Environmental influence on abundance and infection patterns of snail intermediate hosts of liver and intestinal flukes in North and Central Vietnam.

Liver and intestinal flukes (LIF) are important groups of foodborne zoonotic trematodes (FZTs) in Southeast Asia, including Vietnam. Their complex life cycles require specific freshwater snail species as the obligatory first intermediate hosts. In 2019, we conducted a longitudinal study in Yen Bai and Thanh Hoa provinces in North and Central Vietnam, respectively, to investigate the diversity of LIF and their infection prevalence in relation to snail host abundance and environmental factors. Using a combination of morphological and molecular identification techniques, we identified 10 LIF species infecting 11 snail host species. We observed significant seasonal variation in the mean abundance of several snail host species, with the majority of snails collected during the spring. We also detected seasonal changes in LIF species composition, with the highest species richness reported in the spring. Clonorchis sinensis and Fasciola gigantica, two medically important human liver flukes in Asia, were found only in the spring in Yen Bai. Our study revealed that not all snail host species have the same probability of becoming infected, and we recorded seasonal variations in the prevalence of LIF infection in different snail species in relation to water parameters.

Fungal infections in animals: a patchwork of different situations.

The importance of fungal infections in both human and animals has increased over the last decades. This article represents an overview of the different categories of fungal infections that can be encountered in animals originating from environmental sources without transmission to humans. In addition, the endemic infections with indirect transmission from the environment, the zoophilic fungal pathogens with near-direct transmission, the zoonotic fungi that can be directly transmitted from animals to humans, mycotoxicoses and antifungal resistance in animals will also be discussed. Opportunistic mycoses are responsible for a wide range of diseases from localized infections to fatal disseminated diseases, such as aspergillosis, mucormycosis, candidiasis, cryptococcosis and infections caused by melanized fungi. The amphibian fungal disease chytridiomycosis and the Bat White-nose syndrome are due to obligatory fungal pathogens. Zoonotic agents are naturally transmitted from vertebrate animals to humans and vice versa. The list of zoonotic fungal agents is limited but some species, like Microsporum canis and Sporothrix brasiliensis from cats, have a strong public health impact. Mycotoxins are defined as the chemicals of fungal origin being toxic for warm-blooded vertebrates. Intoxications by aflatoxins and ochratoxins represent a threat for both human and animal health. Resistance to antifungals can occur in different animal species that receive these drugs, although the true epidemiology of resistance in animals is unknown, and options to treat infections caused by resistant infections are limited.

Modeling dermatophytosis in reconstructed human epidermis: A new tool to study infection mechanisms and to test antifungal agents.

Dermatophytosis is a superficial fungal infection of keratinized structures that exhibits an increasing prevalence in humans and is thus requesting novel prophylactic strategies and therapies. However, precise mechanisms used by dermatophytes to adhere at the surface of the human epidermis and invade its stratum corneum are still incompletely identified, as well as the responses provided by the underlying living keratinocytes during the infection. We hereby report development of an in vitro model of human dermatophytosis through infection of reconstructed human epidermis (RHE) by arthroconidia of the anthropophilic Trichophyton rubrum species or of the zoophilic Microsporum canis and Arthroderma benhamiae species. By modulating density of arthroconidia in the inoculum and duration of exposure to such pathogens, fungal infection limited to the stratum corneum was obtained, mimicking severe but typical in vivo situation. Fungal elements in infected RHE were monitored over time by histochemical analysis using periodic-acid Schiff-staining or quantified by qPCR-detection of fungal genes inside RHE lysates. This model brings improvements to available ones, dedicated to better understand how dermatophytes and epidermis interact, as well as to evaluate preventive and therapeutic agents. Indeed, miconazole topically added to RHE was demonstrated to inhibit fungal infection in this model.

Relevant Animal Models in Dermatophyte Research.

Dermatophytoses are common superficial fungal infections affecting both humans and animals. They are provoked by filamentous fungi called dermatophytes specialized in the degradation of keratinized structures, which allows them to induce skin, hair and nail infections. Despite their high incidence, little investigation has been performed for the understanding of these infections compared to fungal opportunistic infections and most of the studies were based on in vitro experiments. The development of animal models for dermatophyte research is required to evaluate new treatments against dermatophytoses or to increase knowledge about fungal pathogenicity factors or host immune response mechanisms. The guinea pig has been the most often used animal model to evaluate efficacy of antifungal compounds against dermatophytes, while mouse models were preferred to study the immune response generated during the disease. Here, we review the relevant animal models that were developed for dermatophyte research and we discuss the advantages and disadvantages of the selected species, especially guinea pig and mouse.

