Ringworm in calves: Risk factors, improved molecular diagnosis, and ecacy of Aloe vera gel extract for treatment

Background: Calves dermatophytosis is a major public and veterinary health problem worldwide due to its zoonotic potential and economic losses in cattle farms. However, it has lacked adequate attention; thereby for effective control measures it is worth determining ringworm prevalence, risk factors and direct sample nested-PCR diagnostic indices as compared to conventional methods for dermatophytes identication. Moreover, Aloe vera gel extract (AGE) phenolic composition and its in-vitro and in-vivo antidermatophytic activity in comparison to antifungal drugs were evaluated. Results: Of 760 examined calves, 55.79 % showed ringworm lesions. 84.91% were positive for fungal elements in direct microscopy, and 79.72% were culture positive. Trichophyton verrucosum was the most frequently identied dermatophytes (90.24%). Risk of dermatophytosis is high in 4-6 month than 1-month aged calves (60% versus 41%), in summer and winter compared to spring and autumn seasons (66% and 54% versus 48%). Poor hygienic conditions, intensive breeding system, animals raised for meat production, parasitic infestation, crossbreed, and newly purchased animals were statistically signicant risk factors correlated with dermatophytosis. One-step PCR targeting conserved regions in the 18S and 28S genes achieved unequivocal identication of T. verrucosum and T. mentagrophytes in hair samples. Nested-PCR achieved an excellent performance in all tested diagnostic indices and increased the species-specic detection of dermatophytes by 20 % as compared to culture. Terbinane and miconazole were the most active antifungal agents for dermatophytes. Gallic acid, caffeic acid, chlorogenic acid, cinnamic acid, aloe-Emodin, quercetin, and rutin are the major phenolic compounds of AGE identied by High-performance liquid chromatography (HPLC). These compounds increased and synergized the antidermatophytic activity of AGE. The treated groups showed signicantly lower clinical scores than the control group ( P < 0.05).

dermatophytosis in cattle and can occasionally spread to humans through direct contact with cattle or infected fomites causing highly in ammatory skin and hair dermatophytoses [4][5][6]. As a consequence, a precise laboratory diagnosis for identi cation of dermatophytes species is pivotal for prevention and effective control of dermatophytoses [2]. Considering the previously mentioned contemplations, scant researches have been found so far studying the prevalence, risk factors and treatment of calves' ringworm in Egypt.
Furthermore, there are scarcity publications on the direct molecular diagnostic assays for detection and identi cation of dermatophytes from animals clinical samples [7,8] to surpass the time consuming conventional microscopy and fungal culture that require weeks for a positive identi cation [8]. The nested polymerase chain reaction (PCR) technique proved to be an effective practical approach for dermatophytosis diagnostic, helping clinicians in initiating rapid and targeted as opposed to empirical treatments of animal ringworm [7].
Dermatophytosis in animals remains di cult to eradicate because of the antifungal resistance, the scarcity of accessible and authorized antifungal agents for use in veterinary practice, the restricted systemic treatment in livestock due to the hepatotoxicity, drug residues in products consumed by human [2,9]. Thus, the discovery of natural, less toxic and more speci c therapeutic alternatives is gaining ground. Although the antidermatophytic potentials of natural products promising, are plagued by a lack of in-vivo studies to a rm the antifungal activity of bioactive compounds announced in-vitro [9]. Aloe vera is a plant of Liliaceae family has multiple applications including antifungal, antibacterial, antioxidant, antiseptic and in cosmetics industries [10]. Investigations for in-vitro and in-vivo antidermatophytic potential of Aloe Vera and determination of its bioactive compounds are still meager.
Hence, this work was designed for investigation ofa) the prevalence and risk factors of calves ringworm in Egypt, (b) diagnostic indices of the direct nested PCR for detection and identi cation of dermatophyte species from hair and scale samples in comparison with the conventional microscopic and culture methods, (c) Aloe Vera gel extract (AGE) biological activity and phenolic composition, (d) the antifungal activity of AGE in comparison to the antifungal drugs and (e) its application for treatment of calves ringworm.

