Histopathological survey of free-ranging neotropical bats with dermatitis.

Bats have a fundamental ecological role, and no wildlife disease has decimated more individuals than white-nose syndrome (WNS). This impactful mycosis has raised the importance of monitoring disease threats to bat populations. In this study, we aimed to investigate gross skin lesions in neotropical bats by histopathology to survey the occurrence of dermatitis that could resemble WNS cases in Brazil. Eleven species of free-ranging bats were sampled from the rabies surveillance program in 9 municipalities of Northern Paraná. Members of the Molossidae family were the most frequent ones among the 126 analyzed individuals, and 4 cases of dermatitis in 2 black mastiff bats (Molossus rufus), 1 great fruit-eating bat (Artibeus lituratus), and a big free-tailed bat (Nyctinomops macrotis) were detected. Gross lesions included alopecia, macules, discoloration, and hyperkeratosis. Among the bats with gross lesions, dermal thickening and mild inflammation were observed histologically. Two M. rufus bats had dermal fungal invasion; however, none resembled WNS.

Seroprevalences Against Paracoccidioides cetii: A Causative Agent for Paracoccidiomycosis Ceti (PCM-C) and Coccidioides posadasii; for Coccidioidomycosis (CCM) in Dall's Porpoise (Phocoenoides dalli) and Harbor Porpoise (Phocoena phocoena) Stranded at Hokkaido, Japan.

Paracoccidiodomycosis ceti (PCM-C) is a zoonotic mycosis characterized by chronic granulomatous cutaneous lesions in cetaceans. It is distributed worldwide and is caused by an unculturable fungus; Paracoccidioides cetii. On the other hand, coccidioidomycosis (CCM), caused by Coccidioides spp., is also a zoonotic and highly pathogenic fungal infection endemic in both American continents. Even though the Far East is not an endemic area of CCM, an autochthonous case has been reported in China. Although the seroprevalence against P. cetii in captive dolphins was 61.0%, there is no information on wild dolphins living in cold waters. The present study aimed to investigate the seroprevalence against P. cetii and C. posadasii in 15 Dall's porpoises (Phocoenoides dalli) and 11 harbor porpoises (Phocoena phocoena) stranded in Hokkaido, Japan. The seroprevalence against P. cetii in the above dolphins was 26.9% (7/26), which was recorded only in Dall's porpoises (7/15), and that against C. posadasii was 15.4% (4/26), three in Dall's porpoises and one in harbor porpoise. The present study demonstrated positive seroprevalence against P. cetii and C. posadasii in wild cetaceans living in the subarctic areas of the Far East as the first records, and would issue the warning those who live in the area were exposed to the causative agent of CCM from seawater.

Distinct Pattern of Paracoccidioides lutzii, P. restrepiensis and P. americana Antigens Recognized by IgE in Human Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is caused by the fungi Paracoccidioides spp. The main antigens recognized by IgE are known for P. brasiliensis species complex, but not for P. lutzii. Current research investigated the major P. lutzii (LDR2) antigens recognized by IgE, in comparison to P. restrepiensis and P. americana (former P. brasiliensis species complex), besides IgG recognition. Cell-free antigens (CFA) from P. lutzii (LDR2), P. restrepiensis (B339) and P. americana (LDR3) were analyzed by ELISA and immunoblotting (IB) by detecting specific IgG and IgE from sera from patients with PCM presumable by P. brasiliensis species complex (n = 24). Additionally, somatic antigen (SA) was analyzed by IB. P. lutzii (LDR2) antigens showed significantly lower reactivity than P. restrepiensis (B339) and P. americana (LDR3) in ELISA for both IgE and IgG (p < 0.05). The IgE-IB pattern was different between P. lutzii (LDR2) and the other species, regarding components with ~30 kDa and ~70 kDa in CFA and a ~200 kDa in SA. P. lutzii (LDR2) present at least three antigens recognized by IgE which mainly differ from P. restrepiensis (B339) and, to a lesser extent, from P. americana (LDR3). Current research evidenced for the first time the major P. lutzii (LDR2) antigens recognized by IgE.

