The therapeutic efficacy of Aloe vera gel ointment on staphylococcal pyoderma in dogs.

BACKGROUND AND AIM: Staphylococcus pyoderma is a common problem in dogs that need a novel treatment rather than antibiotic therapy. The aim of this study was to investigate the therapeutic efficacy, anti-inflammatory, and antioxidative properties of Aloe vera (Aloe barbadensis) gel ointment on dogs' Staphylococcus pyoderma compared to gentamicin ointment.
MATERIALS AND METHODS: The inhibition zone of A. vera extract 20% and 40% and gentamicin 1% against Staphylococcus aureus was determined on well diffusion agar. Twenty Baladi local breed dogs were used as control negative group before intradermal inoculation with 105 CFU S. aureus. The animals were classified into four equal groups, control positive group without treatment (n=5), treated groups by 20% A. vera gel ointment (n=5), 40% A. vera gel ointment (n=5), and gentamicin ointment 1% (n=5). Topical application of A. vera and gentamicin ointments was carried out twice daily for 2 weeks until complete healing of dogs' pyoderma. Clinical evaluation was recorded. Inflammatory, oxidant, and antioxidant parameters were measured in serum.
RESULTS: The inhibition zone of A. vera extracts 20% and 40% was 19 mm and 23 mm, respectively, while gentamicin 1% was 18 mm. The half maximal inhibitory concentration (of A. vera 20% and 40% were 13.70 with R2=0.98. Dogs' pyoderma treated with A. vera gel ointment 20% and 40% were more likely to have low haptoglobin and tumor necrosis factor-α concentrations than gentamicin 1% ([odds ratio [OR]=4.6; 95% confidence interval [CI]=1.31-17.40; p<0.05]; [OR=5.2; 95% CI=1.04-22.30; p<0.05]), respectively.
CONCLUSION: It seems evident that A. vera has therapeutic effect, antibacterial, and anti-inflammatory effects against dogs' staphylococcal pyoderma than gentamicin that would support its further use rather than antibiotics in one health arena. Copyright: © Kamr, et al.

Introduction

Aloe vera (Aloe barbadensis) is a medicinal plant that has many functions include wound healing, immunomodulation, and hepatoprotection in addition to its antiviral, skin protective, antioxidant, antidiabetic, anti-inflammatory, antimicrobial, and anticancer properties [1]. A. vera is characterized by the presence of many active compounds, inclusive of amino acids, minerals, anthraquinones, chromones, nutrients, lipids, anthrones, carbohydrates, and flavonoids [2]. Canine superficial pyoderma is described as superficial bacterial contamination of the dermis and hair follicles that caused by Staphylococcus spp. [3]. Pyoderma can be caused by infectious, neoplastic, and/or inflammatory etiologies with consequences of the accumulation of neutrophilic exudates and associated with increased free radical production, oxidative stress [4,5]. Of interest, Staphylococcus aureus, which is considered methicillin-resistant, was isolated from dogs with recurrent pyoderma [6].

Acute-phase response is a distinguished systemic reaction of the body within 24-48 h after local or systemic diseases due to infection, tissues damage, surgery, trauma, and immunological impairment [7-9]. Serum amyloid A (SAA), haptoglobin (HP), and ceruloplasmin (CP) were produced by liver and considered prognostic biomarkers for acute inflammation in dogs as a result of tissue infection or inflammation [10,11]. Cytokines are small glycoproteins (~5–20 kDa) that have autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins (IL), lymphokines, and tumor necrosis factors (TNF) are produced by many types of cells that include macrophages and lymphocytes that are essential in host immune responses to infection, inflammation, trauma, sepsis, cancer, and reproduction [12]. However, the inflammatory and immunomodulatory roles of A. vera in dogs’ pyoderma need further investigation.

This study investigated the beneficial healing effect, antibacterial, anti-inflammatory, and antioxidative roles of A. vera in dogs’ staphylococcal pyoderma. We hypothesized that acute-phase proteins, cytokines, and oxidants will be elevated, while antioxidant biomarkers will be reduced in dog’s staphylococcal pyoderma; furthermore, A. vera gel ointment will modulate the clinical image inflammatory response in a better manner than gentamicin against staphylococcal pyoderma in dogs.

Materials and Methods

Ethical approval

All dogs were used and treated according to Animal Ethics Committee at College of Veterinary Medicine, University of Sadat City (Approval code VUSC-007-1-19).

Study period and location

This study was conducted between September 2018 and May 2019. The experiment was conducted at Animal Medicine and Infectious Diseases Department, teaching hospital and laboratory, University of Sadat City.

