheep with sarcoptic mange (Gurgoze et al., 2003), with psoroptic mange (Dimri et al., 2010), dogs with sarcoptic mange (Camkerten et al., 2009; Singh et al., 2011), with demodectic mange (Dimri et al., 2008a) and buffaloes with sarcoptic mange (Dimri et al., 2008b). The measurements of antioxidant enzymes activity like SOD, GPx and CAT are appropriate indirect ways to assess the status of antioxidant defense. In the present study, we found that the pigs with sarcoptic mange showed the exhaustion of these enzymes due to consistent free radical attack. Lower serum zinc and copper concentrations were observed in pigs suffering from sarcoptic mange. Likewise, blood ascorbic acid concentrations were also lower in pigs suffering from sarcoptic mange. Both zinc and copper are essential components of the antioxidant enzyme Cu–Zn superoxide dismutase. Therefore, overall utilization or sequestration of zinc and copper to neutralize the overproduction of ROS might be responsible for their lower blood concentrations in diseased animals (Singh et al., 2011). Enhanced LPO levels and reduced ascorbic acid concentrations in blood further substantiated increased oxidative stress and compromised systemic antioxidant defense of pigs suffering from sarcoptic mange. Vitamin E (␣-tocopherol) is an important antioxidant nutrient required by the body. Earlier strong correlation was documented between deficiency of this vitamin and sarcoptic mange in animals (Behera et al., 2011; Saleh et al., 2011). Again, vitamin C is a very important intracellular antioxidant, which is thought to be involved in recycling the ␣-tocopheryl radical back to ␣-tocopheral (Halliwell, 2006). The decreased level of the body antioxidant (GSH) and reduced activities of the antioxidant enzymes for instance GPx, CAT and SOD imply that S. scabiei var. suis infested pigs are in a state of significant oxidative stress, and an altered antioxidant defense mechanism is under operation. Potential antioxidants include either natural free radical scavenging antioxidant enzymes or the agents which are capable of augmenting the activities of these enzymes, including the GPx, GST, SOD, and CAT. This is in corroboration with our earlier observations (Dimri et al., 2010, 2008a,b). Significantly higher level of MDA (or TBARS) and reduced activities of SOD, CAT and GST in skin observed in the present study indicates a compromised antioxidant defense and supervened oxidative damage to the dermal tissues of pigs with clinical sarcoptic mange. SOD catalyses the formation of O2 from reactive oxygen species. A coproduct of SOD activity is H2 O2 , which is converted to H2 O by catalase. Free radicals induced cellular damages may be, at least partially, responsible for these

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Please cite this article in press as: Dimri, U., et al., Assay of alterations in oxidative stress markers in pigs naturally infested with Sarcoptes scabiei var. suis. Vet. Parasitol. (2014), http://dx.doi.org/10.1016/j.vetpar.2014.06.015

Assay of alterations in oxidative stress markers in pigs naturally infested with Sarcoptes scabiei var. suis.

The present study aimed to examine the status of antioxidant systems of the pigs naturally suffering from sarcoptic mange. Fifty nine pigs were divide...
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