IL-17-Mediated Immunity Controls Skin Infection and T Helper 1 Response during Experimental Microsporum canis Dermatophytosis.

Despite worldwide prevalence of superficial mycoses, the immune response in dermatophytosis has scarcely been investigated. In this study, we developed a model of superficial skin infection in C57BL/6 mice with Microsporum canis, a highly prevalent human pathogen. This model mimics mild inflammatory human dermatophytosis, characterized by neutrophil recruitment and fungal invasion limited to the epidermis and exhibits the establishment of a specific T helper type 17 immune response during infection. By using IL-17RA- or IL-17A/F-deficient mice we showed that, in the absence of a functional IL-17 pathway, M. canis extensively colonizes the epidermis and promotes an exaggerated skin inflammation and a shift to an IFN-γ-mediated (T helper type 1) response. IL-17 signaling was not involved in neutrophil influx to skin or fungal invasion to deeper tissues. Finally, this study shows that skin langerin-expressing cells contribute to the antifungal T helper type 17 response in vivo. In conclusion, these data directly show a dual function of IL-17 cytokines in dermatophytosis by controlling superficial infection and down-modulating a T helper type 1 antifungal response.

Development of enzyme immunoassays (ELISA and Western blot) for the serological diagnosis of dermatophytosis in symptomatic and asymptomatic cats.

Dermatophytosis is the most common fungal infection in cats worldwide and plays an important role in both animal and human health due to their high zoonotic potential. Effective screening is a strong preventive measure and the fungal culture is quite useful but requires full laboratorial experience and it takes a long time to obtain the result. A rapid and accurate screening test for dermatophytosis in cats is crucial for the effective control of disease outbreaks. The aim of this study was to develop and evaluate the diagnostic efficacy of enzyme immunoassays (ELISA and Western blot [WB]) for the rapid and precise diagnosis of dermatophytosis in cats. Seventy cats of various ages were divided into three groups: S (symptomatic, n = 20), AS (asymptomatic, n = 30), and N (negative, n = 20). All animals were submitted to fungal culture and blood samples for carrying out the serological tests. A significant difference (P < 0.05) was found between IgG-specific levels of sera of Microsporum canis positive and negative animals. There was no statistic difference between groups symptomatic and asymptomatic. The ELISA test showed sensitivity of 94% and specificity of 75%. Receiver operating characteristic (ROC) analysis also showed higher diagnostic accuracy (AUC 0.925). The WB technique detected 13 bands, and the 50 kDa protein was considered the most immunogenic protein, observing reactivity in 83.3% in the symptomatic group and 66.6% in the asymptomatic group. The study concluded that ELISA and WB were useful tools to reliably detect cats that have been exposed to M. canis.

Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs.

Microsporum canis is the most common dermatophyte in pets and is of zoonotic importance but currently there is no effective vaccine available to prevent dermatophytosis. The aim of this work was to assess the immunogenicity and protective efficacy of secreted components (SC) from M. canis adjuvanted with the monophosphoryl lipid-A (MPLA), in a vaccine study using the guinea pig as an experimental model. Animals were vaccinated with either the SC adjuvanted with the MPLA, the MPLA adjuvant alone or PBS three times at two-week intervals, until 42 days prior to M. canis infection. A blind evaluation of dermatophytosis symptoms development and fungal persistence in skin was monitored weekly. The antibody response towards the SC and the levels of Interferon (IFN)γ and Interleukin-4 expressed in peripheral blood mononuclear cells were assessed along or at the end of the study period respectively. The animals that received MPLA had a significantly lower clinical score than those inoculated with PBS. However, no significant difference was observed between the guinea pigs vaccinated with the SC adjuvanted with the MPLA and those having received MPLA alone. The results also showed that vaccination induced a strong antibody response towards the SC and an increase in IFNγ mRNA level. Our results show that the MPLA adjuvant used in this vaccine study can induce per se a partial protection against a M. canis infection. Although they induce a delayed-type hypersensitivity reaction in guinea pigs, the SC do not confer a protection under the present experimental conditions.

Pathogenic fungus Microsporum canis activates the NLRP3 inflammasome.