Parengyodontium album Isolated from Cutaneous Lesions of a Pacific White-Sided Dolphin (Lagenorhynchus obliquidens) During Treatment for Paracoccidioidomycosis Ceti.

The prominence of seafood in Japan motivates close monitoring of its seas and marine lives for potentially pathogenic fungi. During the treatments of the male Pacific white-sided dolphin (Lagenorhynchus obliquidens) for paracoccidioidomycosis ceti (PCM-C), 5 white and floccose colonies showing identical genotype and morphological characteristics were isolated from two skin biopsy samples of cutaneous granulomatous lesions in 2018. The isolates were identified as Parengyodontium album known as one of fungal species having abilities to produce industrially important proteases, and to become a causative agent for emerging mycosis based on morphological and molecular biological characteristics. These lesions consisted of non-malignant pearl-like structures of hyperplastic keratinocytes. Interestingly, although the isolates could grow at 35 °C, their DNA sequences were phylogenetically located in a cluster consisting of environmental and clinical isolates lacking the ability to grow at 35 °C, based on previous reports. The opportunistic infection we observed in the dolphin might be caused by immune disorder due to PCM-C. Notably, although P. album is recognized as non-harmful, and has significant industrial importance and antitumor activity, it has potential to cause not only superficial but also systemic infection, and presents difficulties in treatment because of its high resistance to antifungal compounds.

Seroprevalence of Antibodies Against Paracoccidioides Spp. in Captive Dolphins from Three Aquaria in Japan.

The skin disease paracoccidioidomycosis ceti occurs in several dolphin species globally. Infection by the unculturable fungi Paracoccidioides brasilensis or other Paracoccidioides spp. results in chronic cutaneous and granulomatous lesions. In this study we used immunohistochemistry to investigate the seroprevalence of antibodies to Paracoccidioides spp. in captive dolphins from three aquaria in Japan. We had previously reported that there were serological cross-reactions for Paracoccidioides spp. with related species in the order Onygenales. We hypothesized that the degree of serological cross-reactions for Paracoccidioides spp. might be lower in areas, such as Japan, where the fungal diseases coccidiodomycosis and paracoccidiodomycosis are not endemic. Sera from 41 apparently healthy dolphins, including 20 Atlantic bottlenose dolphins (BD: Tursiops truncatus), 6 Indo-Pacific bottlenose dolphins (IPBD: Tursiops aduncus), 2 F1 generation of a cross between BD and IPBD (F1), 3 Pacific white-sided dolphins (PWD: Lagenorhynchus obliquidens), 2 pantropical spotted dolphins (PSD: Stenella attenuata), 6 false killer whales (FKW: Pseudorca crassidens), and 2 rough-toothed dolphins (RTD: Steno bredanensis) were investigated. Sera from three dolphins with paracoccidioidomycosis ceti were used as a positive control. The yeast-form cells of Paracoccidioides spp. in the cutaneous tissue sample derived from the first Japanese paracoccidioidomycosis ceti case were used as the antigen for the immunohistochemistry. Of the 41 dolphins tested, 61.0% had antibodies against Paracoccidioides spp. This indicates that dolphins of several species in Japanese aquaria have likely been exposed to the pathogen Paracoccidioides spp.

Immunohistochemical Cross-Reactivity Between Arthrographis kalrae and Highly Pathogenic Coccidioides posadasii, Histoplasma capsulatum, and Paracoccidioides Fungal Species.