Are Th17 Cells Playing a Role in Immunity to Dermatophytosis?

Despite their superficial localization in the skin, pathogenic dermatophytes can induce a complex but still misunderstood immune response in their hosts. The cell-mediated immunity (CMI) is correlated with both clinical recovery and protection against reinfection, and CD4+ T lymphocytes have been recognized as a crucial component of the immune defense against dermatophytes. Before the discovery of the Th17 pathway, CMI was considered to be only dependent of Th1 cells, and thus most studies on the immunology of dermatophytosis have focused on the Th1 pathway. Nevertheless, the fine comparative analysis of available scientific data on immunology of dermatophytosis in one hand and on the Th17 pathway mechanisms involved in opportunistic mucosal fungal infections in the other hand reveals that some key elements of the Th17 pathway can be activated by dermatophytes. Stimulation of the Th17 pathway could occur through the activation of some C-type lectin-like receptors and inflammasome in antigen-presenting cells. The Th17 cells could go back to the affected skin and by the production of signature cytokines could induce the effector mechanisms like the recruitment of polymorphonuclear neutrophils and the synthesis of antimicrobial peptides. In conclusion, besides the Th1 pathway, which is important to the immune response against dermatophytes, there are also growing evidences for the involvement of the Th17 pathway.

Diagnosis and treatment of dermatophytosis in dogs and cats.: Clinical Consensus Guidelines of the World Association for Veterinary Dermatology.

BACKGROUND: Dermatophytosis is a superficial fungal skin disease of cats and dogs. The most common pathogens of small animals belong to the genera Microsporum and Trichophyton. It is an important skin disease because it is contagious, infectious and can be transmitted to people. OBJECTIVES: The objective of this document is to review the existing literature and provide consensus recommendations for veterinary clinicians and lay people on the diagnosis and treatment of dermatophytosis in cats and dogs. METHODS: The authors served as a Guideline Panel (GP) and reviewed the literature available prior to September 2016. The GP prepared a detailed literature review and made recommendations on selected topics. The World Association of Veterinary Dermatology (WAVD) provided guidance and oversight for this process. A draft of the document was presented at the 8th World Congress of Veterinary Dermatology (May 2016) and was then made available via the World Wide Web to the member organizations of the WAVD for a period of three months. Comments were solicited and posted to the GP electronically. Responses were incorporated by the GP into the final document. CONCLUSIONS: No one diagnostic test was identified as the gold standard. Successful treatment requires concurrent use of systemic oral antifungals and topical disinfection of the hair coat. Wood's lamp and direct examinations have good positive and negative predictability, systemic antifungal drugs have a wide margin of safety and physical cleaning is most important for decontamination of the exposed environments. Finally, serious complications of animal-human transmission are exceedingly rare.

Overexpression of TLR-2 and TLR-4 mRNA in feline polymorphonuclear neutrophils exposed to Microsporum canis.

BACKGROUND: Polymorphonuclear neutrophils (PMNs), along with macrophages, are the first leukocytes recruited to the site of infection in dermatophytoses and are responsible for the in fine elimination of the fungus. It has been demonstrated that feline PMNs produce pro-inflammatory cytokines after stimulation with Microsporum canis. The activation of these cells results from the recognition of specific PAMPs (pathogen associated molecular patterns) from M. canis by PRRs (pattern recognition receptors) of PMNs. The C-type lectin receptors (CLRs) and toll-like receptors (TLRs) are the two main PRRs in phagocytic cells that recognize fungal components. HYPOTHESIS/OBJECTIVE: The aim of this study was to evaluate the expression of TLR-2, TLR-4 and dectin-1 mRNA in feline PMNs exposed to different components from M. canis. METHODS: Feline PMNs were stimulated for 2 h or 4 h with either live arthroconidia, heat-killed arthroconidia or secreted components from M. canis. The levels of TLR-2, TLR-4 and dectin-1 mRNA were assessed by RT-qPCR. RESULTS: Results showed an increase of TLR-2 and TLR-4 mRNA levels in feline PMNs stimulated with live and heat-killed arthroconidia, but not in those stimulated with the secreted components from M. canis. No significant variation in dectin-1 mRNA expression was observed in PMNs stimulated with the different fungal components. CONCLUSIONS AND CLINICAL IMPORTANCE: The overexpression of TLR-2 and TLR-4 mRNAs in stimulated feline PMNs suggests that these receptors are involved in the host immune response through the recognition of M. canis PAMPs.

Which Fungus Originally was Trichophyton mentagrophytes? Historical Review and Illustration by a Clinical Case.

