Population and collection of clinical samples
From May 2015 to December 2018, a total of 760 Holstein cow calves (597 weaning and 163 suckling calves) raised in different farms in Egypt, were clinically examined for evidence of ringworm infection. Data about age, breed, farm production, breeding system, production management system, origin of calves of the farm were obtained for each calf. For examination of parasitic infestation, fecal samples were examined for enteric parasites and thin blood films were prepared, fixed in absolute methyl alcohol and stained with freshly filtered and diluted 10% Giemsa stain. After cleaning the skin lesion of suspected ringworm-affected calf with 70% ethanol, scales and dull hair samples from the margins were collected using a sterilized plastic hair brush and tweezers, respectively .
Portions of hair and scales were examined microscopically after clearing with 20% potassium hydroxide (KOH), cultured on Mycobiotic agar (Remel™, Thermo Fisher Scientific) slants with 10% thiamine and inositol, incubated at 30 °C for 4–6 weeks and observed for growth at 3 days intervals. Dermatophyte isolates were identified according to their macro-and micromorphological characteristics .
Extraction of DNA from hair and scales samples and PCR amplification
The direct molecular identification of dermatophytes was executed in 150 representative clinical samples that were selected on the basis of direct microscopy and culture results. For high-throughput disruption of samples, 50 mg of hair and scales were placed in a 2 ml safe-lock tube and incubated overnight at 55°C with 360 μl of ATL buffer and 20 μl QIAGEN protease (QIAamp DNA Mini kit, Qiagen, Germany, GmbH). Subsequently, tungsten carbide beads were added and tubes were placed into the TissueLyser adapter set for disruption using the TissueLyser for 2 min at 20–30 Hz two times. Then, DNA extraction was performed utilizing QIAamp DNeasy Plant Mini kit (Qiagen, Germany, GmbH) following the manufacturer’s instructions. DNA was eluted with 50 µl of elution buffer and the concentration was assessed using NanoDrop ™2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
According to Cafarchia et al.  the chitin synthase (chs-1) gene was amplified using DMTFchsF1 (5-CGAGTACATGTGCTCGCGCAC–3) and DMTFchsR1 (5-CGAGGTCAAARGCACGCCAGAG–3) primers to assess the presence of dermatophytes amplifiable DNA in the clinical samples. Then, further one-step PCR was performed using the primers DMTF18SF1 (5-CCAGGGAGGTTGGAAACGACCG–3) and DMTF28- SR1 (5-CTACAAATTACAACTCGGACCC–3) amplifying a 900 bp of the conserved regions in the 18S and 28S genes that includes internal transcribed spacers regions of ribosomal DNA (ITS–1, 5.8S and ITS–2).
A nested PCR was applied to amplify a 400 bp of a conserved region in the dermatophytes 5.8S gene from the ITS+ amplicons of the primary PCR using DMTF18SF1 and DMTFITS1R (5-CCGGAACCAAG AGATCCGTTGTTG–3) primers .
PCR was performed in an amplification reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer (20 pmol), 6 µl of DNA template in case of primary PCR or 1 µl of diluted product from the primary PCR (dilution 1:1 with molecular grade water) for nested PCR and nuclease-free water up to 25µl. T. verrucosum ATCC®28203™ and an amplification reaction without DNA template were utilized as positive and negative control, respectively. The thermo-cycling conditions previously described were employed in an applied biosystems 2720 thermal cycler (Thermo Fisher Scientific, USA).
The amplified products were electrophoresed on ethidium bromide-stained 1.5% agarose gel (Applichem, Germany, GmbH). A gelpilot 100 bp DNA Ladder (Qiagen, Gmbh, Germany) and 100 bp DNA ladder H3 RTU (Genedirex, Taiwan) were used to determine the amplicon sizes. The gel documentation system (Alpha Innotech, Biometra) was used to photograph the gel and the data analysis was done through the computer software.
DNA sequencing and sequence analysis
Tow representative ITS+ PCR products were purified using QIAquick PCR Product extraction kit (Qiagen, Valencia) and then sequenced using Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer) in Applied Biosystems 3130 genetic analyzer (HITACHI, Japan). DNA sequences were compared with those available in NCBI databases (National Center for Biotechnology Information, www.ncbi.nlm.nih.gov) using Basic Local Alignment Search Tool (BLAST). MEGA5 program, product version 5.1 (www.megasoftware.net) was used for sequence analysis. The ITS sequences were available under GenBank accession no. MK918485 and MK918486.
