Antioxidant effects of vitamin E as an adjunct to Marbofloxacin in management of pyoderma in dogs

Introduction

Canine pyoderma, is one of the most frequently diagnosed dermatopathy in dogs, is characterized by a bacterial skin infection. It is widely recognized as a secondary condition caused by underlying factors that disrupt the skin barrier or immune defenses (Bajwa, 2016) [1] . Dogs are particularly predisposed to pyoderma due to the unique characteristics of their skin consisting of a thin stratum corneum, lack of lipid plug in the hair follicles and high skin pH which possess a risk for bacterial invasion, subsequent growth and over colonization (Sasaki et al., 2007) [18] . The condition is most commonly caused by gram positive bacteria (Staphylococcus) (Devriese et al. 2005) [6] which may be superficial, affecting the epidermis or surface or deeper structures of the skin characterized by pustules, papules, pruritus, and alopecia (Bourguignon et al. 2013) [4] . Oxidative stress can be defined as the phenomenon of cell and tissue damage as a result of the imbalance between oxidant/antioxidant substances in body (Puppel et al., 2015) [15] . Reactive oxygen species (ROS) are a group of oxygen based free radicals which gets elevated during oxidative stress, causes biomolecular damage manifested by lipid peroxidation (Mehar et al., 2018) [11] . ROS-induced cellular damage was delayed or prevented by antioxidants (Paulsen and Carroll 2013) [14] . ROS-induced cellular damage was delayed or prevented by antioxidants (Paulsen and Carroll 2013) [14] . Antioxidants include high-molecular-weight antioxidant enzymes represented by glutathione peroxidase, superoxide dismutase, and catalase enzymes (Trouba et al. 2002) [21] . Vitamin E, the most powerful antioxidant, is concentrated in cell membranes. It is considered to be the first line of defence against lipid peroxidation, protecting cell membranes at an early stage of free radical attack through its free radical quenching activity (Kapun et al. 2014) [9] . It is an important constituent of sebum and is constantly secreted to the surface of the skin. Due to the high concentration of fats in the keratinous layer, this lipophilic antioxidant is expected to play an important part in removing ROS in the skin during oxidative stress. This work aimed to assess oxidative stress levels in pyoderma infected dogs and therapeutic response to Vitamin E supplementation as an adjunct treatment along with systemic antibiotic.

Materials and Methods

The proposed work was carried out on the clinical cases of dogs suffering with pyoderma, presented to the Veterinary Clinical Complex, College of Veterinary Science and Animal Husbandry, Rewa, and the Divisional Veterinary Hospital, Bichhiya, Rewa along with private clinics for various dermatological conditions.

Experimental design

A total of 18 dogs diagnosed with pyoderma and exhibiting clinical signs of pyoderma like erythema, alopecia, pruritus, papules, crusts, pustules, epidermal collarettes, Pus in skin etc were randomly allocated into Two therapeutic groups (T 1 , T 2 ,) to evaluate the efficacy of systemic, and systemic plus vitamin E, and In addition, six apparently healthy dogs were included as the control group (T 0 ) and did not receive any treatment. In therapeutic Group (T 1 ) Marbofloxacin was prescribed @ 2mg/kg b.wt. OD, PO for 21 days. Group (T 2 ) Marbofloxacin was prescribed @ 2mg/kg b.wt. OD, PO for 21 days plus vitamin E @ 5mg/kg b.wt. OD, PO for 21 days. In the study, all animals included in the therapeutic trial were provided supportive treatment i.e., Hydroxyzine hydrochloride @ 2 mg/kg, PO OD for one week. and Syrup vitabest derm administered orally at 5-10 ml once daily for one week.

Diagnosing canine pyoderma

Clinical diagnosis of canine pyoderma was based on the presence of erythema, alopecia, pruritus, papules, crusts, pustules, epidermal collarettes, Pus within the skin was assessed, and impression smear examination were taken and examined microscopically by Mendelsohn et al. (2006) [12] Parameters studied The blood of examined animals was drawn using heparinized anticoagulant tubes to prepare plasma and erythrocytes lysates. Briefly, the red cells were collected by centrifugation (3000 rpm for 15 min) and plasma was drawn off, then cells were washed with three times with cold saline, with centrifugation after each wash. The resultant red cell pellets was used for estimation of the levels of LPO (lipid peroxidase) enzymes using method suggested by Rehman (1984) [16] . The resultant red cell pellets were lysed by adding (1:10 dilution) of cold distilled water to the estimated pellet volume and red cell stroma was removed by centrifuging. The resulting clarified supernatant was used for estimation of the levels of superoxide dismutase and catalase enzymes using Madesh and Balasubramanian (1998) [10] and Brannan et al. (1981) [5] , respectively.

