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, and the origin of calves of the farm were obtained for each calf as potential risk factors. For the assessment 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 the 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 [4].
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-day intervals. Dermatophyte isolates were identified according to their macro-and micromorphological characteristics [32].
Extraction of DNA from hair and scale samples and PCR amplification
The direct molecular identification of dermatophytes was executed in 150 clinical samples that were selected based on the results of direct microscopy and culture analyses. For the 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 of QIAGEN protease (QIAamp DNA Mini kit, Qiagen, Germany, GmbH). Subsequently, tungsten carbide beads were added, and the tubes were placed into the TissueLyser adapter set for disruption using the TissueLyser for 2 min at 20–30 Hz twice. DNA extraction was performed using a QIAamp DNeasy Plant Mini kit (Qiagen, Germany, GmbH) according to the manufacturer’s instructions. DNA was eluted with 50 μL of elution buffer and the concentration was assessed using a NanoDrop™2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
One-step PCR
According to Cafarchia et al. [7] the chitin synthase (pchs-1) gene was amplified using DMTFchsF1 (5-CGAGTACATGTGCTCGCGCAC-3) and DMTFchsR1 (5-CGAGGTCAAARGCACGCCAGAG-3) primers to assess the presence of dermatophyte amplifiable DNA in the clinical samples. Subsequently, one-step PCR was performed using the primers DMTF18SF1 (5-CCAGGGAGGTTGGAAACGACCG-3) and DMTF28SR1 (5-CTACAAATTACAACTCGGACCC-3), which amplified a 900 bp fragment of the conserved regions in the 18S and 28S genes, which included the internal transcribed spacer regions of ribosomal DNA (ITS-1, 5.8S, and ITS-2).
Nested PCR
A nested PCR was applied to amplify 400 bp of a conserved region in the dermatophyte 5.8S gene from the ITS+ amplicons of the primary PCR using the DMTF18SF1 and DMTFITS1R (5-CCGGAACCAAG AGATCCGTTGTTG-3) primers [7].
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 the 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 was added up to 25 μL. T. verrucosum ATCC®28203™ and an amplification reaction without DNA template were used as a positive and negative control, respectively. Thermocycling conditions described previously [7] were used in an Applied Biosystems 2720 thermal cycler (Thermo Fisher Scientific, USA).
The amplified products were electrophoresed on ethidium-bromide-stained 1.5% agarose gels (Applichem, Germany, GmbH). A gelpilot 100 bp DNA ladder (Qiagen, Gmbh, Germany) and a 100 bp DNA ladder H3 RTU (Genedirex, Taiwan) were used to determine the amplicon sizes. A gel documentation system (Alpha Innotech, Biometra) was used to photograph the gels and the analysis of the data was performed using a computer software.
DNA sequencing and sequence analysis
Thirty-seven representative ITS+ and pchs-1 PCR products were purified using the QIAquick PCR Product extraction kit (Qiagen, Valencia) and then sequenced using a Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer) in an Applied Biosystems 3130 genetic analyzer (HITACHI, Japan). DNA sequences were compared with those available in the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) database using the 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 the GenBank accession numbers MK918485, MK918486, MT261760–MT261763, MT261765–MT261768, MT260175, MT260404, MT260449, MT260803, MT260878, MT261110, MT261113, MT261177, MT261197, MT261198, MT261202, MT261203, MT261385, MT261616, MT261746, MT261759, and MT269023. In addition, MT273253– MT273262 were for pchs-1 gene sequences.
Antifungal susceptibility testing of dermatophytes isolates
The broth micro-dilution method (according to CLSI M38-A2 guidelines [33]) was used for testing the sensitivity of the dermatophyte isolates to the most commonly used antifungal drugs. Fluconazole was obtained from Pfizer International (New York, NY, USA), itraconazole, and miconazole were obtained from the Janssen Research Foundation (Beerse, Belgium), griseofulvin was purchased from Sigma Chemical Company (St. Louis, MO, USA), and terbinafine was purchased from Novartis (Basel, Switzerland). All drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich), with the exception of fluconazole, which was dissolved 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 serially diluted two-fold to final concentrations of 0.125–64 μg/mL for fluconazole and 0.03–16 μg/mL for the other antifungal agents. MIC values, MIC50, and MIC90 were determined.