Several dermatophytes producing numerous pyriform or round microconidia were called Trichophyton mentagrophytes. Among these dermatophytes are the teleomorph species Arthroderma benhamiae, Arthroderma vanbreuseghemii and Arthroderma simii, and other species such as Trichophyton interdigitale, Trichophyton erinacei and Trichophyton quinckeanum for which only the anamorph is known. Confusion exists about which fungus should be really called T. mentagrophytes and about the rational use of this name in practice. We report a case of beard ringworm (tinea barbae) with A. vanbreuseghemii. According to both clinical signs and the type of hair parasitism, this case was exactly compatible to the first description of a non-favic dermatophytosis by Gruby under the name of "mentagrophyte" from which was derived the dermatophyte epithet mentagrophytes. In addition, the phenotypic characters of the isolated fungus in cultures perfectly matched with those of the first description of a dermatophyte under T. mentagrophytes by Blanchard (Parasites animaux et parasites végétaux à l'exclusion des Bactéries, Masson, Paris, 1896). In conclusion, T. mentagrophytes corresponds to the fungus later named A. vanbreuseghemii. However, because the neotype of T. mentagrophytes was not adequately designated in regard to the ancient literature, we would privilege the use of A. vanbreuseghemii and abandon the name of T. mentagrophytes.

Expression of fungal and host markers in models of dermatophytosis on mice and human epidermis.

Increasing resistance of dermatophytes against antifungals creates global public health problems, rendering essential a better understanding of virulence mechanisms and factors determining host-specificity of dermatophytes. Since dermatophytes switch from a saprophytic to a parasitic lifestyle by reprogramming gene expression, reliable experimental models are needed to investigate the pathogenesis of dermatophytosis. Here, a relevant mouse model of Trichophyton benhamiae dermatophytosis was assessed, together with a model based on reconstructed human epidermis (RHE), allowing their respective validation regarding fungal gene expressed during infection. The use of a standardized inoculum induced a natural-like superficial infection in mice. The severity and persistence of lesions enabled the assessment of infection markers, including mouse-specific pro-inflammatory molecules and fungal genes previously reported as potential virulence factors. Upregulated expression of fungal genes, including those encoding subtilisins, in infected RHE revealed that dermatophytes deploy similar processes as those observed during in vivo infection. The RHE model was then used to compare infections by anthropophilic Trichophyton rubrum and zoophilic T. benhamiae. Therefore, these two models represent complementary analytical tools to study the pathogenesis of acute dermatophytoses. In addition, we have identified certain fungal markers of infection and highlighted the existence of different mechanisms deployed by zoophilic versus anthropophilic dermatophytes.

Prevalence of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Rickettsia spp. and Babesia spp. in cattle serum and questing ticks from Belgium.

BACKGROUND: Anaplasmosis, borreliosis, rickettsiosis and babesiosis are tick-borne diseases of medical, veterinary and economic importance. In Belgium, little is known on the prevalence of these diseases in animals and previous screenings relate only to targeted geographic regions, clinical cases or a limited number of tested samples. We therefore performed the first nationwide seroprevalence study of Anaplasma spp., A. phagocytophilum, Borrelia spp., Rickettsia spp. and Babesia spp. in Belgian cattle. We also screened questing ticks for the aforementioned pathogens. METHODS: ELISAs and IFATs were performed on a representative sample set of cattle sera stratified proportionally to the number of cattle herds per province. Questing ticks were collected in areas where the highest prevalence for the forenamed pathogens in cattle serum were observed. Ticks were analyzed by quantitative PCR for A. phagocytophilum (n = 783), B. burgdorferi sensu lato (n = 783) and Rickettsia spp. (n = 715) and by PCR for Babesia spp. (n = 358). RESULTS: The ELISA screening for antibodies to Anaplasma spp. and Borrelia spp. in cattle sera showed an overall seroprevalence of 15.6% (53/339) and 12.9% (52/402), respectively. The IFAT screening for antibodies against A. phagocytophilum, Rickettsia spp. and Babesia spp. resulted in an overall seroprevalence of 34.2% (116/339), 31.2% (99/317) and 3.4% (14/412), respectively. At the provincial level, the provinces of Liege and Walloon Brabant harboured the highest seroprevalence of Anaplasma spp. (44.4% and 42.7% respectively) and A. phagocytophilum (55.6% and 71.4%). East Flanders and Luxembourg exhibited the highest seroprevalence of Borrelia spp. (32.4%) and Rickettsia spp. (54.8%) respectively. The province of Antwerp showed the highest seroprevalence of Babesia spp. (11%). The screening of field-collected ticks resulted in a prevalence of 13.8% for B. burgdorferi s.l., with B. afzelii and B. garinii being the most common genospecies (65.7% and 17.1%, respectively). Rickettsia spp. was detected in 7.1% of the tested ticks and the only identified species was R. helvetica. A low prevalence was found for A. phagocytophilum (0.5%) and no Babesia positive tick was detected. CONCLUSIONS: The seroprevalence data in cattle indicate hot spots for tick-borne pathogens in specific provinces and highlights the importance of veterinary surveillance in anticipating the emergence of diseases among humans. The detection of all pathogens, with the exception of Babesia spp. in questing ticks, underlines the need of raising awareness among public and professionals on other tick-borne diseases along with lyme borreliosis.