Antifungal susceptibility testing of dermatophytes isolates
Broth micro-dilution method in accordance with the CLSI M38-A2 was employed for testing the sensitivity of dermatophytes isolates to the most commonly used antifungal drugs. Fluconazole were obtained from Pfizer International, New York, NY, USA, itraconazole, and miconazole from Janssen Research Foundation, Beerse, Belgium. Griseofulvin was bought from Sigma Chemical Company, St. Louis, MO, USA, and terbinafine from Novartis, Basel, Switzerland. All drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) except fluconazole in RPMI1640 medium (Sigma Co. St. Louis, USA) buffered at pH 7.0 with 165 mM of 3-(N-morpholino) propanesulfonic acid (MOPS; Sigma) and two-fold serially diluted to final concentrations of 0.125 to 64 μg/mL for fluconazole and 0.03 to 16 μg/mL for the other antifungal agents. Minimum inhibitory and minimum fungicidal concentrations (MIC and MFC) were determined.
Preparation of Aloe vera gel extracts (AGE)
Aloe vera leaves were obtained from Agriculture Faculty, Zagazig University, Zagazig, Egypt. Aloe vera gel was obtained from leaves by scratching. The aqueous extract of gel was prepared utilizing magnetic stirrer (Fisher Scientific) and filtered using Whatman No.1filter paper. The extraction ratio was 1 W: 5 V (gel: solvent). The filtrate was freeze-dried (Thermo-Electron Corporation-Heto power dry LL300 Freeze Dryer), the extract was then weighed to decide the yield and stored at –20°C.
Chemical characterization of AGE
Determination of phenolic compounds
The concentration of total phenols in extract was measured by a UV spectrophotometer (Jenway-UV–VIS Spectrophotometer 6705), based on a colorimetric reduction of the reagent by phenolic compounds, as described by Škerget et al.. Total phenolic content expressed as gallic acid equivalent (GAE) was calculated: y = 0.0228 x + 0.0086 and R² = 0.9969, where (x)is the concentration (µg GAE) and (y)is the absorbance.
Determination of total flavonoids
Total flavonoids content expressed as quercetin equivalent (QE) in AGE at a final concentration of 1 mg mL−1 was calculated: y = 0.0142x - 0.007 and R² = 0.9994, where (x)is the concentration (µg QE) and(y)is the absorbance .
Determination of phenolic compounds by HPLC
HPLC analysis was executed as previously described  with slight modifications using an Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler. The analytical column was Agilent Eclipse XDB C18 (100 x 4.6 µm; 3.5 µm particle size). Diode array detector (DAD) was set to a scanning range of 180–420 nm. The mobile phase consisted of methanol (solvent A) and 0.1% formic acid (v/v) (solvent B). The flow rate was kept at 0.4 mL min–1 and the gradient program was as follows: 10% A - 90% B(0–5 min); 20% A - 80% B (5–10 min); 30% A - 70% B (10–15 min); 50% A - 50% B (15–20 min); 70% A - 30% B (20–25 min); 90% A –10%B (25–30 min); 50% A –50% B (30–35 min) and 10% A - 90% B (35–36 min). There was 5 min of post-run for reconditioning. The injection volume was 10 µL and peaks were monitored simultaneously at 280, 320 and 360 nm for the benzoic acid and cinnamic acid (Sigma, St. Louis, MO, USA) derivatives and flavonoids compound, respectively. All samples were filtered through a 0.45 µm Acrodisc syringe filter (Gelman Laboratory, MI) before injection. Peaks were identified by congruent retention times and UV spectrum and compared with those of the standards (Sigma, St. Louis, MO, USA).
Antioxidant and biological activity of AGE
1, 1-Diphenyl–2picrylhydrazyl (DPPH˙) radical-scavenging activity
The electron donation ability of AGE was measured by bleaching of the DPPH˙ (Sigma, St. Louis, MO, USA) purple colored solution using a UV spectrophotometer (Jenway-UV–VIS Spectrophotometer 6705) . The absorbance was determined against control at 515 nm. Percentage of scavenging activity of free radical DPPH˙ was calculated as follow:
Scavenging activity (Inhibition) % = [(A control—A sample)/A control] × 100
Where A control is the absorbance of the control reaction and A sample is the absorbance in the presence of plant extract. Gallic acid and Tert-butyl hydroquinone (TBHQ) (Sigma, St. Louis, MO, USA) (1 mg / 1mL methanol) were utilized as a positive control. Samples were tested in triplicate.