Animals’ criteria

A total of 20 Baladi local male adult dogs 2-3 years old were housed in individual cages for acclimatization for 3 weeks, with free access to food and water. According to the production company Pharma Swede, Egypt, dogs were dewormed by subcutaneous injection of ivermectin (Paramectin®) at a dosage of 10 mg/50 kg as a routine protocol against external parasites that would interfere with experimental induction of canine pyoderma.

Experimental design

Twenty male adult dogs of Baladi local breed were clinically healthy based on their physical examination. The animals were considered as a control negative group before induction of pyoderma (n=20). The dogs were inoculated intradermally in chest region with 1 mL broth containing 105 CFU staphylococcus [6,13]. The injected dogs were further classified into further subgroups after appearance of pyoderma lesions into control positive group without treatment (n=5), treated groups by 20% A. vera gel ointment (n=5), 40% A. vera gel ointment (n=5), and gentamicin ointment 0.1% (n=5). Topical application of A. vera and gentamicin ointments (Garamycin® 0.1%; Schering-Plough Company, USA) was carried twice daily for 2 weeks until complete healing of dogs’ pyoderma.

In vitro evaluation of antibacterial effect of A. vera extract

Antibacterial activity of A. vera extract was evaluated using agar diffusion test in which 15-20 mL of nutrient agar (Oxoid®) was poured on glass Petri plates of same size and allowed to solidify. Agar surface of each plate was streaked by a sterile cotton swab with S. aureus equivalent to 0.5 McFarland standards. Agar plate was punched with a sterile cork borer of 4 mm size; then, A. vera fresh extract 20% and 40% concentration, gentamycin 0.1%, pure A. vera, and pure ethanol were filled into its wells. The plate was allowed to stand by for 30 min. The plate was incubated at 37°C for 48 h and examined. The diameter of inhibition zones was measured [14].

A. vera gel ointment 20% and 40% preparation

Preparation of A. vera gel ointments 20% and 40% formula was carried out by adding 20 g and 40 g of A. vera gel, respectively, 3 g of wax and 5 mL of paraffin oil with 72 g and 52 g of Vaseline™, respectively. All components were mixed well into mortar until obtaining homogenous ointments. Gentamycin ointment was purchased as a commercial product from the pharmacy (Garamycin® 0.1%; Schering-Plough Company, USA). A. vera concentrations of 5%, 10%, and 15% did not give the optimal results against dogs’ pyoderma, so we used 20% and 40% A. vera concentrations in this study.

Sampling

Blood samples were collected from admitted dogs in EDTA and serum clot tubes for hematobiochemical analysis before induction and on 3rd, 7th, 10th, and 14th day after induction and treatment of Staphylococcus pyoderma. Samples were centrifuged at 2000× g for 10 min at 4°C. Serum and plasma were aliquoted into smaller volumes and stored at −80°C until analyzed.

Clinical examination and complete blood count

Physical and clinical examinations were determined for all dogs according to methods specified by Englar [15]. The hemogram and leukogram examinations were measured and calculated for each dog according to methods described by Turgeon [16]. Dog’s pyoderma lesions diameter (Ø) was measured and expressed as mm.

Oxidant, antioxidant, and inflammatory biomarkers measurements

Serum malondialdehyde (MDA), total antioxidant capacity (TAC), and catalase concentrations were measured by commercial kits (Bio Diagnostics, Ltd., Egypt) according to methods described by Ohkawa et al. [17], Koracevic et al. [18], Aebi [19], respectively. Serum CP, SAA, HP, and ILs; IL-1, IL-6, and IL-10 and TNF-α were determined by commercial ELISA kits (Shanghai Coon Koon Biotech., Ltd.; China) with inter- and intra-coefficient of variations <10 and good linearity (R2=0.95). All procedures were performed as described in the instructions of the manufactures.

Statistical analysis

Data were assessed for normality and were normally distributed based on Shapiro–Wilk test. Data were expressed as mean with standard error. Comparisons between the groups were performed by one-way ANOVA using SPSS Statistics 24.0 version [20]. Receiver operating characteristic curve and the half maximal inhibitory concentration (IC50) were calculated using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). Univariate logistic regression includes odds ratio (OR) and 95% confidence interval (CI) was calculated. Significance was set at p<0.05.