Targeted gene deletion and in vivo analysis of putative virulence gene function in the pathogenic dermatophyte Arthroderma benhamiae.

Dermatophytes cause the majority of superficial mycoses in humans and animals. However, little is known about the pathogenicity of this specialized group of filamentous fungi, for which molecular research has been limited thus far. During experimental infection of guinea pigs by the human pathogenic dermatophyte Arthroderma benhamiae, we recently detected the activation of the fungal gene encoding malate synthase AcuE, a key enzyme of the glyoxylate cycle. By the establishment of the first genetic system for A. benhamiae, specific ΔacuE mutants were constructed in a wild-type strain and, in addition, in a derivative in which we inactivated the nonhomologous end-joining pathway by deletion of the A. benhamiae KU70 gene. The absence of AbenKU70 resulted in an increased frequency of the targeted insertion of linear DNA by homologous recombination, without notably altering the monitored in vitro growth abilities of the fungus or its virulence in a guinea pig infection model. Phenotypic analyses of ΔacuE mutants and complemented strains depicted that malate synthase is required for the growth of A. benhamiae on lipids, major constituents of the skin. However, mutant analysis did not reveal a pathogenic role of the A. benhamiae enzyme in guinea pig dermatophytosis or during epidermal invasion of the fungus in an in vitro model of reconstituted human epidermis. The presented efficient system for targeted genetic manipulation in A. benhamiae, paired with the analyzed infection models, will advance the functional characterization of putative virulence determinants in medically important dermatophytes.

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH.

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.

Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex.

Isolates of the Trichophyton mentagrophytes complex vary phenotypically. Whether the closely related zoophilic and anthropophilic anamorphs currently associated with Arthroderma vanbreuseghemii have to be considered as members of the same biological species remains an open question. In order to better delineate species in the T. mentagrophytes complex, we performed a mating analysis of freshly collected isolates from humans and animals with A. benhamiae and A. vanbreuseghemii reference strains, in comparison to internal transcribed spacer (ITS) and 28S rDNA sequencing. Mating experiments as well as ITS and 28S sequencing unambiguously allowed the distinction of A. benhamiae and A. vanbreuseghemii. We have also shown that all the isolates from tinea pedis and tinea unguium identified as T. interdigitale based on ITS sequences mated with A. vanbreuseghemii tester strains, but had lost their ability to give fertile cleistothecia. Therefore, T. interdigitale has to be considered as a humanized species derived from the sexual relative A. vanbreuseghemii.

Towards a Standardized Procedure for the Production of Infective Spores to Study the Pathogenesis of Dermatophytosis.

Dermatophytoses are superficial infections of human and animal keratinized tissues caused by filamentous fungi named dermatophytes. Because of a high and increasing incidence, as well as the emergence of antifungal resistance, a better understanding of mechanisms involved in adhesion and invasion by dermatophytes is required for the further development of new therapeutic strategies. In the last years, several in vitro and in vivo models have emerged to study dermatophytosis pathogenesis. However, the procedures used for the growth of fungi are quite different, leading to a highly variable composition of inoculum for these models (microconidia, arthroconidia, hyphae), thus rendering difficult the global interpretation of observations. We hereby optimized growth conditions, including medium, temperature, atmosphere, and duration of culture, to improve the sporulation and viability and to favour the production of arthroconidia of several dermatophyte species, including Trichophyton rubrum and Trichophyton benhamiae. The resulting suspensions were then used as inoculum to infect reconstructed human epidermis in order to validate their ability to adhere to and to invade host tissues. By this way, this paper provides recommendations for dermatophytes culture and paves the way towards a standardized procedure for the production of infective spores usable in in vitro and in vivo experimental models.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro -expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

Pets as the main source of two zoonotic species of the Trichophyton mentagrophytes complex in Switzerland, Arthroderma vanbreuseghemii and Arthroderma benhamiae.