β-Carotene/linoleic acid bleaching
The ability of AGE and synthetic antioxidants (gallic aid and TBHQ) to hinder the bleaching of β-carotene (Sigma, St. Louis, MO, USA) was examined according to Dastmalchi et al.  A control sample with no added extract was also analyzed. Antioxidant activity was calculated as follows: Antioxidant activity (%) = [1 − (A0sample − A120sample)/ (A0control − A120control)] × 100
Where A0sample is the absorbance of the AGE or synthetic antioxidant at 0-time, A120sample is the absorbance after 120 min, A0control and A120control are the absorbance of control at 0-time and after 120 min, respectively.
Ferric reducing antioxidant power (FRAP)
The extract reducing power was assessed . Distilled water was employed as a negative control and gallic acid and TBHQ as positive control. Absorbance of this mixture was measured at 700 nm using a UV spectrophotometer (Jenway-UV–VIS Spectrophotometer 6705). Decreased absorbance demonstrates ferric reducing power capability of sample.
TestingAGE antidermatophyte activity
The procedure of Silva et al. was used to test the antidermatophyte activity of AGE. The freeze-dried AGE (3.5 gm) was dissolved and serially two-fold diluted in RPMI–1640 broth to obtain a concentration range of 1000–20000 μg/ml as TPC. A final concentration of 50–1000 μg/ml was obtained by mixing 2 ml of this solution with 18 ml of liquefied Mycobiotic agar medium (Remel™, Thermo Fisher Scientific) at 45 °C in sterile Petri dish. Next, wells of 3 mm diameter were made in the centre of this agar plate and filled with 10µl of fungal spore suspension (106 cfu/ml) that was prepared from freshly cultured isolates. The plates were incubated for 5 days at 25 °C. The assay was carried out in triplicate and growth and drug control were incorporated into the test. The concentration that inhibits the fungal growth was considered as MIC.
Investigation of AGE effectiveness for treatment of calf ringworm
Seventy-five calves showing evident clinical signs of ringworm infection were used for investigation of AGE effectiveness in comparison with antifungal drugs for the treatment of calf ringworm after obtaining informed consent from the farm owners. The enrolled calves proved positive on mycological examination and T. verrucosum was isolated from clinical samples. Calves were randomly allocated into five groups, 15 animals in each group (G). Animals in G1 were treated orally with 250 mg ⁄day terbinafine (Lamisil®; Novartis, Basel, Switzerland). The crust on the skin lesions was removed with a brush, subsequently, topical miconazole (Janssen Research Foundation, Beerse, Belgium) (G2), AGE solution (500 ppm) (G3) or oral terbinafine in combination with AGE (G 4) were applied twice a day for two weeks. Animals in G5 were left untreated as controls. Calves were observed daily for six weeks. At the beginning, during and after treatment, the clinical efficacy was assessed by scoring alopecia, scaling, crusting, the numbers and spreads of lesions on a 0–3 scale. The sum of the scores assigned to each lesion on the evaluated area was divided by the numbers of affected areas yielding the total score for each calf, and the same lesion was assessed on every examination. The mycological examination was performed every week until two consecutive fungal cultures give negative results [32, 43]. The control animals were treated after the observation period.
Antifungal disinfection for the entire stable and all materials with which animals come in contact was performed using 0.2% enilconazole (Clinafarm® EC; Merck Animal Health USA).
Data analysis was performed using IBM SPSS Statistics for Windows, version 24.0 (released 2016), and MedCalc 2014 (MedCalc Software) were used to analyze data. Chi-square test and Odds ratio analyses were performed to determine the association of ringworm infection with different risk factors. Prevalence and risk ratio was used to compare between groups at risk to those not at risk. To confirm the results, random forest non-parametric classification method was done using MetaboAnalystR web server . Briefly, the occurrence of each variable was firstly used to build up random forest classification model (an ensemble of 500 tree trial; out of bag error (OOB) = 0.6) in the respective outcome. The importance of the risk factor was determined by measuring the increase of the OOB error when the respective factor is permuted. The sensitivity, specificity, negative, positive predictive values (NPV, PPV), positive, negative likelihood ratio (LR+, LR-), and diagnostic odds ratio (DOR),that express strength of association between test result and disease, with 95 % confidence intervals (CIs) for direct sample PCR assays were estimated. All diagnostic indices were predestined based on (a) culture and (b) culture and nested PCR as the gold standard for detection/identification of dermatophytes causing calves ringworm. Kappa value was used to test the agreement between test results. Independent samples t-test was run to compare between the mean MIC values ± SD of each antifungal drug for the tested species. Kruskal-Wallis test was used to analyze the differences in clinical score changes within and among the treated and untreated groups over time. Differences in clinical scores between groups were assessed by Mann-Whitney U test after significant Kruskal-Wallis test. P < 0.05 was considered significant.