Prevalence of dermatophytosis among clinically examined calves
On clinical examination, 55.79 % of calves (424/760) showed grayish-white, crusty, circular circumscribed discrete lesions ( Figure 1A) whereas; alopecic, erythematous areas left after removal of raised greasy crusts were occasionally observed ( Figure 1B). The skin lesions were mostly found on the head and neck (46.69%) and all over the body (44.81%). Some cases (8.49%) had lesions on the head, neck and trunk as well. The degree of infection diversi ed from moderate (55.18 %) to severe (44.81%).

Potential risk factors for calves' ringworm
There is highly signi cant association between ringworm infection and the age of calves (χ2 = 19.7, P< 0.001**). Risk of infection in 4-6 month aged animals is higher than younger calves (60% versus 41%).
Risk ratio and odds of disease in 1-month aged animals were 68% and 45% lower than 4-6 month animals, respectively.
Seasonal variation was signi cantly highly associated with skin lesion in the examined animals (χ2 = 19.58, P< 0.01**). The high risk of calves' dermatophytosis was in summer and winter as compared to spring and autumn seasons (66% and 54% versus 48%). Also, there is highly signi cant association between calves ringworm and each of breed type, production system, breeding system, origin of the animals at the farm, ventilation, and pattern of disinfectant used (χ2 = 63.18, P< 0.001**). Risk of infection in cross breed, animals for meat production, parasitic infestation, intensive breeding system, newly purchased animals introduced to the farm, bad ventilation, and irregular use of disinfectant are higher as compared to others, respectively (85 % versus 49%).
The area available per calf highly affects the infection potential. The narrower the area the more skin lesion spread among animals (χ2 = 7.79, P< 0.01**). The relationship is weak (Phi & Cramer's V = 0.16, P< 0.001**). Risk of infection in animals reared in 1.5mr/animal is higher than animals reared in 6mr/animal (62% versus 52%). Ringworm lesion in animals reared in 1.5mr/animal is 1.19 times more likely higher than animals reared in 6mr/animal ( Table 1). The random forest classi cation model con rmed the same observation, where age of calf was the most important risk factor, followed by production system, parasitic infestation, and irregular use of disinfectant was the fourth most risk factor ( Figure 2).

Nested PCR for detection and identi cation of dermatophytes in clinical samples
Pan-dermatophytes, one-step and nested PCR were evaluated for dermatophytes identi cation in 75 direct microscopy and culture-positive samples, 36 microscopy +/culture -, 9 microscopy -/culture + and 30 negative samples. The Pan-dermatophytes PCR had the ability to speci cally detect dermatophytes DNA in 58%, one-step PCR in 62% and nested PCR in 72 % of 150 samples (Table 1) with 440 bp pchs-1 amplicons, ∼ 900 bp ITS+ and 400 bp ITS-1 amplicons, respectively ( Figure 3).
Nested PCR increases the dermatophytes species-speci c detection by 20 and 10 % as compared to culture alone or the combination of culture and direct microscopy, respectively.
Fungal culture identi ed dermatophytes in 56% (84/150), whereas direct microscopy identi ed 74% (111/150) of samples. Out of 66 samples that were negative for dermatophytes by culture, non dermatophyte-moulds were cultured from 21 samples that were test-positive only by one-step PCR. In addition, non dermatophyte-moulds were co-cultured with dermatophytes from six samples that were negative by the pan-dermatophytes, one-step and nested PCRs (Additional table 1).
As depicted in table 2 the performance of nested PCR assay was excellent in all tested diagnostic indices.
Based on fungal culture as a reference standard, a sensitivity of 82.14 %, 71.43 % and a speci city of 72.73 %, 50 % were recorded for pan-dermatophytes and one-step PCRs, respectively. Meanwhile, the respective values were 92.86 and 54.55 % for nested PCR.Whereas using the combination of culture and nested PCR as a gold standard, nested PCR was superior achieving a sensitivity value of 94.74 %, whilst culture and direct microscopy demonstrated 73.68% and 78.95%, respectively. Speci city and PPVs were 100% for nested PCR and dermatophytes culture so they were considered as the gold standard, whilst the competent values were 41.67 and 81.08 % for direct microscopy. Nested PCR was very accurate (AUC = 96%), whereas pan-dermatophytes PCR (82%) and culture (80%) were moderately accurate. Less diagnostic accuracy was recorded for direct microscopy and one-step PCR (50 AUC ≤ 70 %). DOR of nested PCR is much higher than any other test this means that the diagnostic performance of nested PCR is the best and showed strong agreement with culture and nested PCR results (Kappa value = 0. 91and P < 0.001).. Con rmatory ITS sequencing was performed and the BLAST search results for the sequence (GenBank accession no. MK918485) corresponded to various T. verrucosum isolates, with the highest-ranked genetic identity to T. verrucosum isolated from in ammatory tinea capitis (KY623478 (100% identity) and tinea corporis in a 3-years old child in China (KY623476 (99.89% identity). Identity percent for the sequence (MK918486) was 100% with T. mentagrophytes (MK447604 and MK447606) isolated from tinea capitis and tinea faciei in 8 and 44-years old Indian boy and man, respectively.

