Animals
The present study included 48 juvenile male Balady sheep (5.5 ± 0.3 months of age) reared in the eastern part of Assiut governorate, Egypt. Of them, 36 sheep were diagnosed clinically and mycologically with T. verrucosum, and 12 were healthy control subjects. The selected sheep have been reared similarly in small flocks under unorganized farming system without satisfactory standards of feeding and management. Flocks are coalesced and confined by their owners to limited areas for grazing pastures during the daytime. Infected sheep had a history of dermatological lesions for 1.5-3 months before examination, but both infected and control sheep had no history of recent topical or systemic medications. After perfect clinical and routine laboratory diagnosis, the animals were free from bacterial, parasitic (internal and external) and systemic diseases. Healthy sheep were negative for threefold KOH examination and fungal cultures. Lesions in the infected sheep were confined only to the head and ears.
The selected sheep were divided into four groups according to lesions size. Only one of the authors evaluated these lesions. The first group showed 1–2 lesions, each with an approximate diameter of 2–4 cm (n = 14, mild infection, MID). The second contained wider lesions, which approximately cover quarter up to third of the head and ear (n = 12, moderate infection, MOD). Animals in which the lesions cover most of the head and ear were considered severe infection (n = 10, SEV). The remainder (n = 12) were healthy sheep and used as a control group.
Blood sampling
In the early morning, blood was sampled from the jugular vein in 10-ml heparinized vacuumed tubes and sent immediately to the laboratory on crushed ice. After centrifugation (at 2500 ×g for 15 min, at 4°C), clear non-hemolized plasma were stored at − 80°C until analysis within a week.
Mycological investigations
Skin lesions were cleaned with 70% ethyl alcohol. The margins were scraped in a sterile Petri dish to collect hair and skin tissues. In healthy sheep, areas around muzzle and eyes in addition to ears were also sampled. Scraping material was divided into two portions. The first was digested with 10% KOH for 15 min and examined for the presence of spores and hyphae under microscope. The rest of the specimen was inoculated into Sabouraud’s dextrose agar supplemented with chloramphenicol and cycloheximide (Mycobios Selective Medium; Oxoid, Basingstoke, UK). The medium was enriched with inositol and thiamin, and incubated at 28°C and 37°C for 2–6 weeks. Cultures were checked 2–3 times a week during incubation. Lactophenol cotton blue preparation was made for morphological studies of the isolates (Rebell and Taplin 1974).
Biochemical analysis
Superoxide anion (O֗2̄ )
As a major component of free radicals, O֗2̄ was measured according to the procedure of Podczasy and Wei (1988), which based on the reduction of the tetrazolium compound, p-iodonitrotetrazolium (INT) by O֗2̄ generated by xanthine/xanthine oxidase to a water-soluble product (reddish pink) with an absorbance maxima at 505 nm.
Reactive Oxygen Species (ROS):
It is difficult to estimate ROS in vivo because they are rapidly metabolized. Total oxidant status (TOS), also named total peroxides or reactive oxygen metabolites (ROM) is usually used as an indirect method to estimate the reactive oxygen species (ROS) concentration (Erel 2005). Plasma TOS concentration was measured after the method described by (Erel 2005). This method based on the oxidation of Fe+ 2 (as a catalyst) to Fe+ 3 by the various types of peroxides in the presence of xylenol orange in an acidic medium, which binds with Fe+ 3 and forms a colored complex whose optical density is read at 560 nm.
Total Antioxidant Capacity (TAC)
Total antioxidant capacity (TAC) was determined by using the colorimetric ABTS assay kit (Beyotime Institute of Biotechnology, Haimen, China) after the method described by Miller et al. (1993). In this method, the sample antioxidants inhibit peroxidase-mediated oxidation of 2,2ʹ-azino-bis-(3-ethylbenzothiazoline-6-sulfonate, colorless solution) to the radical cation (blue green). TAC was expressed as mmol Trolox equiv/l.
Superoxide dismutase (SOD)
The activity of SOD was estimated according to the method described by Misra and Fridovich (1972). In alkaline medium, SOD inhibits the autoxidation of epinephrine to adrenochrome. OD was measured at 480 nm.
Lipid peroxide (malondialdehyde; MDA)
Lipid peroxidation was determined as thiobarbituric acid reactive substances (TBARS) according to Placer et al. (1966). The method depends on formation of a color complex between the products of lipid peroxidation and thiobarbituric acid (TBA). In this assay, 1,1,3,3-tetramethoxypropane was used as a standard. The absorbance was read at 548 nm against bi-distilled water as a blank.
Protein oxidation (protein carbonyls; PC)
Protein carbonyls were measured by using DNPH (2,4-dinitrophenylhydrazine) according to the method of Levine et al. (1990). In Brief, 10 µl of plasma was incubated with 1 ml of 0.2% DNPH (dissolved in 2.5 M HCl) for 1 h. Then 1 ml of 20% trichloroacetic acid was added to precipitate the protein. After shaking and centrifugation (3000 ×g for 10 min), the supernatant was decanted and the pellets were washed three times with 1 ml of ethanol and ethyl acetate solution (1:1; vol./vol.). The final pellet was re-dissolved in 1.5 ml of 6 M guanidine hydrochloride and the color of the supernatant was read at 370 nm.
Oxidative stress index (OSI)
The TAC unit, mmol Trolox equiv/L, was converted to µmol Trolox equivalent/L. The OSI value (as an indicator of the degree of oxidative stress) was determined as the percentage ratio of TPx to TAS as follows: OSI (Arbitrary unit) = (TPx, µmol/L / TAC, µmol Trolox equv/L) ×100 (Aycicek and Erel 2007)
Statistical analysis:
The packaged SPSS program for windows version 10.0.1 (SPSS, Chicago, IL, USA) was used for statistical analysis. Data were analyzed using one-way analysis of variance (ANOVA) and expressed as mean ± standard error (SE). Differences between groups were determined by means of pairwise multiple comparison procedures (when significant F test was found) using Duncan's new multiple range test. Significance level was set at P < 0.05. Linear regression analysis (R2) and Pearson’s correlation (r) were done on the paired data obtained by the individual cases.