Preparation of Aloe vera gel extracts (AGEs)
Aloe vera leaves were obtained from the Agriculture Faculty, Zagazig University, Zagazig, Egypt. Aloe vera gel was obtained from the leaves by scratching. The aqueous extract of the gel was prepared using a magnetic stirrer (Fisher Scientific) and filtered using Whatman No. 1 filter paper. The extraction ratio was 1:5 (W:V, gel:solvent). The filtrate was freeze-dried (Thermo-Electron Corporation-Heto power dry LL300 Freeze Dryer) and 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 the extract was measured by a UV spectrophotometer (Jenway-UV–VIS Spectrophotometer 6705) based on the colorimetric reduction of the reagent by phenolic compounds, as described by Škerget et al. [34]. The total phenolic content, expressed as GAE, was calculated as follows: 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 flavonoid content, expressed as QE, in AGE at a final concentration of 1 mg mL− 1 was calculated as follows: y = 0.0142 x – 0.007 and R2 = 0.9994, where x is the concentration (μg QE) and y is the absorbance [35].
Determination of phenolic compounds by HPLC
The HPLC analysis was performed as described previously [36], with slight modifications, using an Agilent Technologies 1100 series liquid chromatograph equipped with an autosampler. The analytical column was Agilent Eclipse XDB C18 (100 × 4.6 μm; 3.5 μm particle size). The diode array detector 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). A 5 min post-run was used 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 flavonoid compounds, respectively. All samples were filtered through a 0.45 μm Acrodisc syringe filter (Gelman Laboratory, MI) before injection. Peaks were identified based on congruent retention times and UV spectra and compared with those of the standards (Sigma, St. Louis, MO, USA).
Antioxidant and biological activity of AGE
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) [37]. The absorbance was determined against the control at 515 nm [38]. The percentage of the scavenging activity of the DPPH˙ free radical was calculated as follows:
$$ \mathbf{Scavenging}\ \mathbf{activity}\ \left(\mathbf{Inhibition}\right)\%=\left[\left({\mathbf{A}}_{\mathbf{control}}-{\mathbf{A}}_{\mathbf{sample}}\right)/{\mathbf{A}}_{\mathbf{control}}\right]\times \mathbf{100}\operatorname{} $$
where A control is the absorbance of the control reaction and A sample is the absorbance in the presence of the plant extract. Gallic acid and TBHQ (Sigma, St. Louis, MO, USA) (1 mg/1 mL of methanol) were used as positive controls. 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. [39]. A control sample with no added extract was also analyzed. Antioxidant activity was calculated as follows:
$$ \mathbf{Antioxidant}\ \mathbf{activity}\ \left(\%\right)=\left[\mathbf{1}-\left({\mathbf{A}}_{\mathbf{sample}}^{\mathbf{0}}-{\mathbf{A}}_{\mathbf{sample}}^{\mathbf{120}}\right)/\left({\mathbf{A}}_{\mathbf{control}}^{\mathbf{0}}-{\mathbf{A}}_{\mathbf{control}}^{\mathbf{120}}\right)\right]\times \mathbf{100} $$
where A0sample is the absorbance of the AGE or synthetic antioxidant at time 0, A120sample is the absorbance after 120 min, and A0control and A120control are the absorbances of the control at time 0 and after 120 min, respectively.
Ferric reducing antioxidant power
The reducing power of the extract was assessed [38]. Distilled water was used as a negative control and gallic acid and TBHQ were used as positive controls. The absorbance of this mixture was measured at 700 nm using a UV spectrophotometer (Jenway-UV–VIS Spectrophotometer 6705). A decrease in absorbance indicated the ferric reducing power capability of the sample.