Susceptibility of dermatophytes to antifungal drugs
The mean MICs and MFCs values of the ve antifungal drugs for T. verrucosum and T. mentagrophytes are presented in additional table 2. When the values of the ve antifungals for the two species tested were compared, those for terbina ne were the lowest followed by miconazole (MIC range 0.03-0.5, 0.03-1 µg⁄mL; MFC range, 0.06-1, 0.06-2µg⁄mL, respectively). Mean MIC values ± SD of the tested antifungal agents did not differ for T. verrucosum and T. mentagrophytes (P < 0.05). Fluconazole was the least effective drug with overall MIC range 8-64 µg/ml. Yield, TPC, TF, Phenolic compounds, antioxidant and antifungal activity of AGE As depicted in Table (3) the extract yield was 1.02 g extract 100 g -1 Gel. The amount of total phenolics as mg GAE g −1 AGE was (111.78 mg GAE g −1 ). The avonoid contents of extract as mg quercetin equivalent/g AGE was (45.6 mg QE g −1 ). Flavonoids have an expansive range of chemical and biological activities, including radical-scavenging properties. For this reason, extract was analyzed for total phenolic and avonoid contents.The major phenolic compounds of AGE were identi ed by HPLC as presented in Table 3. These components were gallic acid, caffeic acid, chlorogenic acid, cinnamic acid, aloe-Emodin, quercetin, and rutin. All these compounds increased and synergized the antidermatophyte activity of AGE.
The results of DPPH radical-antioxidant activities of AGE were demonstrated in (Figure 4a) with antioxidant activity 85.3 % for AGE and 92.2% for gallic acid and TBHQ (88.6%) after 2 h. of reaction. The obtained results clearly demonstrate that this extract displayed antioxidant activity. As revealed in Figure  4a AGE inhibited the bleaching of β-carotene by scavenging linoleate-derived free radicals. The decreasing e cacy was ordered as TBHQ>AGE>gallic acid. The results showed a comparable scavenging ability 72.3 % for AGE to the synthetic antioxidants gallic acid 65.7 % and TBHQ 81.22%. AGE displayed a ferric reducing power compared with TBHQ and gallic acid ( Figure 4b). The ferric reducing power of AGE was 1.96 compared with gallic acid (2.23) and TBHQ (2.57). AGE showed an inhibitory effect for T. verrucosum and T. mentagrophytes at MIC values ranged from 300-400 ppm and 400-500 ppm, respectively.

Effectiveness of AGE in the eradication of T. verrucosum from calves
In the treated calves, gradual improvement of the lesions was seen within 7 to 12 days post-treatment. Complete clinical recovery (full hair growth) was observed within 14 -19 days of the study for calves in G2 & G4 and within 21-28 day for animals in G1 and G3 (Additional gure 1). Meanwhile, the lesions on the control animals (G5) progressed and did not heal till 42 day of the study. At day 14, signi cant improvement in the clinical scores (P < 0.05) was detected between miconazole treatment (G2) and untreated control group (G5), G2 and terbina ne treatment (G1) and G2 and AGE (G3).
Neither recurrence, nor grossly side effects were observed throughout the study period and during the clinical follow-up after treatment.