Results

In vitro evaluation of antibacterial effect of A. vera extracts and drug efficacy on dogs’ staphylococcal pyoderma

A. vera 20% and 40% extracts and gentamycin 0.1% inhibition zone diameter were 19 mm, 23 mm, and 18 mm, respectively, compared by raw A. vera gel and pure ethanol, as shown in Figure-1a. A. vera concentrations 20% and 40% had IC50=13.70 with 95% CI=12.54-14.88 with R2=0.98; p<0.05; (Figure-1b).

Figure-1

In vitro evaluation of antibacterial effect of Aloe vera extracts and drug efficacy on dogs’ staphylococcal pyoderma. (a) In vitro evaluation of antibacterial activity of different concentrations A. vera extracts 20% (1) and 40% (2), gentamicin (3), raw A. vera gel (4), and pure ethanol (5); (b) A. vera concentrations 20% and 40% had half maximal inhibitory concentration (IC50) than A. vera 5%, 10%, and 15% concentrations (IC50=13.70; 95% confidence interval=12.54-14.88 with R2=0.98).

Clinical findings between the studied groups of dogs

There was a significant increase in diameter of pyoderma lesions in infected dogs on 3rd-10th day without treatment compared to control negative group (p<0.05; Table-1). In dogs treated with A. vera gel ointment 20% and 40% and gentamicin ointments 0.1%, the diameter of the pyoderma lesions was significantly reduced on the 3rd and 7th day post-treatment (DPT) compared to control positive group dogs (p<0.05; Figure-2a and b and Table-1), while there is no statistical variation between dogs treated with A. vera gel ointment 20% and 40% and gentamicin ointments 0.1% and control ones (p>0.05; Table-1). Control positive group with Staphylococcus pyoderma showed sloughing of epidermis, severe inflammatory cells infiltration in the dermis, necrosis, and collagen degeneration (Figure-3a). A. vera gel ointment treated group with concentration of 40% showed normal epidermis and few inflammatory cells infiltration in the dermis on the 14th day of treatment (Figure-3b). The mean values of body temperature, respiratory, and pulse rates were elevated in infected dogs on the 3rd-10th day without treatment compared to healthy control negative dogs (p<0.05; Table-2), but not with the 14th day without treatment (p>0.05). There was significant increase for the values of body temperature, respiratory, and pulse rates in dogs treated with A. vera gel ointment 20% and 40% and gentamicin ointment 0.1% at different times of treatment on the 3rd DPT (p>0.05; Table-2), then returned to their normal values from the 7th to 14th DPT when compared to control healthy ones (p>0.05; Table-2).

Table-1

Caliber measurement of dog’s pyoderma lesions diameter (Ø) (mm). Values expressed as mean with SE.

Variables	Ø of pyoderma lesion (mm)
Control negative dogs (before induction) (n=20)	No lesions
Infected dogs without treatment Group 1 (n=5)
3 DWT	8.12±0.17b
7 DWT	6.1±0.2c
10 DWT	4.3±0.28d
14 DWT	2.2±0.18e
Dogs treated with Aloe vera gel ointment 20% Group 2 (n=5)
3 DPT	6±0.2c
7 DPT	3.8±0.2d
10 DPT	Cured
14 DPT	Epidermal collarette (lesion with a circular scaly edge)
Dogs treated with Aloe vera gel ointment 40% Group 3 (n=5)
3 DPT	6±0.3c
7 DPT	3.6±0.4d
10 DPT	Epidermal collarette (lesion with a circular scaly edge)
14 DPT	Complete healing
Dogs treated by gentamicin sulfate 0.1% ointment Group 4 (n=5)
3 DPT	6.48±0.4c
7 DPT	3.85±0.11d
10 DPT	Cured
14 DPT	Epidermal collarette (lesion with a circular scaly edge)

SE=Standard error, DWT=Day without treatment, DPT=Day post-treatment, n=Number, mm=Millimeter. Means with different letter superscripts in the same column are significantly different at p<0.05

Figure-2

Measurement of pyoderma lesion diameter (ø) in a dog topically treated with Aloe vera gel ointment 40% (a) lesion ø 6 mm on the 3rd day post-treatment (DPT); (b) lesion ø 3 mm at the 7th DPT.

Figure-3

Histopathological findings. (a) Control positive group was showing sloughing of epidermis, severe inflammatory cells infiltration in the dermis, necrosis, and collagen degeneration; (b) Aloe vera treated group with concentration of 40% was showing normal epidermis and few inflammatory cells infiltration in the dermis on the 14th day post-treatment.

Table-2

Clinical examination of dogs. Values expressed as mean with SE.

