The Faculty of Veterinary Medicine's Ethics Committee gave its approval to the collection method, Mansoura University (Mansoura, Egypt) and completed in accordance with the necessary biosecurity rules, and all laws and recommendations were followed when performing animal research of the "Guide for the Care and Use of Laboratory Animals".

The samples were collected by veterinarians from various private and public veterinary clinics based on Oxford Academic technique to improve molecular diagnosis of dermatophytosis in pet animals. A total of 200 animals (130 cats and 70 dogs) were tested in this research. Veterinarians documented and sequentially categorized as suspected cases of dermatophytosis animals with skin lesions such as alopecia and numerous circular lesions (figure 2. A). No antifungal medication was administered to any of the sampled animals either before or during the sampling. The current investigation was conducted between June 2019 and July 2020 in Dakahlia Government, Egypt.

The clipped hair and skin scrap samples of each animal with probable dermatophytosis were collected from the lesions with both methods; scrubbing and brushing as previously described. In brief, scrubbing method was performed by first cleaning the area with alcohol 70% then scrubbing the skin surface at the edges of the area with the back of sterile scalpel. Brushing method was done to obtain hair samples by using a sterile brush cut after brushing the animal body for 5 min from head to tail. The animal body was brush for 3 min from head to tail. The samples were collected in a sterile tube contains peptone water broth (BioLab, Hungary), and transported to the laboratory within 24 h for further examination.

Skin scraping, clipped hair, and brush samples were inoculated into Sabouraud dextrose agar (SDA) (Biolab) and potato dextrose agar (PDA) (Difco), both of which were supplemented with Modified Dermato Supplement (HiMedia, India). The plates were incubated at 25°C for at least 14 days, and fungal growth was monitored daily. After incubation, lactophenol cotton blue was used to identify each isolate macroscopically and microscopically in terms of hyphae, macroconidia, and microconidia 14. The pure culture was stored on PDA for further analysis.

According to the Clinical Laboratory Standards Institute, agar disc diffusion and broth microdilution assays were used to assess the antifungal susceptibility of dermatophyte isolates 15. Briefly, the inoculum suspensions were made using CLSI recommendations from cultures that were cultivated on PDA without the use of antimicrobial agents and were 7 days old 16. Then, the suspensions were diluted to 1x106 CFU/ml and adjusted using a spectrophotometer (IMPLEN) to optical densities ranging from 0.29 to 0.34 at 635 nm for use in both the broth and agar techniques 17. Agar diffusion test was performed onto Muller Hinton agar plates (Oxoid) and using the following antifungal agents (Oxoid): Itraconazole (10 µg), fluconazole (10-25 µg, Sedico), nystatin (100U), griseofulvin (10 µg, Kahira P harma), amphotericin B (100U), terbinafine (10-25 µg, Global Napi). The plates were observed daily for 5-7 days 18. Microdilution test was applied using the following antifungal drugs: Itraconazole (Global Napi), fluconazole (Sedico), nystatin (Epico), griseofulvin (Kahira Pharma), terbinafine (Global Napi), and acetic acid (SDFCL). In brief, in a 96-well microtitre plate, stock solutions were used to create two-fold drug serial dilutions that were then produced in peptone broth (Oxoid). M. canis and T. mentagrophyte were introduced to each well at a concentration of 1x106each. The plates were incubated for 7 days at 25°C. The results were read by ELISA reader (BioTek, USA) at 560 nm before (0 day) and after incubation (7 days) for comparison 19.

Out of 200 samples from cats and dogs, 90 (45%) samples were positive for Microsporum (Figure 1. a1, a2 & a3) and Tricophyton (Figure 1.b1, b2 & b3) species by conventional mycological technique. Out of 130 samples from cats, M. canis (n=34/63; 53.9%) and T. mentagrophytes (n=29/63; 46%) were isolated from 63 cats (63/130, 48.5%) samples. Among the 70 samples from dogs, 27 (38.6%) samples were positive for M. canis (n=12/27; 44.4%) and T. mentagrophytes (n=15/27; 55.6%) as seen in (Table 1).

The antifungal susceptibility of 88 dermatophyte (M. canis n=44 and T. mentagrophytes n=44) strains to Itraconazole, fluconazole, nystatin, griseofulvin, amphotericin B, and terbinafine was examined by agar disk diffusion test result in (Table 2).