Discussion
An enzootic circumstance of animals' dermatophytosis is the outcome of the con nement of animals in breeding and the viability of the arthrospores in the environment for many months [2]. Prevention is di cult, but periodic surveys on the prevalence and risk factors of cattle ringworm may permit the adoption of increasingly effective prophylaxis and control measures to prevent the infection both to other animals and humans [2,11,12]. In the current study, the prevalence rate of ringworm in calves of 1 to 6 months was 55.79 % that is nearly similar to the 57.5% found in Iran [5]. Furthermore, the infection rate is higher than the 1.6% found in Chitral district of Pakistan [12] but lower than the 87.7% in Tuscany region [4] and 71.7% in nearby Umbria, Central Italy [11]. This discrepancy among different countries perhaps due to cattle breed, production, breeding system, origin of the cattle in the farm and climatic conditions [11]. In accordance with other studies [4,11], random forest classi cation and box plot model indicated that age is the most important risk factors whereas, the risk of infection in 4-6 month aged calves is higher than the younger suckling calves (60% versus 41%) and this could be attributed to the stressors of weaning and rapid growth. Furthermore, there is a highly signi cant correlation of several risk factors found in the examined calves population and ringworm infection, mainly season, bad ventilation, overcrowding and irregular use of disinfectants. This participate in reinforcing the broadly accepted concept that high humidity, close contacts between calves, poor hygienic conditions of the stable play a signi cant role in the ringworm prevalence [4,5,11,12]. Hence, repeated topical treatment of all infected animals, together with good ventilation, thorough disinfection of the stable, halters, fences, cleaning tools and all the materials in contact with the animals is the basis for cattle ringworm effective control [11,13]. Of interest, there is highly signi cant (P < 0.001) association between the risk of dermatophytosis and the newly introduced animals to the farm. In support of this nding, [4] debate that the newly introduced calves in the herd spread the infection to both calves and humans as they are carriers of dermatophytes before the development of clinical signs.
As described in the literature [2,5,11,14], the detected clinical signs of cattle dermatophytosis were crusty lesions on the head, neck regions and other parts of the body. Though, other study conducted in Tanzania [15] occasionally reported the widespread lesion of alopecia and erythema that were observed herein as well. The detection rates were 84.91 % by direct microscopy and 79.72% by fungal culture. In such a context, inadequate scraping of the lesion and the slow and poor growth of T. verrucosum that hampered its detection probably the reasons for direct microscopy and culture false-negative results, respectively [4].
According to Nweze; Agnetti et al. [11,14]T. verrucosum is the main dermatophytes causing cattle ringworm, although T. mentagrophytes that is usually associated with the presence of small rodents in the farm has been isolated. The present ndings showed that calves ringworm caused by T. verrucosum (90.24%) and T. mentagrophytes (9.76%). Nevertheless, Aghamirian and Ghiasian [5] exclusively isolated T. verrucosum from 352 infected cows in Iran.
To date, direct molecular assays are used for the detection of T. verrucosum in clinical samples and also as culture con rmation tests [16]. Wollina and coauthors have failed to cultivate T. verrucosum from clinical sample and para n-embedded skin tissue from a patient with severe tinea barbae. But, real-time PCR and subsequent sequencing of the ITS2 region of rRNA could successfully identify T. verrucosum. No previous studies attempted to identify dermatophyte species from hair samples of calves using direct nested PCR assay. The obtained ndings revealed that one-step PCR could correctly identify T. verrucosum and T. mentagrophytes in samples, that were culture positive (n = 72/150), with an amplicon size of 900 bp and 872 bp, respectively. Meanwhile, nested-PCR ampli ed ITS+ of both species in 108 samples producing 400 bp ITS-1 amplicons. This coincides with another study [7] that the one step-PCR accurately identi ed Microsporum canis in hair samples from canine and felines at 922 bp, but ∼ 851 to ∼ 872 bp ITS+ amplicons were obtained for T. mentagrophytes, T. terrestre or M. gypseum, whereas nested PCR achieved unequivocal identi cation. The highly sensitive nested PCR also had high speci city and positive predictive value and detecting additional dermatophyte-positive samples that were missed by culture (n = 30/150) or both microscopy and culture (n = 15). Other studies highlighted the importance of direct sample PCR incorporation in the laboratory diagnosis of onychomycosis for increasing the detection of dermatophyte-positive samples that were negative by culture [17,18]. Despite direct microscopy and /or culture positive (n = 