Variables	Body temperature (°C)	Respiratory rate (cycle/min)	Pulse rate (beat/min)
Control negative dogs (before induction) (n=20)	37.96±0.14a	24.6±1.3a	80±1.7a
Infected dogs without treatment Group 1 (n=5)
3 DWT	38.9±0.1b	32.5±1.04b	91.25±1.49b
7 DWT	38.4±0.08c	29±1.29c	85±0.4c
10 DWT	38.35±0.2c	27.75±1.03c	84.25±0.85c
14 DWT	38.2±0.85a	26±0.4a	82.4±1.6a
Dogs treated with Aloe vera gel ointment 20% Group 2 (n=5)
3 DPT	38.7±0.24b	28.6±1.8c	85.3±2.06c
7 DPT	38.2±0.1a	25.4±0.8a	83.4±0.8a
10 DPT	38.3±0.4a	26±1.4a	82±1.2a
14 DPT	38.2±0.08a	25.2±1.06a	80.8±1.4a
Dogs treated with Aloe vera gel ointment 40% Group 3 (n=5)
3 DPT	38.6±0.2b	29.5±0.6c	86.4±1.8c
7 DPT	38.2±0.14a	24.8±1.54a	82±1.34a
10 DPT	37.8±0.3a	25±0.8a	80.6±0.88a
14 DPT	38±0.2a	23.3±1.6a	81.4±2.08a
Dogs treated by gentamicin sulfate 0.1% ointment Group 4 (n=5)
3 DPT	38.6±0.09b	30.2±0.8c	88.4±0.85c
7 DPT	38±0.4a	26.3 ±1.2a	80.4±2.4a
10 DPT	38.2±0.1a	24.2±0.8a	82±0.88a
14 DPT	37.8±0.2a	25.5±1.4a	82.5±1.4a

SE=Standard error, DWT=Day without treatment, DPT=Day post-treatment, n=Number. Means with different letter superscripts in the same column are significantly different at p<0.05

Hematological findings between the studied groups of dogs

The mean values of the red blood cells (RBCs) and hemoglobin (Hb) were significantly decreased in infected control positive dogs on the 3rd-14th day without treatment compared to control negative ones (p<0.05; Table-3). Packed cell volume (PCV) values were significantly decreased in infected control positive dogs on the 3rd-10th day without treatment compared to control negative ones (p<0.05; Table-3), but not on the 14th day without treatment (p>0.05). In A. vera ointment gel 20% treated group, RBCs and Hb values were decreased on the 3rd DPT (p<0.05), then returned to their baseline on the 7th-14th DPT when compared to control ones (p>0.05). No significance was recorded in PCV values between groups treated with A. vera gel ointment 20% and healthy ones (p>0.05). No significant change for RBCs values in A. vera 40% treated group at different time points of treatment when compared with healthy ones before induction (p>0.05). In A. vera 40% treated group, Hb values were decreased on the 3rd DPT, while PCV values were elevated on the 10th DPT (p<0.05), then returned to their normal values on the 14th DPT (p>0.05). In gentamicin 0.1% treated group, no significant change for RBCs values at different time points of treatment when compared with healthy ones (p>0.05), but Hb and PCV values were decreased on the 3rd DPT than healthy ones (p<0.05), then returned to their normal values when compared to control ones (p>0.05; Table-3). Moreover, white blood cells (WBCs) and neutrophils were significantly increased in infected dogs on the 3rd-14th day without treatment, while in dogs treated with A. vera gel ointment 20%, WBCs and neutrophils were elevated on the 3rd, 7th, and 10th DPT (p<0.05) and returned to their normal values on the 14th day when compared to healthy ones (Table-3). In dogs treated with A. vera gel ointment 20% and gentamicin ointment 0.1%, WBCs were increased on the 3rd, 7th, and 10th DPT (p<0.05) and returned to their normal values on the 14th day, except for neutrophils which were elevated on the 3rd and 7th day (p<0.05) then returned to their normal values on the 10th and 14th DPT when compared to healthy ones. Lymphocytes, monocytes, and eosinophils were decreased in all treated groups (p<0.05) and returned to their baseline on the 14th DPT when compared to healthy groups (p>0.05; Table-3). Furthermore, mean corpuscular volume, mean corpuscular hemoglobin (MCH), and MCH concentration values were not significantly different between all groups except on the 3rd DPT at which these values were decreased when compared with control ones (p<0.05; Table-3).

Table-3:

Hematological profile of control and infected dogs. Values expressed as mean with SE.