Recently, we have reported serological cross-reactivity between paracoccidioidomycosis ceti and paracoccidioidomycosis, histoplasmosis, and coccidioidomycosis. However, data on the interaction of Arthrographis kalrae with the above pathogenic fungal infections are lacking. A. kalrae is a widely occurring ascomycetous fungus; causes superficial and deep mycoses; shows thermally dependent dimorphism; and has a genomic profile related to the above-mentioned fungal species. Our study aims to investigate cross-reactivity using eight murine sera, obtained from experimental infection with two A. kalrae isolates. The murine sera were incubated with fungal cells of A. kalrae, Coccidioides posadasii, Histoplasma capsulatum, Paracoccidioides sp., and P. brasiliensis. Thirty murine sera, obtained from experimental infection with six isolates of H. capsulatum, sera from three cases of dolphin paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum sample from a healthy person with a history of coccidioidomycosis, were also incubated with A. kalrae fungal cells and the respective fungal cells that caused the infection as positive controls. Sera derived from the mice infected with A. kalrae reacted strongly when incubated with the Paracoccidioides sp., P. brasiliensis, and C. posadasii, but no positive reaction was observed against the fungal cells of H. capsulatum. The murine sera infected with three out of six isolates of H. capsulatum, and all cetacean and human serum samples reacted positively with the fungal cells of A. kalrae. The present study demonstrated serological cross-reactions among A. kalrae infection, coccidioidomycosis, paracoccidioidomycosis, paracoccidioidomycosis ceti, and histoplasmosis.

Polyclonal antibodies to Paracoccidioides brasiliensis are able to recognise antigens from different strains from Paracoccidioides species complex, including Paracoccidioides lutzii LDR2.

Since the new species Paracoccidioides lutzii emerged in 2009, much has been researched on strains previously considered atypical. Still, there is no consensus about recognition of antigens from P. lutzii by antibodies directed to other Paracoccidioides species, which can have great impact on Paracoccidioidomycosis (PCM) diagnosis. Current research investigated soluble protein/carbohydrate epitopes from P. lutzii LDR2, Paracoccidioides restrepiensis B339 and Paracoccidioides americana LDR3 recognised by IgG directed to Paracoccidioides brasiliensis. Cell free antigens (CFA) were analysed by: (a)silver and periodic acid-Schiff staining of SDS-PAGE; (b)immunoblot (IB) with rabbit IgG anti-P. brasiliensis Pb18; (c)IB and ELISA with a pool of PCM patients' sera before and after treatment with sodium metaperiodate (SMP) to oxidise carbohydrate epitopes. Both rabbit and human immune sera recognised several antigens of P. lutzii LDR2, P. restrepiensis B339 and P. americana LDR3. P. lutzii's gp43 was not observed in IB or silver/PAS staining. SMP treatment affected reactions with all 3 CFAs, but more intensely with antigens from P. lutzii LDR2. In conclusion, antibodies directed to P. brasiliensis recognised antigens from P. lutzii LDR2. The use of any of the recognised antigens in a broad spectrum diagnostic model for Paracoccidioides species complex needs to be further investigated.

Immunohistochemical Cross-Reactivity Between Paracoccidioides sp. from Dolphins and Histoplasma capsulatum.

Paracoccidioidomycosis ceti is a cutaneous disease of cetaceans caused by uncultivated Paracoccidioides brasiliensis or Paracoccidioides spp. Serological cross-reactions between paracoccidioidomycosis ceti and paracoccidioidomycosis, paracoccidioidomycosis and histoplasmosis, and paracoccidioidomycosis and coccidioidomycosis have been reported before. The present study aimed to detect immunohistochemical cross-reaction between antibodies to Paracoccidioides sp. and Histoplasma capsulatum, and vice versa. Thirty murine sera, obtained from experimental infections of 6 isolates of H. capsulatum, were reacted with paraffin-embedded yeast-form cells of Paracoccidioides sp. derived from a case of paracoccidioidomycosis ceti in Japan. The murine sera were also reacted with human isolates of H. capsulatum yeast cells, with P. brasiliensis yeast cells, and with fungal cells of Coccidioides posadasii. Three dolphins' sera from cases of paracoccidioidomycosis ceti, two human sera from patients with paracoccidioidomycosis, and a serum from a healthy person with a history of coccidioidomycosis were used in order to determine that the tested fungal cells reacted properly. Sera derived from mice infected with an isolate of H. capsulatum reacted positively against yeast cells of Paracoccidioides sp., yeast cells of P. brasiliensis, and fungal cells of C. posadasii, while those derived from other strains were negative. The present study recorded for the first time the cross-reaction between the yeast cells of H. capsulatum and antibodies against Paracoccidioides spp., the yeast cells of Paracoccidioides sp. and antibodies against H. capsulatum, the yeast cells of Paracoccidioides sp. and antibodies against Coccidioides sp., and fungal cells of C. posadasii and antibodies against Paracoccidioides spp.