In cases of highly inflammatory dermatophytosis in humans, it is important to identify the possible source of animal transmission in order to prevent recurrence, family outbreaks or rapidly progressing epidemics. A survey of dermatophytes in pets during a 14-month period in Switzerland revealed, in addition to Microsporum canis, two different species of the Trichophyton mentagrophytes complex, Arthroderma benhamiae and Arthroderma vanbreuseghemii, all causing inflammatory dermatophytoses. Arthroderma benhamiae was only and frequently isolated from guinea pigs. Arthroderma vanbreuseghemii was isolated mainly from European short hair cats, but also from dogs and in one case from a pure-bred cat. Ninety-three percent of the cats carrying A. vanbreuseghemii were hunters and all had skin lesions. In contrast, cats with skin lesions that were strictly indoors were found to be almost exclusively infected by M. canis. Therefore, it can be suspected that infection with A. vanbreuseghemii occurred during hunting and that the natural source of this dermatophyte is either soil or an animal other than the cat, most probably a rodent.

Responses of Reconstructed Human Epidermis to Trichophyton rubrum Infection and Impairment of Infection by the Inhibitor PD169316.

Despite the threatening incidence of dermatophytosis, information is still lacking about the consequences of infection on epidermal barrier functions and about the keratinocyte responses that alert immune components. To identify the mechanisms involved, arthroconidia of the anthropophilic dermatophyte Trichophyton rubrum were prepared to infect reconstructed human epidermis (RHE) in vitro. Integrity of the barrier was monitored during infection by measurements of transepithelial electrical resistance and dye-permeation through the RHE. Expression and release of pro-inflammatory cytokines and antimicrobial peptides by keratinocytes inserted into the RHE were assessed, respectively, by quantitative reverse transcriptase-PCR (to analyze mRNA content in tissue extracts) and by ELISA (to detect proteins in culture media). Results reveal that infection by T. rubrum is responsible for disruption of the epidermal barrier, including loss of functional tight junctions. It additionally causes simultaneous expression and release of cytokines and antimicrobial peptides by keratinocytes. Potential involvement of the p38 mitogen-activated protein kinase signaling pathway was evaluated during infection by targeted inhibition of its activity. Intriguingly, among several p38 mitogen-activated protein kinase inhibitors, PD169316 alone was able to inhibit growth of T. rubrum on Sabouraud agar and to suppress the process of infection on RHE. This suggests that PD169316 acts on a specific target in dermatophytes themselves.

Th1 and Th17 Immune Responses Act Complementarily to Optimally Control Superficial Dermatophytosis.

Dermatophytoses are among the most common fungal infections worldwide, but little is known about the immune response in them. By comparing Trichophyton benhamiae acute superficial dermatophytosis in wild-type and Rag2-/- mice, we showed that TCR-mediated immunity is critical for fungal clearance and clinical recovery. In WT mice, CD4+ T cells isolated from the skin-draining lymph nodes exhibit both T helper type (Th) 1 and Th17 differentiation during infection, with regard to produced cytokines or mRNA levels of transcription factors. Using IL-17A- and IFN-γ-deficient mice, we showed that IL-17A and IFN-γ are individually dispensable but together contribute to the optimal resolution of dermatophytosis. Furthermore, we generated and infected IL-17A and IFN-γ double-deficient mice and showed that both fungal clearance and clinical recovery were much lower in these mice than in single-deficient mice, suggesting the complementary roles of the two cytokines in dermatophytosis resolution. Thus, our data suggest that TCR-mediated immunity is critical for the optimal control of superficial dermatophytosis and that adaptive immunity is polarized to both Th1 and Th17 responses, with the Th17 antifungal response acting on dermatophyte clearance and the Th1 response being involved in both fungal clearance and Th17-inflammation down-modulation.

Secreted subtilisins of Microsporum canis are involved in adherence of arthroconidia to feline corneocytes.

Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis, mainly in cats and humans. The mechanisms involved in adherence of M. canis to epidermis have never been investigated. Here, a model was developed to study the adherence of M. canis to feline corneocytes through the use of a reconstructed interfollicular feline epidermis (RFE). In this model, adherence of arthroconidia to RFE was found to be time-dependent, starting at 2 h post-inoculation and still increasing at 6 h. Chymostatin, a serine protease inhibitor, inhibited M. canis adherence to RFE by 53%. Moreover, two mAbs against the keratinolytic protease subtilisin 3 (Sub3) inhibited M. canis adherence to RFE by 23%, suggesting that subtilisins, and Sub3 in particular, are involved in the adherence process.