Microsporum canis showed high susceptibility to griseofulvin (75%), followed by terbinafine 25µg (70.5%), nystatin (68.2%), and terbinafine 10µg (68.1%), while Trichophyton mentagrophytes revealed high sensitivity to griseofulvin(61.4%), followed by nystatin (56.8%), terbinafine 25µg (54.5%), terbinafine 10µg (45.5%), and Itraconazole (27.3%). On the other hands, no antifungal activity of fluconazole and amphotericin B was observed against dermatophytes. In particular, Itraconazole showed no sensitivity to all examined Microsporum species.

The MICs of fluconazole, nystatin, griseofulvin, terbinafine, and acetic acid for 10 dermatophyte isolates, including M. canis (n=5) and T. mentagrophytes (n=5), were evaluated using the broth microdilution assay. MIC range, MIC, IC50 range, IC50 and geometric mean MIC were computed (Table 3).

In general, the information demonstrated that the most effective antifungal agent tested against dermatophytes was terbinafine and acetic acid. For M. canis strains, the lowest MIC was observed to terbinafine (MIC = 0.98 µg/ml; IC50 = 0.54 µg/ml) followed by acetic acid (MIC = 1.05 µg/ml; IC50 = 0.58 µg/ml), nystatin (MIC = 1.08 µg/ml; IC50 = 0.6 µg/ml), griseofulvin (MIC = 1.24 µg/ml; IC50 = 0.69 µg/ml), and fluconazole (MIC = 1.3 µg/ml; IC50 = 0.72µg/ml). While among T. mentagrophytes strains, the lowest MIC was detected to acetic acid (MIC = 0.28 µg/ml; IC50 = 0.16 µg/ml) followed by terbinafine (MIC = 0.295 µg/ml; IC50 = 0.16 µg/ml), nystatin (MIC = 0.4199 µg/ml; IC50 = 0.23 µg/ml), fluconazole (MIC = 0.587 µg/ml; IC50 = 0.33 µg/ml), and griseofulvin (MIC = 4.6875 µg/ml; IC50 = 2.6 µg/ml).The viability of M. canis strains was decreased after treatment with nystatin, and acetic acid, followed by fluconazole, terbinafine, and griseofulvin in the lowest concentration and reached 20-30%. For T. mentagrophytes, the lowest viability was detected to acetic acid followed by nystatin, and fluconazole ranges from 60-80%, while the highest viability (90-100%) to terbinafine and griseofulvin was observed (Figure 3).

Clinical signs and culture after dermatophytes infection

All of the guinea pigs (G2 & G3) exposed to the fungus during the experimental infection with M. canis or T. mentagrophytes developed lesions (Figure 2).

The first symptoms of infection appeared in all affected animals on the seventh day following inoculation and ranged from dandruff-like scalps to hair loss. Some animals showed pruritus with itching and scratching of the lesion. Around the 14th day, these alterations became more evident with the development of hair rarefaction and squamosis. The lesions gradually increased in diameter with the development of complete hair loss and crust formation between the 10th and 14th days. All infected animals' skin scrapings and hair from days 7 and 14 were used to re-isolate M. canis and T. mentagrophytes. All of the animals that had received the inoculation tested positive for mycological cultures (Figure 4).

G1, Control group; G2, M. canis infected group; G3, T. mentagrophytes infected group; the results represent significant results (mean ± SD) (P > 0.002).

The total leukocytes count is significantly elevated in all infected groups (4-6x10⁹/µl for G2 and 6-8_X10⁹/µl for G3) at 14 days post infection compared to G1 group (approximately 2_X10⁹/µl). The differential leukocytes count showed that lymphocyte and neutrophil were raised in comparison with monocyte. The neutrophil level was significantly elevated to 2-4.5 _X10⁹/µl in G2 and 5.4-6.3 x10⁹/µl in G3 in comparison to G1 at 14^th days post infection. The lymphocyte level was raised to 1.3-2.2 _x10⁹/µl in G2 and 0.9-1.2 _x10⁹/µl in G3 compared to G1. Also, the monocyte level was increased to 0.4-0.5 _x10⁹/µl in G2 and 0.2-0.8 _x10⁹/µl in G3 compared to G1. The result was tabulated below (Table 4).