Variables	RBCs (106/µL)	Hb (g/dl)	PCV (%)	MCV (fi)	MCH (pg)	MCHC (%)	WBCs (103/µL)	Neutrophil (%)	Lymphocyte (%)	Monocyte (%)	Eosinophil (%)
Control negative dogs (before induction) (n=20)	5.96±0.15a	13.5±0.3a	33.8±0.66a	56.75±0.7a	22.7±1.7a	40.1±1.7a	18.89±0.5a	61.75±0.62a	30.75±0.85a	5.5±0.28a	2±0.4a
Infected dogs without treatment Group 1 (n=5)
3 DWT	5.5±0.11b	12.3±0.1b	31.5±0.6b	56.8±2.2a	22.1±0.2a	39.1±1.2a	30.47±0.6b	68.25±0.8b	26±0.57b	4.25±0.2b	1.5±0.28b
7 DWT	5.6±0.08b	12.6±0.08b	32±0.4c	57.1±0.12a	22.5±0.3a	39.4±0.5a	28.4±0.3b	65.7±0.4c	28±0.4b	4.5±0.28b	1.75±0.25a
10 DWT	5.6±0.06b	12.5±0.1b	32.7±0.4c	58.2±0.6a	22.2±0.2a	38.2±0.3a	26.39±0.4c	65±0.4c	28.5±0.6b	5±0.0a	1.5±0.28b
14 DWT	5.7±0.06b	12.7±0.09b	33±0.4a	56.9±1.5a	21.6±0.4a	38.4±0.7a	22.9±0.59d	63.5±0.6d	30.5±0.6a	4.75±0.25b	1.25±0.25c
Dogs treated with Aloe vera gel ointment 20% Group 2 (n=5)
3 DPT	5.5±0.06b	11.3±0.2c	33±0.4a	59.1±0.17b	21.7±0.5a	36.7±0.7b	28.32±0.4b	66.25±0.47c	28.25±0.25b	4.2±0.25b	1.2±0.25c
7 DPT	6±0.09a	13.1±0.16a	33±1.08a	55.1±2.52c	21.2±0.58a	40±0.9a	25.15±0.38c	64.25±0.25c	29.75±0.25a	4.5±0.28b	1.5±0.28b
10 DPT	6.2±0.1a	13.47±0.2a	33.5±0.6a	54.1±1.82c	21.75±0.5a	40.23±0.5a	23.4±0.35d	62.75±0.47a	30.75±0.47a	5±0.4a	1.5±0.28b
14 DPT	6.06±0.05a	13.07±0.1a	33.75±0.6a	54.48±1.4c	21.1±0.44a	38.7±0.45a	20.34±0.57a	62.75±0.47a	30.75±0.47a	5±0.4a	1.5±0.28b
Dogs treated with Aloe vera gel ointment 40% Group 3 (n=5)
3 DPT	6±0.09a	12.17±0.2b	32.5±0.9a	54.1±1.12c	20.26±1.2b	37.4±0.4b	28.2±0.35b	66.75±0.47c	27.25±0.25b	4.7±0.25a	1.2±0.25c
7 DPT	6.1±0.09a	13±0.09a	33.2±0.62a	54.5±0.98c	21.3±0.39a	39.1±0.86a	26.75±0.21c	65±0.47c	29±0.4a	4.5±0.288b	1.5±0.28b
10 DPT	6.15±0.13a	13±0.18a	35.2±0.47d	57.39±1.56a	21.17±0.6a	36.88±0.3b	24.7±0.44d	62±0.4a	31.25±0.47a	5±0.4a	1.75±0.25a
14 DPT	6.25±0.14a	13.05±0.1a	33.7±0.85a	54.4±2.12c	20.8±0.35a	38.76±1.3a	20.95±0.28a	61.5±0.28a	31.75±0.25a	5.25±0.25a	1.5±0.28b
Dogs treated by gentamicin sulfate 0.1% ointment Group 4 (n=5)
3 DPT	5.7±0.19a	12.17±0.24b	30.5±0.64b	53.65±2.7c	21.36±0.9a	39.8±0.6a	30.1±0.38b	64±0.4d	30±0.4a	4.5±0.28b	1.5±0.28b
7 DPT	6.1±0.1a	13.3±0.29a	32.7±0.47a	53.7±1.38c	21.9±0.78a	40.8±1.2a	27.6±0.21b	63±0.4d	29.75±0.47a	5.25±0.25a	2±0.25a
10 DPT	6.12±0.08a	12.8±0.17a	32.25±0.8a	52.6±0.82c	21.01±0.2a	39.9±0.5a	23.125±0.5d	61.75±0.6a	31.25±0.47a	5.25±0.25a	1.75±0.25a
14 DPT	6.35±0.1a	13.1±0.19a	31.5±0.64b	49.6±1.7d	20.7±0.18b	41.9±1.3a	20.9±0.51a	62.5±0.2a	30.75±0.47a	5±0.4a	1.75±0.25a

