Evaluation of Enzymatic Activity of Microsporum canis Isolates from Pet Cats by API-ZYM Test

Microsporum canis is a crucial filamentous dermatophyte species because it can generate cutaneous zoonosis lesions, frequently in domestic animals and particularly cats  (Chupia et al., 2022). The diagnosis of dermatophytosis based on clinical findings is unreliable, not just because the dermatological findings are inconsistent (Neves et al., 2018), but also because a number of different skin conditions might resemble the characteristic fungal lesion (circular lesions with alopecia) (Pasquetti et al., 2017). To identify dermatophytes, several morphological traits and presence of microconidia may be satisfying (Fawzi et al., 2023). Diagnosis of M. canis by PCR test is important since, detection and species identification is relevant to the choice of treatment and to understand a probable source of infection also it may supply data for epidemiological studies (Sattasathuchana et al., 2020; De Meb et al., 2022). The enzymatic activity of dermatophytes isolated from companion animals has not been thoroughly studied. However, the enzymatic and antifungal characteristics of dermatophytes are significant contributors to infections in both humans and animals (Aneke et al., 2021). Also, the mechanism of infection by pathogenic fungi may vary in severity depending on the host (Pratibha et al., 2022).
       
The role of enzymes is essential in the pathogenesis and survival strategies of dermatophytes, produce keratinases to degrade keratin, a tough protein found in the outer layer of skin, hair and nails (Cafarchia et al., 2010). Elastases break down elastin, another protein component of skin and connective tissues, DNases hydrolyze DNA, which can be released from damaged host cells during infection, Collagenases break down collagen, a major structural protein in connective tissues and Lipases hydrolyze lipids (Viani et al., 2007). These enzymes are not only important for the virulence and pathogenicity of dermatophytes but also serve as targets for potential antifungal therapies. Understanding their biochemical and molecular characteristics helps in developing strategies to combat dermatophyte infections effectively (Alhasan et al., 2022).
       
API ZYM is an efficient enzyme testing method. Its applicability in filamentous fungal identification has been proven (Mohammed et al., 2020). Secreted hydrolytic enzymes can aid in the invasion of host tissue, enhancing adhesion by breaking down host surface chemicals or breaking down host immune system cells and molecules to fend off or resist antimicrobial action (Al-abedi et al., 2020); Putriningsih et al., 2017). The aim of the present study is to identify isolated M.canis from suspected dermatophytosis in pet cats confirming it by PCR and evaluate the ferocity of the isolates by studying their enzymatic activities using set hydrolases API ZYM test.

Ethical approval
 
All samples were collected from housed cats after getting approval from their owner; euthanized ethically, according to the Animal Welfare Committee in Mosul University- College of Veterinary Medicine No. UM.VET.2023.118 in 23-11-2023.
 
Sample collection
 
One hundred and forty-seven hair and skin sample from pet cats with typical clinical signs for dermatophytosis and positive  green fluorescence on UV light exposure were collected during the period between December 2023 to March 2024 in Mosul/ Iraq. Wood’s lamp examination (Fig 1),  all samples are collected by mechanical debridement method (skin scrapping)  (Sudad et al., 2011) and transported in sterile conditions to the Veterinary Mycological lab. In Mosul university,  Where the research is conducted.


 
Mycological examination
 
The samples were treated with KOH 10% for 10 min, they viewed under a microscope to note fungal hyphy, then cultured on Sabouraud dextrose agar (SDA)  with cycloheximide 0.5 g/ml and chloramphenicol 0.05 g/ml, incubated at 25-30^oC for up to 4 weeks (Alhasan et al., 2022).  The isolated Microsporum canis that showed a positive result macroscopically for growth characteristics (roughly wooly colonies with feathery texture) were picked and cultured again on SDA 7days at 3^oC. for more purification. The isolates were identified to species level according to the microbiological appearance of conidia.
 
Molecular identification by polymerase chain reaction (PCR)
 
The identification of suspected M. canis isolates was confirmed by amplifying a segment of internal transcribed spacer (ITS) 1 and 4 universal primers with a size of 550 bp for PCR amplicons. M. canis isolates were maintained at 4^oC for subsequent experiments, The genomic DNA was extracted using Zymo Research DNA MiniPrep kit /USA (Cat No: D6005). ITS Region was detected by conventional PCR Using the forward and reverse primers targeting ITS1 and ITS2 genes provided from  IDT Company, Canada (Table 1) with 550 bp amplicon size (Hamied et al., 2024).