Microsporum canis is a pathogenic fungus with worldwide distribution that causes tinea capitis in animals and humans. M. canis also causes invasive infection in immunocompromised patients. To defy pathogenic fungal infection, the host innate immune system is the first line of defense. As an important arm of innate immunity, the inflammasomes are intracellular multiprotein complexes that control the activation of caspase-1, which cleaves proinflammatory cytokine pro-interleukin-1ß (IL-1ß) into its mature form. To determine whether the inflammasome is involved in the host defense against M. canis infection, we challenged human monocytic THP-1 cells and mouse dendritic cells with a clinical strain of M. canis isolated from patients with tinea capitis. We found that M. canis infection triggered rapid secretion of IL-1ß from both THP-1 cells and mouse dendritic cells. Moreover, by using gene-specific shRNA and competitive inhibitors, we determined that M. canis-induced IL-1ß secretion was dependent on NLRP3. The pathways proposed for NLRP3 inflammasome activation, namely, cathepsin B activity, K(+) efflux, and reactive oxygen species production, were all required for the inflammasome activation triggered by M. canis. Meanwhile, Syk, Dectin-1, and Card9 were found to be involved in M. canis-induced IL-1ß secretion via regulation of pro-IL-1ß transcription. More importantly, our data revealed that M. canis-induced production of IL-1ß was dependent on the NLRP3 inflammasome in vivo. Together, this study unveils that the NLRP3 inflammasome exerts a critical role in host innate immune responses against M. canis infection, and our data suggest that diseases that result from M. canis infection might be controlled by regulating the activation of inflammasomes.

Feline polymorphonuclear neutrophils produce pro-inflammatory cytokines following exposure to Microsporum canis.

The mechanisms involved in the establishment of the specific immune response against dermatophytes remain unknown. Polymorphonuclear neutrophils (PMNs) are recruited early during the infection process and participate in the elimination of dermatophytes. They could therefore be involved in the induction of the immune response during dermatophytoses by producing specific cytokines. The aim of this work was to assess the in vitro cytokine production by feline PMNs exposed to living arthroconidia from the dermatophyte species Microsporum canis or stimulated with either a secreted or a structural component of M. canis, the latter consisting of heat-killed arthroconidia. The levels of specific cytokines produced by PMNs were determined by capture ELISA and/or quantitative RT-PCR. Results showed that PMNs secrete TNFα, IL-1ß and IL-8 following exposure to M. canis living arthroconidia and stimulation with both a secreted component and heat-killed arthroconidia. The level of IL-8 mRNA was also increased in PMNs stimulated with M. canis living arthroconidia. In conclusion, infective M. canis arthroconidia induce the production of pro-inflammatory cytokines by feline PMNs that can be activated either by secreted or structural fungal components. Our results suggest that these granulocytes are involved in the initiation of the immune response against M. canis.

Use of western blot to study Microsporum canis antigenic proteins in canine dermatophytosis.

Western blotting was used to describe the Microsporum canis proteins with antigenic activity in dogs with dermatophytosis. Electrophoretic separation of whole fungal strain extract cultured from a cat was performed under denaturing conditions. The proteins were blotted onto nitrocellulose and probed with sera collected from 22 dogs with dermatophytosis (18 M. canis, 3 M. gypseum, 1 Trichophyton mentagrophytes; group A), 20 dogs with skin diseases other than dermatophytosis, and 22 dogs with no clinical cutaneous signs (group B, n = 42). Nine principal IgG-binding proteins with apparent molecular weights of 180, 144, 130, 120, 102, 96, 80, 68, and 48 kD were visualised on group A blots. For these proteins, serological cross-reactivity with different strains of M. canis may be indirectly confirmed, whereas additional proteins were found to react with sera from individual dogs. The proteins visualised in this study may represent diagnostic markers of dermatophyte infection. The proteins should be further evaluated for their role in the cellular immune response of dogs with dermatophytosis.

Binding of DC-HIL to dermatophytic fungi induces tyrosine phosphorylation and potentiates antigen presenting cell function.