SE=Standard error, DWT=Day without treatment, DPT=Day post-treatment, n=Number. Means with different letter superscripts in the same column are significantly different at p<0.05

Oxidant and antioxidant concentrations between dogs’ studied groups

Serum MDA concentrations were higher in infected dogs on the 3rd-14th day without treatment compared to control ones (p<0.05; Table-4), while dog treated with A. vera gel ointment 20% and 40% and gentamicin 0.1%, serum MDA concentrations were elevated on the 3rd and 7th DPT (p<0.05) and returned to its baseline on the 10th and 14th DPT compared to control ones (p>0.05; Table-4). Serum MDA concentrations were a diagnostic biomarker for Staphylococcus pyoderma in dogs (area under the curve=0.92; p<0.05; Figure-4). Serum TAC and catalase concentrations were decreased in infected dogs on the 3rd-14th day without treatment compared to control group (p<0.05). In dogs treated with A. vera gel ointment 20%, serum TAC and catalase concentrations were decreased on the 3rd and 7th DPT (p<0.05) and returned to their normal concentrations on the 10th and 14th DPT compared to healthy ones (p>0.05). In dogs treated with A. vera gel ointment 40%, serum TAC and catalase concentrations were decreased on the 3rd DPT (p<0.05) and returned to their baseline from the 7th to14th DPT when compared to healthy ones (p>0.05). In dogs treated with gentamicin 0.1%, serum TAC concentrations were decreased on the 3rd and 7th DPT (p<0.05) and returned to their baseline on the 10th and 14th DPT when compared to healthy ones (p>0.05), while catalase concentrations were not changed all over the time points than healthy ones (p>0.05; Table-4).

Table-4

Oxidant and antioxidant status of control and infected dogs. Values expressed as mean with SE.

Variables	MDA (nmol/mL)	TAC (mM/L)	Catalase (U/L)
Control negative (before induction) (n=20)	10.35±0.46a	1±0.06a	72.26±0.95a
Reference range of negative control	(3.20-17.80)	(0.40-1.30)	(52.3-100.40)
Infected dogs without treatment Group 1 (n=5)
3 DWT	21.8±0.45b	0.41±0.1b	55.4±0.8b
7 DWT	22.2±0.14b	0.35±0.09c	53.7±1.2c
10 DWT	22.4±0.8b	0.32±0.04c	51.2±1.4c
14 DWT	23.8±0.23c	0.30±0.08c	50.8±0.54c
Dogs treated with Aloe vera 20% Group 2 (n=5)
3 DPT	20.12±0.37b	0.597±0.03d	65.47±1.27d
7 DPT	19.78±0.54b	0.6±0.03d	67.3±0.5d
10 DPT	11.8±0.56a	0.83 ±0.07a	69.44±0.37a
14 DPT	12.45±0.29a	0.877±0.033a	69.71±0.3a
Dogs treated with Aloe vera 40% Group 3 (n=5)
3 DPT	19.56±0.42b	0.707±0.033d	66.97±0.63d
7 DPT	16.4±0.4d	0.905±0.055a	68.99±0.56a
10 DPT	11.73±0.37a	0.916±0.032a	70.36±0.4a
14 DPT	11.8±0.22a	0.882±0.047a	69.45±0.44a
Dogs treated by gentamicin sulfate 0.1% Group 4 (n=5)
3 DPT	16.24±0.29d	0.67±0.025d	70.125±0.64a
7 DPT	15.13±0.42d	0.64±0.04d	71.03±0.56a
10 DPT	12.43±0.26a	0.88±0.04a	70.10±0.2a
14 DPT	12.12±0.19a	0.98±0.04a	71.5±0.58a

SE=Standard error, MDA=Malondialdehyde, TAC=Total antioxidant capacity, DWT=Day without treatment, DPT=Day post-treatment, n=Number. Means with different letter superscripts in the same column are significantly different at p<0.05

Figure-4

Receiver operating characteristic curve analysis for SAA, HP, TNF-α, and MDA concentrations in dogs with Staphylococcus pyoderma (p<0.05). SAA=Serum amyloid A, HP=Haptoglobin, TNF-α=Tumor necrosis factor-α, MDA=Malondialdehyde.

