Ringworm in calves: risk factors, improved molecular diagnosis, and therapeutic efficacy of an Aloe vera gel extract

Abstract

Background: Dermatophytosis in calves is a major public and veterinary health concern worldwide because of its zoonotic potential and associated economic losses in cattle farms. However, this condition has lacked adequate attention; thus, to develop effective control measures, we determined ringworm prevalence, risk factors, and the direct-sample nested PCR diagnostic indices compared with the conventional methods of dermatophytes identification. Moreover, the phenolic composition of an Aloe vera gel extract (AGE) and its in vitro and in vivo antidermatophytic activity were evaluated and compared with those of antifungal drugs.

Results: Of the 760 calves examined, 55.79% (424/760) showed ringworm lesions; 84.91% (360/424) were positive for fungal elements in direct-microscopy, and 79.72% (338/424) were positive in culture. Trichophyton verrucosum was the most frequently identified dermatophyte (90.24%). The risk of dermatophytosis was higher in 4-6-month-old vs. 1-month-old calves (60% vs. 41%), and in summer and winter compared with spring and autumn seasons (66 and 54% vs. 48%). Poor hygienic conditions, intensive breeding systems, animal raising for meat production, parasitic infestation, crossbreeding, and newly purchased animals were statistically significant risk factors for dermatophytosis. One-step PCR targeting the conserved regions of the 18S and 28S genes achieved unequivocal identification of T. verrucosum and T. mentagrophytes in hair samples. Nested-PCR exhibited an excellent performance in all tested diagnostic indices and increased the species-specific detection of dermatophytes by 20% compared with culture. Terbinafine and miconazole were the most active antifungal agents for dermatophytes. Gallic acid, caffeic acid, chlorogenic acid, cinnamic acid, aloe-Emodin, quercetin, and rutin were the major phenolic compounds of AGE, as assessed using high-performance liquid chromatography (HPLC). These compounds increased and synergized the antidermatophytic activity of AGE. The treated groups showed significantly lower clinical scores vs. the control group (P < 0.05). The calves were successfully treated with topical AGE (500 ppm), resulting in clinical and mycological cure within 14-28 days of the experiment; however, the recovery was achieved earlier in the topical miconazole 2% and AGE plus oral terbinafine groups.

Conclusions: The nested PCR assay provided a rapid diagnostic tool for dermatophytosis and complemented the conventional methods for initiating targeted treatments for ringworm in calves. The recognized antidermatophytic potential of AGE is an advantageous addition to the therapeutic outcomes of commercial drugs.

Keywords: Aloe vera gel extract; Antifungal drugs; Calves dermatophytosis; Direct-sample nested PCR; Risk factors; Treatment.

Fig. 1

(a) Random forest classification showing the most important risk factor (y-axis) as a classifier differentiating between diseased and non-diseased calves when it was clinically examined. The X-axis refers to the predictive accuracy of the studied risk factors. The mini heatmap shows the frequency distribution of each factor across the two outcomes (ringworm lesion and without lesion). Each dot refers to the value of mean decrease accuracy of one risk factor, (b) Box plot for a normal distribution of age (as a continuous variable) across the examined calves (n = 760), each dot represents one case and the horizontal line refers to the median of age distribution

Fig. 2

Agarose gel electrophoresis for amplicons of direct PCR assays for testing dermatophytes DNA from calves’ hair samples. (a) Pan-dermatophytes PCR amplicons of pchs-1gene at 440 bp, (b) One-step PCR ITS+ amplified products for T. verrucosum at 900 bp (lanes 2–8) and T. mentagrophytes at 872 bp (lanes 11, 12), and (c) ITS-1 amplicons of nested PCR at 400 bp. Lanes M: 100 bp molecular size marker, lane Pos.: positive control and lane Neg.: negative control

Fig. 3

(a) The antioxidant activity of Aloe vera gel extract (AGE) against 1,1-Diphenyl-2picrylhydrazyl (DPPH˙) radical and β-Carotene/linoleic emulsion compared with gallic acid and tert-butyl hydroquinone (TBHQ), (b) The absorbance of ferric reducing power of AGE against gallic acid and TBHQ

Fig. 4

Medians for clinical scores of ringworm lesions on treated groups and control untreated group from day 0 to 42 days of the study. There is a non-significant difference between the clinical scores of groups on days 0 and 7, while the treated groups displayed significantly (P < 0.05) lower clinical scores than the control group on days 14, 21, 28, and 42. Clinical scores carrying asterisks with the same color was statistically different. † indicating a high significant difference between the control untreated group and all other groups within the same days