APCs express receptors recognizing microbes and regulating immune responses by binding to corresponding ligands on immune cells. Having discovered a novel inhibitory pathway triggered by ligation of DC-HIL on APC to a heparin/heparan sulfate-like saccharide of syndecan-4 on activated T cells, we posited DC-HIL can recognize microbial pathogens in a similar manner. We showed soluble recombinant DC-HIL to bind the dermatophytes Trichophyton rubrum and Microsporum audouinii, but not several bacteria nor Candida albicans. Dermatophyte binding was inhibited completely by the addition of heparin. Because DC-HIL contains an ITAM-like intracellular sequence, we questioned whether its binding to dermatophytes can induce tyrosine phosphorylation in dendritic cells (DC). Culturing DC with T. rubrum (but not with C. albicans pseudohyphae) induced phosphorylation of DC-HIL, but not when the tyrosine residue of the ITAM-like sequence was mutated to phenylalanine. To examine the functional significance of such signaling on DC, we cross-linked DC-HIL with mAb (surrogate ligand), which not only induced tyrosine phosphorylation but also up-regulated expression of 23 genes among 662 genes analyzed by gene-array, including genes for profilin-1, myristoylated alanine rich protein kinase C substrate like-1, C/EBP, LOX-1, IL-1beta, and TNF-alpha. This cross-linking also up-regulated expression of the activation markers CD80/CD86 and heightened APC capacity of DC to activate syngeneic T cells. Our findings support a dual role for DC-HIL: inhibition of adaptive immunity following ligation of syndecan-4 on activated T cells and induction of innate immunity against dermatophytic fungi.

Persistence of antibodies in blood and body fluids in decaying fox carcasses, as exemplified by antibodies against Microsporum canis.

To assist in evaluating serological test results from dead animals, 10 silver foxes (Vulpes vulpes) and 10 blue foxes (Alopex lagopus), 6 of each species previously vaccinated against and all challenged with Microsporum canis, were blood sampled and euthanased. Fox carcasses were stored at +10 degrees C, and autopsy was performed on Days 0, 2, 4, 7, and 11 post mortem during which samples from blood and/or body fluid from the thoracic cavity were collected. Antibodies against M. canis were measured in an enzyme-linked immunosorbent assay (ELISA) as absorbance values (optical density; OD). To assess the degradation of antibodies, the ratio between post mortem and ante mortem absorbance was calculated. The mean absorbance from samples collected during autopsy was generally lower than from samples from live animals. In blood samples, this difference increased significantly with time (P = 0.04), while in body fluid samples the difference decreased (not significant; P = 0.18). We suggest that a positive serological result from testing blood or body fluid of a dead animal may be regarded as valuable, although specific prevalences obtained by screening populations based on this type of material may represent an under-estimation of the true antibody prevalence. Negative serological test results based on material from carcasses may be less conclusive, taken into account the general degradation processes in decaying carcasses, also involving immunoglobulin proteins.

Eighty-four consecutive cases of tinea faciei in Siena, a retrospective study (1989-2003).

Consecutive cases of tinea faciei diagnosed in Siena between 1989 and 2003 were studied retrospectively for differences in clinical form, demographic data and species of dermatophyte isolated. The series consisted of 84 cases (59 females, 25 males) with a mean age of 27 years. Mean age of females (32.4 years) was significantly greater than that of males (14.2 years). The dermatophytes most frequently isolated were Microsporum canis (38 cases) and Trichophyton rubrum (31 cases). Clinical form was typical of tinea in 54 subjects (64.3%) and was tinea incognito because of inappropriate therapy in the other 30 (35.7%) subjects. The mean age of patients with the typical form (19.2 years) was significantly lower than that of those with tinea incognito (41.1 years). All cases in the age range 6-15 years had typical tinea, whereas the maximum frequency of cases with tinea incognito was 46-50 years. In the group with tinea incognito there was a majority of women and the dermatophytes isolated differed with gender. No such difference was observed in the group with typical tinea.

[Incidence, immunity and treatment of feline dermatophytosis]. / Vorkommen, Immunität und Behandlung der felinen Dermatophytose.

