Subtilisin 3 production from Microsporum canis is independent of keratin substrate availability.

Dermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. We investigated a critical adhesion protein, subtilisin 3, utilized by Microsporum canis during initial stages of infection, analyzing its production and expression under varying growth conditions. Additionally, as this protein must be expressed and produced for dermatophyte infections to occur, we developed and optimized a diagnostic antibody assay targeting this protein. Subtilisin 3 levels were increased in culture when grown in baffled flasks and supplemented with either l-cysteine or cat hair. As subtilisin 3 was also produced in cultures not supplemented with keratin or cysteine, this study demonstrated that subtilisin 3 production is not reliant on the presence of keratin or its derivatives. These findings could help direct future metabolic studies of dermatophytes, particularly during the adherence phase of infections.

Current Topics in Dermatophyte Classification and Clinical Diagnosis.

Dermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. Classification of many of these species has recently changed due to genetic analysis, potentially affecting clinical diagnosis and disease management. In this review, we discuss dermatophyte classification including name changes for medically important species, current and potential diagnostic techniques for detecting dermatophytes, and an in-depth review of Microsporum canis, a prevalent zoonotic dermatophyte. Fungal culture is still considered the "gold standard" for diagnosing dermatophytosis; however, modern molecular assays have overcome the main disadvantages of culture, allowing for tandem use with cultures. Further investigation into novel molecular assays for dermatophytosis is critical, especially for high-density populations where rapid diagnosis is essential for outbreak prevention. A frequently encountered dermatophyte in clinical settings is M. canis, which causes dermatophytosis in humans and cats. M. canis is adapting to its primary host (cats) as one of its mating types (MAT1-2) appears to be going extinct, leading to a loss of sexual reproduction. Investigating M. canis strains around the world can help elucidate the evolutionary trajectory of this fungi.

Genetic Characterization of Microsporum canis Clinical Isolates in the United States.

Microsporum canis is the primary agent causing dermatophytosis in cats, and also infects humans, dogs, and other species. Assessment of genetic variation among M. canis isolates in the United States has not been conducted. Further, M. canis mating type and assessment of disease severity associated with genotypic characteristics have not been rigorously evaluated. We therefore isolated M. canis from 191 domestic cats across the US and characterized genotypes by evaluation of ITS sequence, MAT locus, and microsatellite loci analysis. The genes SSU1 and SUB3, which are associated with keratin adhesion and digestion, were sequenced from a subset of isolates to evaluate potential genetic associations with virulence. Analysis of microsatellite makers revealed three M. canis genetic clusters. Both clinic location and disease severity were significant predictors of microsatellite variants. 100% of the M. canis isolates were MAT1-1 mating gene type, indicating that MAT1-2 is very rare or extinct in the US and that asexual reproduction is the dominant form of replication. No genetic variation at SSU1 and SUB3 was observed. These findings pave the way for novel testing modalities for M. canis and provide insights about transmission and ecology of this ubiquitous and relatively uncharacterized agent.

Two novel species of Arthroderma isolated from domestic cats with dermatophytosis in the United States.

Dermatophytosis is a superficial fungal infection of keratinized tissues that can occur in humans and other animals. In domestic cats, the majority of cases are caused by Microsporum canis and can spread to other animals and humans via arthrospores. Between 2019 and 2021, 164 cases of suspected dermatophytosis were recorded in animals from a high-volume shelter in California. Samples (hair, nail, and skin scraping) were collected for routine screening from these individuals. One hundred and twenty-six of these were diagnosed as M. canis by culture and internal transcribed spacer (ITS) sequence. In four suspected dermatophytosis cases occurring in kittens in 2019, cultures grown at 20°C yielded fungi with colony morphology more similar to Arthroderma species than Microsporum. Morphologic and microscopic examinations were conducted, and gene segments for the ITS, ß-tubulin, and translation elongation factor 1-alpha (TEF1) regions were sequenced from DNA extracted from these cultures. Sequences were aligned to other dermatophytes using maximum likelihood and neighbor-joining trees and were compared to previously described fungal species to assess nucleotide homology. We identified two previously undescribed fungal species, herein proposed as Arthroderma lilyanum sp. nov. and Arthroderma mcgillisianum sp. nov. M. canis co-cultured in two of the four cases. Other physiologic tests supported this diagnosis. These species have significance as potential pathogens and should be considered as rule-outs for dermatophytosis in cats. The potential for infection of other species, including humans, should be considered. LAY SUMMARY: Two novel fungal species were cultured and characterized from four cases of suspected ringworm in cats at an animal shelter in CA, US. These species were genetically distinct from other dermatophytes and are herein described as Arthroderma lilyanum sp. nov. and Arthroderma mcgillisianum sp. nov.

Serum Samples from Co-Infected and Domestic Cat Field Isolates Nonspecifically Bind FIV and Other Antigens in Enzyme-Linked Immunosorbent Assays.

We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates (e.g., shelter or privately owned cats). Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen.

Detection of glucosamine as a marker for Aspergillus niger: a potential screening method for fungal infections.

Several species of fungus from the genus Aspergillus are implicated in pulmonary infections in immunocompromised patients. Broad screening methods for fungal infections are desirable, as cultures require a considerable amount of time to provide results. Herein, we developed degradation and detection methods to produce and detect D-glucosamine (GlcN) from Aspergillus niger, a species of filamentous fungus. Ultimately, these techniques hold the potential to contribute to the diagnosis of pulmonary fungal infections in immunocompromised patients. In the following studies, we produced GlcN from fungal-derived chitin to serve as a marker for Aspergillus niger. To accomplish this, A. niger cells were lysed and subjected to a hydrochloric acid degradation protocol. Products were isolated, reconstituted in aqueous solutions, and analyzed using hydrophilic interaction liquid chromatography (HILIC) in tandem with electrospray ionization time-of-flight mass spectrometry. Our results indicated that GlcN was produced from A. niger. To validate these results, products obtained via fungal degradation were compared to products obtained from the degradation of two chitin polymers. The observed retention times and mass spectral extractions provided a two-step validation confirming that GlcN was produced from fungal-derived chitin. Our studies qualitatively illustrate that GlcN can be produced from A. niger; applying these methods to a more diverse range of fungi offers the potential to render a broad screening method for fungal detection pertinent to diagnosis of fungal infections.