Statistical analysis

The data generated during the study were analysed using standard statistical procedures as described by Snedecor and Cochran (1994) [20] . Results were expressed as mean ± standard error, and differences between groups and treatment days were evaluated for statistical significance. [8] . Folliculitis damages hair follicles causing alopecia (Santaro et al., 2015) [17] and is exacerbated by pruritus and self-trauma. Pustules rupture oozing serum and WBCs along with bacteria forming dried crusts and further perpetuating the infection (Beco et al., 2013). Healing pustules leave circular scaling as epidermis regenerates causing formation of epidermal collarettes (Millier et al., 2013). Deep bacterial invasion may lead to tissue necrosis and further ulcer formation. In chronic inflammations, it may stimulate melanocytes causing post inflammatory hyper-pigmentation (Santaro et al., 2015) [17] .

Results and Discussion

Impression smear examination

Impression smears from the lesions of superficial pyoderma affected dogs were stained with Giemsa stain and EMB (Eosin methylene blue) and observed under oil immersion (100 x) which revealed the presence of cocci and rod bacteria in most of the cases along with degenerated neutrophils (Plate 02 A 100x). impression smear revealing large no. of rods (bacilli) (Plate no. 02 B 100x), were recorded during microscopic examination of stained impression smears Similar observations as in the present study have been previously recorded by Bloom and Rosser (2001) [3] , Hiller et al. (2006) [7] and Veeranki (2015) [22] .

Oxidative stress parameters Lipid peroxidase (µmol/L)

The Mean ± S.E. values of LPO before and after treatment in the control and different treatment groups across different days are presented in Table 1 A significantly (P ≤ 0.05) higher LPO value was recorded in group T 1 (4.29 ± 0.09 bC ) and group T 2 (4.56 ± 0.06 cD ) compared with the healthy control group (T 0 ) on day 0. These findings are consistent with Mehar et al. (2018) [11] and Sivel et al. (2022) [19] . Who found a significant increase in mean values of LPO in dogs affected with pyoderma when compared with healthy control animals. Bacterial infection may lead to increased liberation of reactive oxygen species (ROS) causing lipid peroxidation reflected in form of increased lipid peroxidase activity (Miller et al., 2013) [13] . Similarly, cytokines and neutrophils might also release ROS causing oxidative damage. Skin inflammation also leads to cellular membrane damage and increased lipid peroxidation. (Santoro et al., 2015) [17] . Treatment groups (T 1 -T 2 ) exhibited progressive declines in LPO levels from Day 7 onwards (P ≤ 0.05), attaining values approach to the control group by Day 21, which were significant (P ≤ 0.05) when compared to Day 0 values. Group T 2 showed the most rapid reduction by Day 14, stabilizing thereafter and becoming nearly comparable to controls. Vitamin supplemented antibiotic group (T 2 ) performed best in a synergistic fashion as compared to other groups in reducing the oxidative stress and resultant skin healing in pyoderma by reducing bacterial load along with reduction in oxidative stress. Similarly, Verma (2023) [23] also recorded improvements after different treatment with like clindamycin along with Vit E as compared to clindamycin alone.

The Mean ± S.E. values of SOD before and after treatment in the control and different treatment groups across different days are presented in Table 2 A significantly lower SOD values were observed in group T 1 (1009.5 ± 67.31 dA ) and group T 2 (959.17 ± 54.96 aA ) compared with the healthy control group (T 0 ) on day 0. These findings are consistent with Mehar et al. (2018) [11] and Sivel et al. (2022) [19] . Treatment groups (T 1 -T 2 ) exhibited progressive increases in SOD levels from Day 7 onwards (P ≤ 0.05), attaining values approaching the control group by Day 21, which were significant when compared to Day 0 values. Group T 2 showed the most rapid and substantial elevation, becoming nearly comparable to controls by Day 14 and stabilizing. In contrast, T 1 showed moderate progressive improvements but remained somewhat below control levels. Similar Improvements were also recorded by Verma (2023) [23] after regimens like clindamycin with vitamin E.

The Mean ± S.E. values of Catalase before and after treatment in the control and different treatment groups across different days are presented in Table 3 catalase values were significantly lower in group T 1 (936.5 ± 25.34 aA ) and group T 2 (931.5 ± 32.56 aA ) compared with the healthy control group (T 0 ) on day 0 These findings were in correspondence with the reports of Mehar et al. (2018) [11] , Sivel et al. (2022) [19] Treatment groups (T 1 -T 2 ) exhibited progressive increases in SOD levels from Day 7 onwards (P ≤ 0.05), attaining values approaching the control group by Day 21, which were significant when compared to Day 0 values. Group T 2 showed the most rapid and substantial elevation, surpassing other treatment and nearing control levels by Day 14. In contrast, T 1 showed moderate progressive improvements but remained below control levels. Similarly, Verma (2023) [23] also recorded improvements in catalase values in pyoderma dogs when treated with clindamycin alone or along with Vit. E. Adjunctive Vitamin E enhanced antibiotic efficacy, reduced oxidative damage, and accelerated healing, supporting its role as a valuable antioxidant in canine pyoderma management.

Acknowledgement

AcknowledgmentsThe author is very much grateful to the Dean, College of Veterinary Science and A.H., Rewa, Madhya Pradesh India for providing the facilities for conducting this research.

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