Gel electrophoresis of the cDNDs of the dermatophyte (M. canis and T. mentagrophytes from skin scrap samples (n=6, each) obtained from experimentally infected guinea pigs revealed characteristic banding pattern with single protease targeted cDNA band in (Figure 5).

Dermatophyte is one of the most widely spread diseases in the world particularly countries with poor sanitation and dense populations. Though it is not a fatal disease but it is rapidly spread and causes yearly huge economic losses in antifungal treatment 25. The fact that this disease is transmitted from animals to man and vice versa makes its proper diagnosis and control of great importance to help eradicate the disease 18. Especially that most cases goes miss diagnosed or not reported in Egypt as most owners do not seek veterinary service in skin diseases 26. Cats and dogs are regarded as one of the principal carriers of disease in a number of European nations 27. This study looked into the frequency of dermatophytes in Egyptian cats and dogs with dermatophytosis. In this study, standard mycological testing revealed that 90 (45 %) of the 200 samples analyzed contained fungal components. The prevalence of infections is typically more than 20% in cats, but it is between 4% and 20% in dogs, according to epidemiological studies on cats and dogs with probable dermatophytosis lesions ^28-30. In this study, in particular, dermatophytes were identified from 48.5% of the cats and 38.6% of the dogs. In Spain and Egypt, previous studies revealed a prevalence that was two times higher in feline than in canines 263132 detected the isolation rates of dermatophyte species (19.3%) from cats (20.1%) and dogs (18.7%). In a recent study performed on 10,678 and 15,684 cats and dogs samples only 23% were found positive for dermatophytes 33. In fact, our findings indicated that dermatophytosis can be prevalent feline and canine illnesses in Dakahlia Government, Egypt. Therefore, the higher isolation rate of dermatophytes among cats and dogs in the present study may be alarming in these areas of Egypt.

Numerous investigations have proven that systemic and topical antifungal treatments for dermatophytosis are ineffective 34, therefore, it is crucial to assess and standardize in-vitro assays that are straightforward and repeatable to ascertain the antifungal effectiveness of various medications against dermatophytes. Antifungal agent in-vitro activity studies are intermittent and primarily focus on filamentous fungi 35. The creation of a standardized disc diffusion agar-based assay is needed because it is highly straightforward, simple to use, easy to perform, and affordable, making it suited for use in routine testing. In this study, agar disc diffusion antifungal susceptibility tests were used to determine how susceptible 88 isolates of dermatophytes were to six commonly used antifungal medications (Itraconazole, fluconazole, nystatin, griseofulvin, amphotericin B, and terbinafine) for the treatment of dermatophytosis. Relative to the tested agents, griseofulvin, nystatin, and high concentration of terbinafine (25 µg) exhibited the best antifungal efficacy against those dermatophytes. On the other hands, no antifungal activity of fluconazole and amphotericin B was observed against dermatophytes. Also, Itraconazole showed no susceptibility to all examined Microsporum species. Our data concurred with previous study indicating the high susceptibility of examined dermatophytes to griseofulvin (46.4%) and high resistance to fluconazole (94.5%) 18 from clinical samples in Egypt. The research showed that fluconazole was the least potent antifungal agent 36. Another study found that dermtophyte strains showed higher susceptibility to terbinafine than griseofulvin, while Itraconazole and fluconazole had no effect on T. mentagrophytes24. In contrast, Fowora et al.37 detected the high sensitivity of T. mentagrophytes to Itraconazole, whereas griseofulvin had no effect in Nigeria. The development of resistance to fluconazole and Itraconazole, which were previously more effective on dermatophytes, may be due to their commonly and safely use on the contrary of other azole compounds 24.