A new marker sequence for systematics of medically important fungi based on amino acid sequence of the largest subunit of RNA polymerase I.

Molecular evolution has dominated taxonomic studies for decades, replacing traditional methods for identification and classification. However, there is a need for better markers to resolve the problems that have limited their usefulness. In this report, we introduce the protein tag (Ptag) sequence, a highly polymorphic amino acid sequence within the C-terminal region of the largest subunit of RNA polymerase I, as a new systematic tag sequence for delineating the evolutionary history of medically important fungi. As Ptag sequences are highly polymorphic between species and low within species, 42 fungal species representing the main taxonomic groups in the phyla Ascomycota and Basidiomycota were tested. The phylogenetic tree inferred from the Ptag sequences showed high consistency with the accepted classification of the Assembling the Fungal Tree of Life (AFTOL) project. Moreover, it could resolve the interspecies phylogenetic relationships of the tested taxa. In contrast, the phylogeny inferred from the nucleotide tag (Ntag) sequence, encoding the Ptag peptide, displayed lesser discriminatory power in resolving the phylogenetic relationships among distantly related taxa. In the case of closely related fungal species, the phylogenetic trees for Ptag and Ntag sequences were consistent with the tree for ITS1 sequences of 11 dermatophytic species. Taken together, the Ptag sequences should contribute to inferring phylogeny among species whereas the Ntag sequences should be useful to analyze variations among closely related species to resolve taxonomic issues in fungi.

Trichosporon asteroides Isolated from Cutaneous Lesions of a Suspected Case of "paracoccidioidomycosis ceti" in a Bottlenose Dolphin (Tursiops truncatus).

"Paracoccidioidomycosis ceti" is a rare zoonotic fungal infection affecting dolphins and is endemic worldwide. The causative agents are Paracoccidioides species; however, it is impossible to isolate the fungal species. We isolated Trichosporon asteroides from multifocal, irregularly raised skin lesions on a female bottlenose dolphin (Tursiops truncatus) captured off coast of Japan, which was suspected to have "paracoccidioidomycosis ceti." An abundance of round, yeast-like cells was detected in a potassium hydroxide direct-mount specimen of the skin samples; however, nested PCR targeting the partial sequence of 43-kDa glycoprotein-coding gene correspondent to Paracoccidioides sp. was negative. Biopsied tissue samples were cultured on brain heart infusion agar plates supplemented with chloramphenicol, 1% yeast extract, and 4% sodium chloride (4% NaCl-BHI), on Mycosel agar with 4% sodium chloride (4% NaCl-Mycosel), and on potato dextrose agar supplemented with chloramphenicol (CPDA) at 35 °C for 4 weeks. Cream-colored and wrinkled colonies consisting of hyphae and arthroconidia grew on 4% NaCl-BHI and CPDA, while film-like colonies composed of arthroconidia and round yeast-like cells developed on 4% NaCl-Mycosel. Although these primary cultures resembled fresh isolates of P. brasiliensis, they were identified as Trichosporon asteroides based on routine mycological studies and the internal transcribed spacer regions of ribosomal RNA sequences. The results suggested that trichosporonosis caused by T. asteroides might remain latent among cases of "paracoccidioidomycosis ceti" diagnosed without cultures and molecular biological analysis.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

Immunoblotting of soluble antigens in Paracoccidioides brasiliensis culture.