Acute-phase proteins and cytokines concentrations between studied groups of dogs

There was a significant increase in the concentrations of acute-phase proteins (CP, SAA, and HP) and ILs (IL-1, IL-6, IL-10, and TNF-α) in infected dogs on the 3rd-14th day without treatment and in dogs treated with A. vera gel 20% and 40% and gentamycin ointments 0.1% on the 3rd and 7th DPT when compared to the control negative ones (p<0.05; Table-5), then started to return to their baseline on the 14th DPT in dogs treated with A. vera gel ointment 20% and 40% and gentamicin 0.1% when compared to control ones (p>0.05). SAA, HP, and TNF-α concentrations were significant biomarkers for Staphylococcus pyoderma in dogs (p<0.05; Figure-4). Serum HP and TNF-α concentrations were significantly lower on the 10th and 14th DPT in dogs treated with A. vera gel 20% and 40% compared to dogs treated with gentamycin 0.1% (p<0.05; Table-5). Dogs’ pyoderma treated with A. vera 20% and 40% were more likely to have low HP and TNF-α concentrations than gentamicin ([OR=4.6; 95% CI=1.31-17.40; p<0.05]; [OR=5.2; 95% CI=1.04-22.30; p<0.05]), respectively, as shown in Table-6.

Table-5

Acute-phase protein biomarkers and cytokines expression in control and infected dogs. Values expressed as mean with SE.

Variables	Ceruloplasmin (µg/mL)	Serum amyloid A (g/mL)	Haptoglobin (µg/mL)	Interleukin 1 (pg/mL)	Interleukin 6 (ng/L)	Interleukin 10 (ng/L)	Tumor necrosis factor-α (ng/L)
Control negative (before induction) (n=5)	25.8±0.24a	0.62±0.04a	67.25±2.2a	59.3±0.6a	29.5±0.8a	40.5±0.4a	12.04±0.2a
Reference range of negative control	(10.2-40.2)	(0.2-0.76)	(58.30-85.20)	(42.3-70.50)	(21.3-40.2)	(35.2-48.4)	(10.25-15.35)
Infected dogs without treatment Group 1 (n=5)
3 DWT	57.38±0.2b	1.8±0.8b	132.7±1.8b	101.4±1.6b	42.4±0.6b	58.5±0.8b	25.3±0.2b
7 DWT	51.51±0.3c	1.6±0.1b	128.4±2.4b	100.8±0.78b	40.7±1.2b	57.4±1b	20.8±0.4c
10 DWT	50.8±0.08c	1.5±0.06b	125.6±1.6b	90.8±0.5c	38.8±0.6b	48.8±0.8c	18.8±0.2c
14 DWT	44.54±0.5d	0.8±0.03c	124.7±1.2b	85.2±2.2c	35.2±0.8c	45.6±0.6c	18.2±0.5c
Dogs treated with Aloe vera gel ointment 20% Group 2 (n=5)
3 DPT	48.45±0.65c	1.4±0.2b	118.8±2.2b	88.8±0.8c	38.2±1.4b	54.3±1.4b	18.2±0.4c
7 DPT	42.8±0.4d	0.9±0.08c	82.54±1.2c	74.2±1.4d	35.8±0.5c	42.4±0.6a	15.8±0.6d
10 DPT	29.3±0.8a	0.85±0.05c	72.2±1.8c	64.5±0.6a	32±0.8a	42.5±0.8a	13.2±0.8a
14 DPT	28.12±0.54a	0.68±0.1a	70.6±1.1a	60.3±1.2a	30.8±0.6a	41.8±0.5a	12.5±0.2a
Dogs treated with Aloe vera gel ointment 40% Group 3 (n=5)
3 DPT	52.4±0.3c	1.4±0.1b	115.4±2.4b	85.2±0.95c	38±0.38b	55.3±0.7b	19.2±0.5c
7 DPT	44.48±0.38d	1.08±0.06c	80.7±1.2c	73.6±0.8d	32.4±0.8a	42.8±0.3a	15.7±0.2d
10 DPT	30.2±0.4a	0.8±0.0c	73.8±0.8a	65.2±0.4a	30.4±0.4a	42±0.6a	13.6±0.8a
14 DPT	28.7±0.6a	0.66±0.04a	70.85±1.2a	61.4±1.2a	30±1.3a	40.6±0.9a	12.2±0.6a
Dogs treated by gentamicin sulfate 0.1% sulfate 0.1% ointment Group 4 (n=5)
3 DPT	48.12±0.2c	1.28±0.1c	116.6±1.4b	87.9±0.4c	39.2±0.6b	55.8±0.6b	18.4±0.2c
7 DPT	44.2±0.4d	1.18±0.06c	112.9±0.8b	74.8±1.2d	38.5±0.8b	45.8±0.8c	16.2±0.3d
10 DPT	42.42±0.5d	0.85±0.02c	111.4±1.2c	68.4±0.58a	32.2±0.5a	40.6±1.08a	19.6±0.4d
14 DPT	30.4±0.6a	0.70±0.04a	112.4±2.2c	63.42±0.6a	31.4±1.4a	40.2±0.7a	19.8±0.2d