Feline dermatophytosis is a superficial skin infection characterized by the invasion of cornified tissues such as hair and nails. This infection is nearly always caused by Microsporum canis. Infected animals release infective spores in the environment which will then contaminate other animals or humans. Infected animals usually develop immunity so the infection will spontaneously disappear after a few weeks to months. Long haired and immunocom-promised cats do not have the same ability to acquire resistance and spontaneous recovery does usually not occur. The treatment of such an infection will require topical and systemic treatment of all contaminated and in-contact cats. The use of desinfectants such as bleach or enilconazole has been proven effective to destroy the spores in the environment. In addition, the efficacy of topical and systemic treatments with azole derivates or allylamines has also been demonstrated in several studies. On the contrary, dermatophyte vaccination has never been proven effective in well controlled studies. Regular follow-up and fungal cultures are mandatory to ensure succesfull treatment.

Development of an enzyme-linked immunosorbant assay (ELISA) for the serodiagnosis of canine dermatophytosis caused by Microsporum canis.

Abstract In dogs, dermatophytosis should be considered in any case of alopecic, papular or pustular lesion. The aim of this study was to develop an enzyme-linked immunosorbant assay (ELISA) as an aid in the diagnosis of canine dermatophytosis. The antigen used was a whole fungal extract obtained from an isolate of Microsporum canis cultured on a liquid medium from the parasitized hair of a cat with patches of alopecia. To assess the ELISA performances, sera from 18 dogs with dermatophytosis caused by M. canis (group A, n = 18), 20 dogs with skin diseases other than dermatophytosis and 22 healthy dogs (group B, n = 42) were tested. Four further animals were tested: three with dermatophytosis caused by M. gypseum and one by T. mentagrophytes. A significant difference (P < 0.01, Wilcoxon's test, w = 364) was found between IgG-specific levels of sera of recently M. canis-infected dogs (infection < 15 days) and controls (although three dogs had negative titres at this stage). A highly significant difference (P < 0.001, w = 462) was noted between controls and dogs with infection of longer duration (> 30 days). All dogs had positive titres at this stage. A highly significant correlation (P < 0.001, Spearman's test, rho = 0.86) between duration of infection and IgG concentration was noted. The test has good sensitivity (83.3%) and high specificity (95.2%) but some dogs retained positive titres after elimination of infection. The sensitivity is higher than that of direct microscopic hair examination and similar to that of fungal culture with DTM (dermatophyte test medium).

Evaluation of immunogenicity and protective efficacy of a Microsporum canis metalloprotease subunit vaccine in guinea pigs.

In order to identify protective immunogens against Microsporum canis infection, a purified recombinant keratinolytic metalloprotease (r-MEP3) was tested as a subunit vaccine in experimentally infected guinea pigs. Both humoral and cellular specific immune responses developing towards r-MEP3 were evaluated, by enzyme-linked immunosorbent assay and by in vitro lymphocyte transformation tests respectively. Vaccination induced a strong antibody response, and a significant but transient lymphoproliferative response against the protein. However, the protocol failed to prevent fungal invasion or development of dermatophytic lesions. These results show that under the present experimental conditions, r-MEP3 specific antibodies are not protective against a challenge exposure. They also suggest that in the same model, the induction of cell-mediated immunity towards r-MEP3 is not sufficient, indicating the need for further research in the field of specific immune mechanisms involved in M. canis dermatophytosis.

A recombinant 31.5 kDa keratinase and a crude exo-antigen from Microsporum canis fail to protect against a homologous experimental infection in guinea pigs.

A Microsporum canis recombinant 31.5 kDa keratinase and a M. canis crude exo-antigen were tested as vaccines in an experimental infection model in guinea pigs. Animals were vaccinated subcutaneously three times at two-week intervals with either the keratinase, the exo-antigen or the adjuvant alone. Cutaneous challenge was performed blindly. Both humoral and cellular-specific immune responses to M. canis antigens were evaluated every 14 days, while a blind evaluation of clinical lesion development and fungal persistency in skin were monitored weekly. Vaccination induced very high and significant (P < 0.01) antibody responses towards both antigens. High cell-mediated immune responses to both immunogens were also induced by vaccination. After challenge, however, scores reflecting the severity of dermatophytic lesions did not differ significantly between vaccinated and control groups at any time after challenge. These results suggest that, in the guinea pig, the induction of specific immune responses against the M. canis-secreted antigens used in this study are not protective against challenge exposure.

