Cats can develop skin diseases caused by fungal organisms called dermatophytes, generally known as ringworms. These fungi reside on the feline dermis, fur, and nails and can invade the epidermis, resulting in symptoms such as circular areas of alopecia, inflammation, flaking, and pruritus [1]. Dermatophytes are categorized into 3 distinct families based on their ecological niche, consisting of anthropophiles (found in humans), zoophiles (seen in animals), and geophiles (found in soil) [2]. The primary molecular techniques for identifying dermatophyte species include Multiplex, Nested, real-time polymerase chain reaction (PCR), quantitative PCR, PCR-enzyme-linked immunosorbent assay, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA, and mass spectrophotometry. PCR-based methods have significantly enhanced the accuracy of dermatophyte identification at the inter and intra-specific levels [3].

Many advanced techniques, such as confocal reflection microscopy, are being used to observe the cellular characteristics of the skin during infection, potentially allowing for the detection of dermatophyte fungi [4]. The primary advantage of this test is its non-invasive nature and great sensitivity [5]. Researchers are utilizing immunochromatography, a technology, to identify dermatophytes in skin and nails [6]. Amplification of the universal genes, such as chitin synthetases, or the specified internal transcribed spacer (ITS) region followed by sequencing is the most popular and straightforward way to understand the genomic diversity and molecular distinctions among the isolates of animal and human dermatophytosis [7]. Phylogenetic analysis and the identification of dermatophyte species frequently rely on sequencing the ITS region. Using the ITS region as a target for study, along with complementary fingerprinting techniques such as amplified fragment length polymorphism (AFLP) or RFLP, can provide more valuable insights into the reorganization and taxonomy of Trichophyton. Mentagrophytes, also called M. canis and Trichophyton [8]. The ITS region can be amplified and analyzed using RFLP with a single enzyme, such as MvaI, to identify the species [9].

AFLP is a method used for DNA fingerprinting. This technique combines restriction enzymes to cut DNA into fragments and the selective amplification of these fragments using PCR [10]. Very limited information exists on dermatophytosis in cats in Basrah, Iraq. Therefore, this study isolated and diagnosed the fungi causing dermatophytosis in cats in Basra Governorate, Iraq.

Of the 80 samples clinically positive for infection with dermatophytosis, 69 (86.3%) samples tested positive after culturing on SDA and DTM. Morphological (macroscopic and microscopic) approaches were first applied to identify growth, followed by molecular identification. The growth on SDA revealed colonies of M. canis that were characterized by the white to yellow color, while on DTM, they were characterized by the white color and the soft and fluffy appearance in the center with the red agar color (converted from orange to red color). These were critical for confirming the diagnosis of dermatophytosis because these media are designed specifically to support the growth of fungi while inhibiting the growth of bacterial contaminants (Figs. 1, 2).

Dermatophytosis is a ubiquitous and contagious superficial skin disease of cats. In animals, the disease is commonly caused by M. canis and Trichophyton species [22-25]. In the present study, 2 different media, SDA and DTM, were used to grow and diagnose dermatophytes. These media help distinguish between dermatophytes and non-pathogenic fungi because DTM contains a pH indicator that changes to red when dermatophytes grow, facilitating the diagnosis. The results showed that 69 out of 80 (86.3%) samples exhibited dermatophyte growth, reflecting the widespread prevalence of an infection among the tested samples. This percentage indicates the familiar presence of dermatophytes in cats, supporting the need for regular screening, especially in environments with large animals. Similar results have been reported elsewhere [26,27]. On the other hand, 11 (13.8%) samples showed no growth, possibly due to contamination with other fungi Spp, which inhibit or prevent the pathogenic dermatophytosis of infection, or the samples were affected by different factors such as 5 which may hinder the growth of fungi, such as the lack of fungi in the sample itself, which appears to agree with previous studies [26,28].

The colonies of dermatophytes, such as M. canis, have distinctive shapes when grown on culture media such as SDA and DTM. Some of the characteristics of these colonies are as follows. M. canis color colonies are white to yellow and usually exhibit a smooth, velvety surface. The surface appears velvety or cottony and is often dense. The underside appears dark yellow to orange, which agrees with previous work [29,30].

Trichophyton spp. colonies are thinner than M. canis and may range in color from white to gray. The surface often appears dendritic or velvety. The color on the underside varies from dark brown to yellow or orange. DTM medium contains a pH indicator, which turns the medium from yellow to red when dermatophytes are growing because of their alkaline secretions. This color change is an essential indicator for distinguishing dermatophytes from other fungi [31,32]. These phenotypic characteristics are used to rapidly identify dermatophytes and aid in early diagnosis, especially when a cat infection is suspected [33-36].

In the present study, 57 out of 69 samples tested positive for ITS1, while only 41 samples tested positive for chitin synthase 1 (specific for M. canis). The percentage of the ITS1 gene supported the results of cultured media in this study, while the low number of isolates identified using the chitin synthase 1 gene may be due to the low DNA concentration, which was less than 10 μg/μL in the samples tested. The nanodrop results also produced a negative result, and the chitin layer in the dermatophyte wall was very thick, which may have resulted in low concentrations of DNA extracts (4 to 5 μg) being overlooked. Therefore, only 12 (ITS1) and 28 (Chs1) samples of the 69 samples showed low concentrations. Accordingly, the molecular method has higher sensitivity and accuracy in distinguishing Microsporum species and is more accurate than the culture test. Moreover, its widespread use in diagnosis will be of great benefit. In addition to demonstrating the importance of the ITS1 region from a taxonomic perspective, agarose gel electrophoresis (1.5% agarose) images of amplicons obtained from gradient PCR (between 50°C to 60°C) targeting the ITS1 genes (600 bp in size) are shown. Hence, all temperatures used were optimal for DNA amplification. The molecular marker sequence database (ranging between 1,000-300 bp) also showed that morphologically identifiable fungi and morphologically unidentifiable fungal strains could be identified using this system. This ITS1-based system is accurate and applicable even to strains with atypical morphological features, and these results are consistent with previous studies [21,37-39].

The 6 local strains ranged from 99% to 100% homology because of point mutations at various positions. Although all strains were from the same area, the phylogenetic tree consisted of 6 isolates based on the sequence numbers obtained, where the present M. canis isolates (PQ578761, PQ578762, PQ578763, PQ578764, PQ578765, and PQ578766) are located close to the Iraq isolate (from another study) and other isolates from India and Japan [40-42]. The phylogenetic data extracted from the partial sequencing of the 18s rRNA gene showed that M. canis is located on the same node (with a reassignment value ranging from 99%-100%). Accordingly, the source of a dermatophyte infection in cats may have been due to the importation of animals from neighboring and non-neighboring countries to Iraq because a strong match was observed between the sequences described in the literature and the sequences identified in the present study. These results are consistent with previous studies [34,43,44].

The results according to NCBI-BLAST Homology Sequence identity (100%) in local M. canis (partial chitin synthase 1 gene) with deposited accession numbers (PQ588604, PQ588605, PQ588606, PQ588607, PQ588608, and PQ588609) are located in USA and Italy only, indicating that the present strain has been documented for first time in Iraq. This strain may have entered Iraq via infected cats imported from Europe or the USA because this gene has not been observed previously in Iraq but has been reported elsewhere [45-47].