This study investigated the major soluble antigens produced by Paracoccidioides brasiliensis (Pb339) cultured in solid Sabouraud (pH 5.6 and 8.5), Sabouraud plus brain heart infusion and liquid tomato juice-enriched complex medium media at intervals of 3 days over 30 days by immunoblotting and concluded that, to optimize the source of each antigen, both time and growth conditions should be considered.

Isolation of dermatophytes and related species from domestic fowl (Gallus gallus domesticus).

We investigated 793 bird combs [645 chickens and 148 fighting cocks (Shamo)] to determine the prevalence of dermatophytes and their related fungal species. The targeted fungal species were recovered from 195 of the 793 examined birds (24.6 %). Prevalence ratios were compared in temperate (the mainland) and subtropical (Nansei Islands) areas, genders, strains, breeding scale (individual and farm), and housing system (in cage and free ranging). The frequency of the fungal species in the mainland, males, fighting cocks, breeding scale by individual nursing, and free-range housing system exhibited significantly higher positive ratios than that in the other groups. A total of 224 dermatophytes and related species were isolated, including 101 Arthroderma (Ar.) multifidum, 83 Aphanoascus (Ap.) terreus, five Uncinocarpus queenslandicus, two U. reesii, two Ap. pinarensis, one Amauroascus kuehnii, one Ar. simii, one Gymnoascus petalosporus, one Microsporum gallinae, and 28 Chrysosporium-like (Chrysosporium spp.) isolates, which were identified using internal transcribed spacer regions of ribosomal RNA gene sequences. The predominant fungal species in the mainland was Ap. terreus and that in the Nansei Islands was Ar. multifidum. Pathogenic fungal species to humans and animals were limited to M. gallinae and Ar. simii, which corresponded to 0.025 % of the isolates in this study.

An atypical case of chronic paracoccidioidomycosis in a dog caused by a fungus from the Paracoccidioides brasiliensis complex.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin American countries and one of the most important fungal diseases regarding incidence and mortality in humans. PCM has also been described in some animal species such as dogs. In this study we describe a new case of PCM disease in a dog that differed from previous records in the literature which includes a progressive evolution of fungal dermatitis causing a deforming lesion in the nose, like those found in human patients, and humoral response against gp70 instead of gp43, the major diagnostic antigen for human PCM. The clinical isolate through the ITS and partial gp43 gene phylogenetic analysis was grouped in the Paracoccidioides brasiliensis complex. This case describes several features which may contribute to improving diagnosis and understanding of canine paracoccidioidomycosis.

Isolation of Microsporum gallinae from a fighting cock (Gallus gallus domesticus) in Japan.

A case of tinea corporis caused by Microsporum gallinae was found in 2011 in Okinawa, located in the southern part of Japan. The patient was a 96-year-old, otherwise healthy, Japanese man, who had been working as a breeder of fighting cocks for more than 70 years. He was bitten on his right forearm by one of the cocks and a few weeks later, two erythematous macules appeared on the right forearm, accompanied by a slight itchy sensation. While the first isolate of this dermatophyte was recovered from the region by Miyasato et al. in 2011, it was not obtained from the same fighting cock owned by the patient. However, frequent exchanges of fighting cocks and special domestic breeds of chickens related to fighting, mating, and/or bird fairs are common among the fans and breeders. We investigated 238 chickens and 71 fighting cocks in Okinawa and in the suburbs of Tokyo (Chiba, Tokyo, Ibaraki, and Sizuoka). One isolate of M. gallinae from a fighting cock in Chiba Prefecture in the Tokyo metropolitan area exhibited a different genotype, with a single base difference from the patient isolate based on the internal transcribed spacer 1-5.8s-ITS2 regions (ITS1-5.8S-ITS2) of the ribosomal RNA gene sequence. The isolation of M. gallinae from a fighting cock on the mainland of Japan is the first such finding in animals in our country.

Re-Evaluation of the Cross-Reactions of the Antibody against the Causative Agent for Paracoccidioidomycosis Ceti; Paracoccidioides ceti and the Related Fungal Species.