The prevalence of immediate and delayed type hypersensitivity reactions to Microsporum canis antigens in cats.

Spontaneous recovery from Microsporum canis infections in cats is thought to be dependent on the development of a competent immune response. The purpose of this study was to determine the prevalence of positive delayed type hypersensitivity reactions in cats with and without dermatophytosis. Four groups of cats were intradermally skin tested with M canis extract and test sites were evaluated both subjectively and objectively at 0, 24 and 48 h after injection. Delayed intradermal testing (IDT) reactions were absent in cats not exposed to dermatophytosis (n=20); infected-recovered cats (n=38 culture negative lesion negative and n=43 lesion negative but culture positive) had significantly larger IDT reactions than unexposed cats and cats that were still actively infected (n=18). Based on the results of this study, IDT with M canis extract can be used to assess the cellular immune response of cats with dermatophytosis.

Humoral and cellular immune response to a Microsporum canis recombinant keratinolytic metalloprotease (r-MEP3) in experimentally infected guinea pigs.

In order to better understand the host-fungus relationship in Microsporum canis dermatophytosis and to identify major fungal antigens, the immune response to a crude exoantigen preparation and to a purified recombinant keratinolytic metalloprotease (r-MEP3) was evaluated in guinea pigs experimentally infected with M. canis. Humoral and cellular immune responses were assessed from day 0 to day 57 post-infection (PI), the former by enzyme-linked immunosorbent assay (ELISA) and the latter via a lymphocyte proliferation assay. Infected guinea pigs developed humoral and cellular responses to both M. canis exoantigen and r-MEP3, while no specific immune response to these antigens was observed in control animals. This is the first report on the development of both humoral and cell-mediated immune responses to a purified keratinase in M. canis dermatophytosis.

Safety and immunologic effects after inoculation of inactivated and combined live-inactivated dermatophytosis vaccines in cats.

OBJECTIVE: To determine antidermatophyte immunologic effects of an experimental combined live-inactivated dermatophytosis vaccine (CLIDV) and a commercial inactivated dermatophytosis vaccine (IDV) in cats and to evaluate adverse effects associated with administration of these vaccines. ANIMALS: 20 healthy juvenile domestic shorthair cats. PROCEDURE: Cats were injected with 2 doses of CLIDV at the standard dosage or 1 dose of CLIDV at 10 times the standard dosage; IDV was administered at the manufacturer-recommended dosage. Cats were observed for illness and reactions at inoculation sites. Periodically, samples were obtained for fungal culture, lymphocyte blastogenesis test (LBT) as an indicator of cell-mediated immunity against dermatophyte antigens, and antidermatophyte IgG titers. Following vaccination, cats were challenge-exposed by topical application of Microsporum canis macroconidia and examined weekly for clinical signs of dermatophytosis. RESULTS: of 10 cats given CLIDV developed focal crusts at the injection site that resolved without treatment; these were areas of dermatophyte infection with the vaccine strain. Antidermatophyte IgG titers increased significantly with all vaccination protocols. Cellular immunity against M canis increased slightly and variably during the vaccination period and did not differ significantly between vaccinated and control cats. All cats developed dermatophyte infection after challenge exposure. Vaccination with CLIDV or IDV was associated with slightly reduced severity of initial infection. CONCLUSIONS AND CLINICAL RELEVANCE: Noculation with IDV or CLIDV did not provide prophylactic immunity against topical challenge exposure with M canis. Inoculation with either vaccine did not provide a more rapid cure of an established infection.

[A method for group identification of hair infected with Microsporum]. / Sposob opredeleniia gruppovoi prinadlezhnosti volos, porazhennykh mikrosporiei.

A method for identifying the group appurtenance of biological objects from subjects suffering from various diseases is developed. The method can be used in examination of putrefactive objects (blood, secretions, hair, etc.) and in cases when the group appurtenance cannot be identified by other methods.