According to these results of broth microdilution studies, the most effective drugs against M. canis and T. mentagrophytes were terbinafine and acetic acid, respectively, whereas the least effective drugs were fluconazole and griseofulvin, respectively. The antifungal efficiency of fluconazole was previously detected using microdilutiion test 38. Acetic acid is a commonly used house hold remedy (water vinegar mixture) for the fastest method of Dermatophyte treatment, particularly in cats and dogs. The lowest MIC values for ketoconazole, itraconazole, and amphotericin-B against M. canis, T. mentagrophytes, and M. gypseum isolated from cats and dogs, respectively, were found by Debnath et al. 39 in India. Fluconazole had the highest MIC activity (0-24 µg/ml), whereas itraconazole had the lowest MIC activity (0.03-0.5 µg/ml) against human dermatophyte isolates 40. The previous study showed low MIC of griseofulvin (2-4 μg/ml) and high efficacy of terbinafine (0.06-0.5 μg/ml) against dermatophytes isolates involving M. canis and T. mentagrophyte 41. Bossche 42 reported higher MIC result with IC50 of griseofulvin (0.5 µg/l) against M. canis. Nystatin is less effective towards dermatophytosis that leads to the unpopularity of this antifungal agent to local veterinarians. Zaman and Gupta 24 reported the following MIC values for griseofulvin (1 μg/ml) and terbinafine (0.004 μg/ml) against T. mentagrophytes. Recently, a study on T. mentagrophytes showed high antifungal activity of nystatin and griseofulvin in Nigeria 37. Our research demonstrates that in-vitro antifungal susceptibility testing is essential for the routine clinical laboratory assessment of dermatophyte susceptibility to antifungal drugs. It enables comparisons between various antifungal medications and can aid in improving the dermatophytosis treatment plan.

Most of Microsporum and Trichophyton species are saprophytic and found naturally in the environment and not necessary active or capable of inducing disease. Dermatophytes, which infect keratinized tissue in both people and animals, cause dermatophytosis, a superficial fungal infection. Dermatophytosis lesions display an inflammatory response that is brought on by the host's natural immunological response to get rid of the invading fungi 20. Due of its high receptivity, ease of handling, and reproducibility, and the guinea pig has been utilized as a model extensively. In our investigation, guinea pigs that were experimentally infected with either M. canis or T. mentagrophytes developed a variety of lesions that started to appear on day seven after infection and became more noticeable around day 14 in all exposed animals. There were several clinical signs among model animals infected by a dermatophyte ranging from erythema and red papules to typical signs of T. mentagrophytes of thick scales, and crust that reach its peak at day 14th day post infection 2043. These results support the observations made by 44, who used guinea pigs to test the immunogenicity and protective efficacy of a Microsporum canis metalloprotease subunit vaccine. They noted slight but characteristic dermatophytosis-like symptoms on the seventh day after inoculation. Additionally, 87.5 % of the G. pigs exhibited the first symptoms around the fifth day following M. canis inoculation, as shown by the presence of moderate edema, erythema, and mild shedding 12.

The leukogram is unspecific indicative for the infection. In the current study, the leukocytes mass finding of the infected G. pigs at 14 days post infection revealed significant leukocytosis, particularly lymphocytosis which may be due to Th1 response towards the proteases secreted by the fungi 4. In the present study, the infected groups showed a significant neutrophilic leukocytosis as the stress and the severity of the inflammation in response to infection causes attraction of neutrophils to the site of fungal infections 45. Additionally, the monocytosis profile observed in infected G. pigs provides evidence that M. canis and T. mentagrophytes consistently stimulated monocytes and polymorphonuclear neutrophils, two types of leucocytes 46. DNA identification tools are expensive, not always available and not accurate as it may not be active or the sole cause of the disease. The need to find a new tool to diagnose active fungi that are capable of disease production and can be used on both Microsporum and Trichophyton species becomes a necessity. Keratin lytic producing genes are encoded within the fungal genome and only expressed in the presence of keratin (skin, nails, and hair keratin rich parts) as a nitrogen source of energy. Although there are numerous published studies on the use of Reverse Transcriptase -PCR on specimens from experimentally infected G. pigs, there are few published studies on the identification of Microsporum and Trichophyton spp. using PCR 47. The current work detected the presence of gene responsible for the protein lysis in all examined samples using Reverse Transcriptase -PCR. The use of Reverse Transcriptase -PCR contributed in the identification of dermatophytes (Microsporum and Trichophyton species) and detection of their most virulence keratinase genes were recently stated ^48-50. The infectivity of fungi is associated with its ability to produce keratinase enzyme 51.