Paracoccidioidomycosis ceti (PCM-C) is a chronic granulomatous keloidal dermatitis in cetaceans that has been reported worldwide and is caused by Paracoccidioides ceti. Serological cross-reactions among highly pathogenic fungal infections and related diseases have been reported. However, the true cross-reaction of antibodies against P. ceti has remained unknown due to the use of positive control sera from infected dolphins. This study aimed to re-evaluate antibodies from mechanically dislodged fungal cells in the infected tissue of a PCM-C case and demonstrate the actual cross-reaction. The results revealed a limited cross-reaction between PCM-C and paracoccidioidomycosis, while the antibodies did not react with other pathogens such as Coccidioides posadasii, Histoplasama capsulatum, and Arthrographis kalrae. Thus, the method for evaluation of the antibody against PCM-C is reliable, and there is potential for epidemiological study.

Involvement of serine protease and proteinase-activated receptor 2 in dermatophyte-associated itch in mice.

We investigated the involvement of serine protease and proteinase-activated receptor 2 (PAR(2)) in dermatophyte-induced itch in mice. An intradermal injection of an extract of the dermatophyte Arthroderma vanbreuseghemii (ADV) induced hind-paw scratching, an itch-related behavior. ADV extract-induced scratching was inhibited by the opioid receptor antagonists naloxone and naltrexone, the serine protease inhibitor nafamostat mesylate, and the PAR(2) receptor antagonist FSLLRY-NH(2). ADV extract-induced scratching was not inhibited by the H(1) histamine receptor antagonist terfenadine or by mast cell deficiency. Heat pretreatment of the ADV extract markedly reduced the scratch-inducing and serine protease activities. Proteolytic cleavage within the extracellular N terminus of the PAR(2) receptor exposes a sequence that serves as a tethered ligand for the receptor. The ADV extract as well as tryptase and trypsin cleaved a synthetic N-terminal peptide of the PAR(2) receptor. The present results suggest that serine protease secreted by dermatophytes causes itching through activation of the PAR(2) receptors, which may be a causal mechanism of dernatophytosis itch.

[Fungal dacryocystitis with cacosmia after penetrating keratoplasty--taxonomy and identification of pathogenic fungi based on DNA sequence analysis].

CASE: A 76-year-old woman with a history of penetrating keratoplasty had cacosmia associated with dacryocystitis. Two species of yeast-like fungi were isolated from the contents of her lacrimal sac. Each cultured fungus had a unique, distinctive odor. Althogh treated with an oral antifungal agent and washing of the nasolacrimal duct, the cacosmia was not improved. The continuous dacryocystitis with cacosmia was treated by dacryocystectomy. Two yeast-like fungi were again isolated from the contents of the lacrimal sac. Pathological examination confirmed a diagnosis of fungal dacryocystitis based on the fungal hyphae observed on the excised lacrimal sac wall. The cacosmia promptly disappeared. The fungi isolated from the contents of lacrimal sac were identified by DNA sequencing as Wickerhamomyces anamalus (Pichia anomala-Candida pelliculosa) and Galactomyces geotrichum (Geotrichum candidum). CONCLUSION: The cause of cacosmia in the present case was fungal dacryocystitis. Antibiotic eye drops and steroid eye drops for the treatment of penetrating keratoplasty can cause atypical fungal presentation in the inconsistently treated lacrimal system and can induce dacryocystitis. Careful usage and consideration is necessary in the long-term use of antibiotics and steroids following corneal transplantation.

In vitro Arthrographis kalrae biofilm formation: Scanning electron microscopy and cytotoxic analysis.

To our knowledge, this study represents the first demonstration of Arthrographis kalrae biofilm formation in vitro by scanning electron microscopy and the distinct cytotoxic activity between planktonic and biofilm extracts on RAW 264.7 cell line. Higher activity was observed with biofilm. It could impact host immune response, that require furthers study.