27/150), or negative samples in both (n = 15), negative PCR results, except 21 samples that were positive by one-step PCR and with non-dermatophytes mould grew in culture, could be explained byPCR inhibition by components in samples from non-dermatophytes contaminant/colonizer or the existence of smaller amounts of dermatophytes DNA in samples [7,17]. A possible reason for the low speci city and accuracy of one-step PCR than pan-dermatophytes and nested PCRs is employing the universal fungal regions of rDNA. Additionally, the low sensitivity of culture could be attributed to overgrowth of non-dermatophytes contaminating moulds in the culture or dermatophytes cultures were not yet positive after 4 weeks of incubation [4,7]. Other reasons are the non-viable fungal material in specimens from treated calves or that the DNA extraction step ease overcoming the impediment of trapping fungus in the keratin [18].
As previously reported [19,20], terbina ne and miconazole were effective antifungal drugs for dermatophytes followed by itraconazole and griseofulvin whereas; uconazole was the least active antifungal agent. Recently, Pal [21] recommended the conduction of further research for the development of cheap, safe and potent chemotherapeutic agents for cattle dermatophytosis management. AGE is a cheap, easily obtainable and safe natural product. Besides an endless source of bioactive compounds that clearly recognized for having antifungal activities that correlated with the antioxidant activities [22].
So, in this study, antioxidant activities were measured by three methods (DPPH • free radical, βcarotene/linoleic emulsion bleaching, and FRAP). DPPH • is a model of a stable nonpolar radical. A chain reaction of radicals is initiated by lipid autoxidation. Antioxidants react with DPPH • , reducing the number of DPPH • free radicals to the number of their available hydroxyl groups [23]. The antioxidant activity of AGE against DPPH • was concentration-dependent. In β-carotene/linoleic emulsion bleaching assay, oxidation of linoleic acid results in free radicals derived from hydroxide that assault the chromophore of β-carotene, leading to bleaching the emulsion of the reaction. An extract can inhibit/retard the oxidation of β-carotene might be portrayed as a free radical scavenger and initial antioxidant [24]. Antioxidant compounds cause the reduction of ferric (Fe +3 ) form to the ferrous (Fe +2 ) form because of their reductive capabilities [25]. According to our results, ferric reducing power and TPC content are related to one another. Fe (III) reduction is commonly utilized as a marker of electron-donating activity, which is a signi cant mechanism of the phenolic antioxidant action [26]. Considering the results of all three assays, phenolic compounds can explain high antioxidant capacity [27]. The antioxidant activity of phenolic compounds is chie y because of their redox properties, which can assume a signi cant role in the absorption and neutralization of free radicals, decomposing peroxides or triplet oxygen, quenching singlet and reductive heavy metals with two or more valence states [28]. The phenolic compounds are the active antimicrobial constituents of various plants extract. However, the whole extract has more noteworthy antifungal activity. Accordingly, AGE might be more advantageous than the isolated component, since a bioactive individual constituent can change its properties in the existence of other compounds [29]. The additive and synergistic impacts of phenolic compounds in fruits and vegetables are accountable of their e cient bioactive properties, which clari es the purpose behind which no single antimicrobial can supplant the combination of these natural components to achieve the antifungal activity [30]. The recognized anti-dermatophytic activity of AGE was in consistent with previous report [31] that the water extract of Aloe vera was effective on T. mentagrophytes. However, no reports were found about AGE activity on T. verrucosum. Nonetheless, most investigations are performed on fungal isolates, thereby it hard to extrapolate the ndings to real conditions. Therefore, more in-vivo studies are needed to ensure reliable results [9]. The e cacy of 2 weeks' twice-daily topical application of AGE was compared with topical miconazole 2%, oral terbina ne with topical AGE application and once-daily oral terbina ne in proven T. verrucosum infected calves. There were signi cantly lower clinical scores in all treated groups after 14 day than untreated group (P < 0.05) but complete clinical recovery was achieved earlier in miconazole group and AGE with oral terbina ne than both oral terbina ne group and AGE alone. This con rms the high e cacy of AGE for the treatment of calves dermatophytosis and suggests that the combination of AGE with oral terbina ne was highly effective. The achieved results were comparable with the treatment ndings of calves dermatophytosis with topical applications of propolis and whit eld's ointment [32] and polyherbal lotion, along with levamisole and griseofulvin [33].