       
The rapid fungal DNA extraction and gene detection were performed as described by (Chupia et al., 2022). The gene amplification was performed using an Applied Biosystems thermocycler with the following amplification conditions: 95^oC for 5 min (initial denaturation) and 35 thermocycler (denaturation for 30 s at 95^oC, annealing for 30 s at 52^oC for ITS universal gene and extension for 1 min at 72^oC) followed by final extension for 7 min at 72^oC. The PCR product (5 μl) was electrophoresed against 3 μl of DNA marker (100 pb, Promega, USA) for 60 min at 100 volts using 2% agarose gel supplemented with ethidium bromide (0.5 μg/mL), then DNA bands were visualised with a UV illuminator system (Fisher Scientific, UK).
 
Detection of hydrolytic enzymes by API ZYM Kit
 
API ZYM test is utilized to profile the enzymatic activities of M. canis isolates (BioMerieux, France) API ZYM test strips containing various substrates specific for 19 hydrolytic enzymes evaluating the virulence of the isolated dermatophytes. The suspension of fungal material with 2 ml of distilled water prepared and its turbidity was adjusted to match McFarland tube No. 5, (65) µl of the specimen suspension was dispensed into each capule of the API ZYM test strip. The inoculated API ZYM strips were covered with a plastic lid to prevent exposure to bright light, after incubation (4 hours at 37^oC), a drop of ZYM A (tensioactive agent) and ZYM B (diazonium salt) reagents  added into each cupule of the API ZYM strip, The inoculated strips were briefly exposed to a powerful light for ten seconds. The color of reactions in each cupule was compared with standard chart provided (Wawron et al., 2011; Hawrra et al., 2019).
 
Scoring
 
Each enzyme reaction was scored on a scale of 0 to 5: 0: Negative reaction (no color change). 1-4: Intermediate reactions of increasing intensity. 5: Maximum intensity reaction (17).

Identification of isolated Microsporum canis
 
From 174 cat hair and skin samples, 30 of Microsporum canis (17.2%, 30/174) were isolated and identified based on the cultural characteristics depending on laboratory process described by (Matloob et al., 2019) ,and microscopic examination as shown in (Fig 2) A,these findings were enhanced by (Murmu et al., 2017) which include the infected pet cats by dermatophytes as the most predominant host (55.5%) while the most isolated species was M.canis.


       
Molecular identification by amplifying a segment of ITS universal gene using specific primers for Microsporum canis revealed a single clear amplified DNA band with 550 bp fragment size, as depicted in Fig 3.


       
Currently, most human infections are caused by microorganisms transmitted from animals, for many reasons, including the increase in the ownership of pets. (Demirbilek et al., 2022). Our results relating with the prevalence of dermatophytosis in pet cats showed similarities with percentages of infection caused by M. canis of some epidemiological researches reported in other areas like Southern Brazil (10.2%) and Thailand (20.5%)  (Copetti et al., 2006), also Like many studies conducted on dermatophytes, M.canis  species was the most prevalent (60.0%)  among the other isolated species like M. gypseum, T. mentagrophytes and T. rubrum from pet cats and dogs (Murmu et al., 2016). While the frequency of M. canis was lower than those reported in Iraq (71.6%) (Sattasathuchana et al., 2020)  which was (27.5%), also in Kurdistan of Iraq (44.36), this variation may be due to the difference in climatic, environmental and economic conditions between different regions in the same country which effect the growth of the pathogenic fungi.
       
In Brazil similar results found in which the frequency of Microsporum canis was (76.9%) (Mohammad and Yassien, 2022). Determining the molecular epidemiology of dermatophytes  in pets will be greatly aided by PCR-based detection (Maharana* et al., 2019). Accurate and clear results were obtained with PCR for detecting Microsporum canis, which gives confirmatory results and enhances traditional methods. PCR is used in this research to support the mycological methods for isolation, thus they were identical. (Chupia et al., 2022).
 
API- ZYM enzymatic activities
 
Thirty Microsporum canis isolates were used to assess the activity of 19 hydrolases using the API ZYM test. Table 2 list these hydrolases along with naphthol-AS-BI-phosphohydrolase, acid phosphatase, α-galactosidase, β-galactosidase, N-acetyl-β-glucosaminidase, α-mannosidase, leucine arylamidase, valine arylamidase, cystine arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. The enzymatic activity was measured by observing the color reaction after 5 minutes, with the intensity of the reaction corresponding to nanomoles of hydrolyzed substrate on a 5-point scale: zero (no reaction), 1-5 nanomoles, 2-10 nanomoles, 3-20 nanomoles, 4-30 nanomoles and 5-40 nanomoles as shown in (Fig 4).