SE=Standard error, n=Number. Means with different letter superscripts in the same column are significantly different at p<0.05

Table-6

Univariate logistic regression of variables in dogs’ staphylococcal pyoderma.

Variables	Tested drugs	Range	OR	95% CI	p-value
HP (µg/mL)	Aloe vera gel ointment 20% and 40% Gentamicin ointment 0.1%	58.3-85.2 >85.2	4.6*	1.31-17.40	0.03
TNF-α (ng/L)	Aloe vera gel ointment 20% and 40% Gentamicin ointment 0.1%	10.25-15.35 >15.35	Referent 5.2*	1.04-22.30	0.02

HP=Haptoglobin, TNF-α=Tumor necrosis factor-α, OR=Odds ratio, CI=Confidence interval.

p<0.05 compared to referent

Discussion

In the present study, the antibacterial effect of A. vera gel ointments 20% and 40% against dogs’ staphylococcal pyoderma had been evaluated in vitro. A. vera extract showed clear inhibition zone for S. aureus growth in well diffusion agar test. The inhibition zone of A. vera extract 40% was wider than the ones that caused by A. vera extract 20% and gentamicin 0.1%. Our results were in the same line with previous studies that recorded the antibacterial effect of A. vera extract against S. aureus [21,22]. The antibacterial activity of A. vera could be attributed to its components that include p-coumaric, ascorbic, pyrocatechol, and cinnamic acid that could be used as an alternative herbal antibacterial therapy to subside the antibiotic resistance and reduce wound healing time [22,23].

In the present study, RBCs, Hb, PCV and WBCs were returned to their normal values after topical application of different concentrations of A. vera gel ointments 20% and 40% on dogs’ pyoderma thus could be explained by A. vera may increase erythropoiesis process and also increase leukocyte infiltration as a part of immunomodulation [24].

We have shown that the concentrations of acute-phase proteins are in positive correlation with the severity of dog’s pyoderma that could be attributed to increase in their synthesis and release from the liver in response to tissue injury of skin [25]. Furthermore, we have documented that ILs were elevated in infected dogs than control ones. It is possible that pyoderma is suggested to increase an inflammatory response by releasing cytokines into the bloodstream [26,27]. Interestingly, the serum concentrations of MDA were significantly increased, while the TAC and catalase concentrations were decreased in infected dogs with staphylococcal pyoderma compared to control ones. It has been reported that dogs with skin pathological conditions have significant oxidative stress with elevated MDA and decreased TAC and catalase concentrations as reported in our study [28-30].

In the current study, topical application of A. vera gel ointment 20% and 40% modulates the inflammatory response through reduction of acute-phase proteins and IL concentrations and enhances skin healing of dogs’ Staphylococcus pyoderma. The previous studies documented that topical application of different doses of A. vera extract on injured skin resulting in reduction of the acute-phase response [31,32]. Moreover, the positive effect of A. vera gel on oxidant and antioxidant status has been shown in this study through elevation TAC and catalase concentrations and reduction of MDA concentrations that could be explained potentially by A. vera had antioxidant effects through glutathione peroxidase activity [33]. From that prospective, one could speculate that A. vera gel ointment has anti-inflammatory and antioxidative roles against dogs’ staphylococcal pyoderma.

Conclusion

Increased acute-phase proteins, cytokines, and oxidants and reduced antioxidant biomarkers were frequent findings in dog’s pyoderma. Furthermore, topical application of A. vera gel ointments 20% and 40% had antibacterial, anti-inflammatory, and antioxidative actions against dogs’ staphylococcal pyoderma that will support its further use rather than antibiotics in the health arena.

Authors’ Contributions

HH designed the experiment. AA, AhE, AE, and HK performed the experiment. AK performed the statistical analysis of results. HH, AK, and AA drafted and revised the manuscript. All authors read and approved the final manuscript.