Conclusion
This study highlights the need for good hygienic conditions, regular disinfection of holdings, rapid treatment of infected calves and examination of the incoming calves so as to prevent dermatophytic epizoonoses in calves and human as well. Implementation of nested PCR assay providing a rapid diagnostic tool for dermatophytosis augments and complement the conventional methods for dermatophytes species-speci c detection for initiating targeted treatment which would reduce the burden of economic losses due to the ringworm infection. The recognized anti-dermatophytic potential of AGE is advantageous countenance to commercial drugs to go used in therapeutics. Further studies are recommended to use the AGE for large scale treatment of calves' dermatophytosis.

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 lms were prepared, xed in absolute methyl alcohol and stained with freshly ltered 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 [4].
Portions of hair and scales were examined microscopically after clearing with 20% potassium hydroxide (KOH), cultured on Mycobiotic agar (Remel™, Thermo Fisher Scienti c) 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 identi ed according to their macro-and micromorphological characteristics [34].

Extraction of DNA from hair and scales samples and PCR ampli cation
The direct molecular identi cation 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 Scienti c, Waltham, MA, USA).

Nested PCR
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 [7].
PCR was performed in an ampli cation 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 ampli cation reaction without DNA template were utilized as positive and negative control, respectively. The thermo-cycling conditions previously described [7] were employed in an applied biosystems 2720 thermal cycler (Thermo Fisher Scienti c, USA).
The ampli ed 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 puri ed 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 [35]was employed for testing the sensitivity of dermatophytes isolates to the most commonly used antifungal drugs. Fluconazole were obtained from P zer 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 terbina ne from Novartis, Basel, Switzerland. All drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) except uconazole 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 nal concentrations of 0.125 to 64 μg/mL for uconazole 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 Scienti c) and ltered using Whatman No.1 lter paper. The extraction ratio was 1 W: 5 V (gel: solvent). The ltrate 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.

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.[36]. 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 avonoids
Total avonoids content expressed as quercetin equivalent (QE) in AGE at a nal 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 [37]. Determination of phenolic compounds by HPLC HPLC analysis was executed as previously described [38] with slight modi cations 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 ow rate was kept at 0.4 mL min -1 and the gradient program was as follows: Where A 0 sample is the absorbance of the AGE or synthetic antioxidant at 0-time, A 120 sample is the absorbance after 120 min, A 0 control and A 120 control are the absorbance of control at 0-time and after 120 min, respectively.

Ferric reducing antioxidant power (FRAP)
The extract reducing power was assessed [40]. 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. [42] 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 nal concentration of 50-1000 μg/ml was obtained by mixing 2 ml of this solution with 18 ml of lique ed Mycobiotic agar medium (Remel™, Thermo Fisher Scienti c) at 45 °C in sterile Petri dish. Next, wells of 3 mm diameter were made in the centre of this agar plate and lled with 10µl of fungal spore suspension (10 6 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 were left untreated as controls. Calves were observed daily for six weeks. At the beginning, during and after treatment, the clinical e cacy 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
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 con rm the results, random forest non-parametric classi cation method was done using MetaboAnalystR web server [44].
Brie y, the occurrence of each variable was rstly used to build up random forest classi cation 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, speci city, 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 % con dence 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/identi cation 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 signi cant Kruskal-Wallis test. P < 0.05 was considered signi cant.  Tables   Table (1): Calves' dermatophytosis prevalence and risk ratio of different risk factors † Risk ratio and ‡ odds of summer to winter season. **, *** significant P-values.      Score lesions (mean and standard deviations) for treated groups and control untreated group from 0 day to 42 day of experiment. There is a non-signi cant difference between the clinical scores of groups on days 0 and 7, while the treated groups displayed signi cantly (P < 0.05*) lower clinical scores than the control group on days 14, 21, 28, and 42. Clinical scores carrying asterisks within the same day was statistically different. ** indicating signi cant difference between control untreated group and all other groups within the same days.

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