       
The enzymatic activity profile revealed the highest activities for lipase, arylamidase, leucine, naphthol-AS-BI-phosphohydrolase and esterase lipase (C8) each showing the percentage was 100% activity. Twenty four isolates (80%) with high activity of Acid phosphatase with 6 isolates (3.3%) with no activity, followed by Valine arylamidase in which 5 isolates (16.6) produce this enzyme strongly while 25 isolates (83.3%) produced it moderately. One isolates (3.3) with high activity of Cystine arylamidase and 29 (96.6) isolates with moderate activity. Twenty four isolates (80%) with high activity of Acid phosphatase and only 5 isolates (16.6%) with moderate activity and 1 isolates (3.3%) with no activity, In case of α-glucosidase 28 isolates (93.3) produce this enzyme with strongly activity and only 2 isolates (6.6%) produce this enzyme moderately.
   
The observed geographical variations in species distribution discrepancies may stem from several factors, including differences in sample sizes across regions or variations in diagnostic methodologies employed (Jarjees and Issa, 2022). Factors such as environmental conditions, regional veterinary practices and the genetic diversity of local animal populations could also contribute to these discrepancies (Minnat et al., 2019). Thus, understanding these nuances is crucial for accurate epidemiological assessments and effective management strategies for fungal infections in animal populations.
       
Hydrolases API ZYM test showed the activity of enzymes for lipase, arylamidase, leucine, naphthol-AS-BI-phosphohy drolase and esterase lipase (C8) each showed the percentage as 100% activity in all tested M canis isolates. Twenty four isolates (80 %) with high activity of Acid phosphatase in six isolates (3.3%) with no activity, followed by Valine arylamidase in 5 isolates (16.6) produce this enzyme strongly while 25 isolates (83.3%) produced it moderately. One isolate (3.3%) with high activity of Cystine arylamidase and 29 (96.6%) isolates with moderate activity. These results were parallel to Hawraa et al., 2019. WhileTwenty four isolates (80 %) show high activity of Acid phosphatase and only 5 isolates (16.6%) with moderate activity and one isolates (3.3%) with no activity, In case of α-glucosidase 28 isolates (93.3) produce this enzyme with strongly activity and only two isolates (6.6%) produce this enzyme moderately this may indicate the ferocity of the isolates and the difficulty of controlling it therapeutically.
       
The current study found differences in enzymatic activity compared to what was reported in the study referenced as (Papini and Mancianti, 1995), who noticed patterns of extracellular enzymatic activity 70 feline M. canis isolates for all enzymatic activity, significant intensity fluctuations were found. There was activity for leucine arylamidase in 35 samples (50%) and esterase lipase (C8) in 31 samples (44%), out of 57 samples (81%). Seven (10%) of the samples had valine and cystine arylamidases, while 64 (91%) had acid phosphatase, 60 (86%), alpha-galactosidase in 5, 7%, beta-galactosidase in 6, 8%, alpha-glucosidase in 25, 36%, N-acetyl-beta-glucosaminidase in 41 (58%) and alpha-mannosidase in 51 (73%), were found to have naphthol-AS-BI-phosphohydrolase. The discrepancy between this study and others could be attributed to geographic variances that impact species distribution and, in turn, their capacity to produce enzymes. Studying the pathogens of dermatophytes opens up the horizons for finding alternative treatment solutions, which helps to limit the spread of these transmissible diseases (Nanavare et al., 2024).
               
Dermatophytes cause superficial infections by penetrating the keratinous layer of the epidermis and through hairs and nails. Proteases and lipases are produced by dermatophytes,  aiding in their invasion of the host’s tissues. The lipid and protein components of lipid cell membranes are structurally disturbed by the enzymes, which impairs or completely destroys the membranes’ ability to function (Kobierzycka and Cisto,  2005; Yassein and Zghair, 2020). The interplay of virulence factors which is sometimes represented by a number of enzymes in fungal pathogens plays an important role in pathogenicity and severity of infection . It may also cause transformation of saprophytic fungal agents into life-threatening pathogens. 

The rates of M.canis isolation within skin lesions among pet cats in Vet clinics were determined using traditional and molecular methods which have proven effective in dealing with and diagnosing transmissible pathogens such as dermatophytes., variable hydrolytic enzymes which is known to be putative virulence factors in M. canis pathogenesis has been investigated  by API ZYM test . API ZYM test proven effective as a good method for detection the hydrolytic enzymes activities   and it is an additional procedure to diagnose and classify isolates.

Although the present study was completed through individual efforts by the researchers, we want to thank the staff of Department of Microbiology, College of Veterinary Medicine, University of Mosul ,and the Veterinary clinics  of pet animals in Mosul city.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors have disclosed that they do not have any conflicts of interest. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.