Testing the antidermatophyte activity of AGE
The procedure of Silva et al. [40] was used to test the antidermatophyte activity of AGE. The freeze-dried AGE (3.5 g) was dissolved and serially two-fold diluted in RPMI-1640 broth, to obtain a concentration range of 1000–20,000 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 a sterile Petri dish. Subsequently, wells with a diameter of 3 mm were made in the center of this agar plate and filled with 10 μL of the 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 controls were incorporated into the test. The concentration that inhibited the fungal growth was considered as the MIC.
Investigation of AGE effectiveness for the treatment of calf ringworm
Seventy-five calves showing evident clinical signs of ringworm were used for the investigation of AGE effectiveness in comparison with antifungal drugs for the treatment of this condition after obtaining informed consent from the farm owners. The enrolled calves were positive on mycological examination and T. verrucosum was isolated from clinical samples. Sample size calculation at a 0.05 significance level and 80% power revealed that 15 calves per group (G) would have been required. Calves exhibiting an equivalent severity of lesions distributed on the head, neck, and body were allocated randomly into five groups by using random number generator. Animals in G1 were treated orally with 250 mg/day of terbinafine (Lamisil®; Novartis, Basel, Switzerland). The crust on the skin lesions was removed with a brush and topical miconazole (Janssen Research Foundation, Beerse, Belgium) (G2), AGE solution (500 ppm) (G3), or oral terbinafine in combination with AGE (G4) were applied twice a day for 2 weeks. Animals in G5 were left untreated (as controls). Calves were observed daily for 6 weeks. In the beginning, during and after the treatment, the clinical efficacy was assessed by scoring dermatophytosis lesions on a 0–3 scale using previously published criteria [41,42,43] (Additional Table 2). The scoring was performed by the same investigator who was blinded to the treatment groups. The scores for each evaluated area (e.g. head, neck, and body) were averaged as follows: Sum of scores assigned to all lesions on the area/ Number of lesions on this area. Average total score of each animal = Sum of scores assigned to all evaluated areas/ Number of affected areas [44]. The lesions were assessed on every examination. The mycological examination was performed every week until two consecutive fungal cultures gave negative results [30, 44]. The control animals were treated after the observation period.
Each animal group was housed in a separate well ventilated, open sided pen with sheltered area. All the pens received similar management conditions. Area per calf in each pen was not less than 4m2 to avoid the overcrowding. The pens were bedded with straw that was changed every 1–2 weeks and antifungal disinfection for the entire pen and all materials with which animals come in contact was performed using 0.2% enilconazole (Clinafarm® EC; Merck Animal Health USA).
Data analysis
Various risk factors that were recorded on the whole set of the 760 clinically examined calves for ringworm lesions were included as independent variables in a multiple step-wise logistic regression model (PROC LOGISTIC, SAS Institute Inc. [45]). An approximate measure of relative risk was determined using odds ratio (the antilogarithm of the coefficient) with 95% confidence intervals. To confirm results and to identify the most important risk factor as a classifier that differentiated between infected and non-infected calves, a random forest non-parametric classification analysis was performed using the MetaboAnalystR web server [46]. Briefly, the occurrence of each variable was first used to build up a random forest classification model (an ensemble of 500 tree trials; out-of-bag (OOB) error = 0.6) in the respective outcome. The importance of the risk factor was determined by measuring the increase in the OOB error when the respective factor was permuted. The sensitivity, specificity, negative and positive predictive values, positive and negative likelihood ratio, and diagnostic odds ratio, which express the strength of the association between the test results and disease, with 95% confidence intervals 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 the detection/identification of dermatophytes causing calves’ ringworm. The Kappa value was used to test the agreement between test results. Student’s t-test was used to compare 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 among the treated and untreated groups over time after confirming the significance of Shapiro-Wilk test results [47]. The differences in clinical scores between groups were assessed by the Mann–Whitney U test after a significant result on the Kruskal–Wallis test. Significance was set at P < 0.05.
