亀井 克彦

人獣共通皮膚糸状菌症原因菌の1種Arthroderma vanbreuseghemiiによる感染は我が国でも散見される。本菌種はげっ歯類(ドブネズミなど)が保菌し、まず猫がネズミを捕獲することにより感染し、その猫に人が接触して感染すると推測されている。今回、ドブネズミなど33頭の被毛を調べたところ、千葉県で捕獲されたドブネズミ1頭より本菌種が分離され、その遺伝子型は既知の人症例由来株と同一であったので、本菌種の感染にドブネズミの関与が示唆された。(著者抄録)

We investigated the effects of high protein intake on host resistance to Paracoccidioides brasiliensis. Two-d fasted mice were infected with P. brasiliensis and refed on diets with three different levels (54%, 20%, and 5%) of casein. The mice refed the 54% casein diet showed reduced antifungal activity in the spleen and liver as compared with the mice refed the 5% or the 20% casein diet. After infection, increases in spleen and liver mRNA levels of myeloperoxidase, cathepsin-G, and elastase-2 were more profound in the mice refed the 54% casein diet as compared with the mice refed the 5% or the 20% casein diet. Infected mice refed the 54% casein diet exhibited greater interferon (IFN)-gamma production in the spleen and liver and higher levels of thiobarbituric acid reactive substances (TBARSs) in the liver as compared with those refed the 5% casein diet. These results indicate that high protein intake impairs host resistance to P. brasdiensis.

An atypical isolate of Paracoccidioides brasiliensis (IFM54648), recovered from the sputum of a Brazilian man, was not detected in immunodiffusion tests for paracoccidioidomycosis and in species-specific PCR for the major antigen 43-kDa glycoprotein coding gene (gp43). The mycological characteristics of the isolate were similar to those of a typical P. brasiliensis. A total of 8 genes were sequenced from IFM54648, and the sequences were compared between the new isolate and other reference isolates and database sequences. We analyzed fragments of the gene sequences that code for gp43, the internal transcribed spacer regions of ribosomal RNA, the D1/D2 domains of the large subunit ribosomal RNA, glucan synthase, chitin synthase, glyoxalase I mRNA, 70-kDa heat-shock protein mRNA and urease. The gene sequences were 98.9-100% identical between IFM54648 and Pb01 (another atypical isolate). When compared to the other typical isolates, the identities were generally lower than 98%. A phylogenetic tree constructed using gp43 sequences showed that IFM54648 clustered with Pb01 at a considerable distance from other isolates. Therefore, this isolate is likely related to Pb01, which has recently been shown to be genetically distinct from other isolates of this species.

Genotypes ofCandidaspp. isolated from exhalation of 20 dolphins, 11 water samples from captive pools, and 24 oral cavities of staff members in an aquarium using a combination of multiple drug resistance 1 gene (MDR1) and the internal transcribed spacer (ITS) 1 5.8s-ITS 2 regions of ribosomal RNA gene (ITS rDNA) sequences were studied. The holding ratios of the dolphins, captive pools, and staff members were 70, 90, and 29%, respectively. Isolated pathogenic yeast species common to the dolphins and environments wereCandida albicansandC. tropicalis. Identical genotypes in bothCandidaspp. based on the combination ofMDR1andITSrDNAwere found in some dolphins, between a dolphin and a staff, among dolphins and environments, and among environments. The results indicated the diffusion and exchange of pathogenic yeasts at the aquarium among dolphins and environments. The isolates at the aquarium showed higher rates of resistance to azole antifungals compared to reference isolates.

In eukaryotes from yeasts to human, DNA double-strand breaks are repaired by nonhomologous end-joining (NHEJ) or homologous integration (HI). In the human pathogenic yeast Cryptococcus neoformans, gene manipulation by HI does not occur frequently because ectopic integration by NHEJ is predominant, and it has been necessary to screen 30-100 transformants per experiment to obtain transformants with the desired genotypes. To overcome this problem, we constructed a strain in which one of the NHEJ-related genes, CnLIG4, was deleted. CnLIG4 encodes a homologue of the human DNA ligase IV involved in the last step of DNA repair by NHEJ. Gene targeting in the URA5 locus of a URA5-lacking strain TAD1 with URA5 gene fragments having 1-kb flanking sequences achieved 80% HI efficiency, which is higher than that of the wild-type control (50%). Growth phenotypes and virulence were not attenuated by deletion of the CnLIG4 gene. Such results suggest that the CnLIG4 knockout strain created in this study provides an additional alternative for the molecular genetics study of C. neoformans.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

Histoplasmosis, caused by Histoplasma capsulatum, is an endemic mycosis in many countries of the world except for Japan. Outbreaks of histoplasmosis among Japanese people are very rare and are mainly imported by travelers. We report an outbreak of histoplasmosis among healthy Japanese people who traveled to a resort area in Southeast Asia. Three young Japanese women traveled to Langkawi island, Malaysia and stayed on the island for five days without visiting caves, a known reservoir of H. capsulatum. All three individuals developed flu-like symptoms with multiple nodule shadows on chest X rays or chest CT scans at around ten days after their return to Japan. Serum samples obtained from the three subjects were positive for anti-Histoplasma antibody and specific PCR for H. capsulatum on lung biopsy specimens and the serum from one patient was positive. The clinical course of all three patients improved without the use of anti-fungal agents and no recurrence has been confirmed. Clinical attendants should consider histoplasmosis when they see patients with flu- like symptoms with abnormal chest X-rays after visiting H. capsulatum endemic areas, especially Southeast Asia.

This report describes Exophiala infection in cultured striped jack, Pseudocaranx dentex, in Japan in 2005. One hundred out of 35 000 fish died per day and mortalities continued for 1 month. Diseased fish showed swelling of the abdomen and kidney distension. Numerous septate hyphae, pale brown in colour, were seen in kidney in squash preparations. Histology revealed abundant fungal hyphae and conidia in gill, heart and kidney. Fungal hyphae were accompanied by cell necrosis and influx of inflammatory, mainly mononuclear cells. The fungus isolated from the diseased fish had septate hyphae, pale brown in colour and 1.8-3.0 μm in diameter. Conidiogenous cells were conspicuous annellides, short or cylindrical or fusiform in shape. Conidia were one-celled, ellipsoidal with smooth walls, accumulated in balls at the apices of annellides that tended to slide down, 1.5-2.0 μm in width and 3.0-5.0 μm in length. The fungus was classified into the genus Exophiala based on its morphology and as Exophiala xenobiotica based on the sequences of the ITS 1-5.8S-ITS 2 regions of rDNA. This is the first record of this fungus in a marine fish. © 2009 Blackwell Publishing Ltd.

Endotracheal intubation in mice is both a common and important technique. However, it is a difficult procedure because of the small orotracheal size and the success rate is variable. There have been many reports of refined techniques that facilitate intubation but only a few reports have proposed how to verify the proper placement of the endotracheal tube. We describe a novel, safe and reliable method to confirm endotracheal intubation in mice using an extension tube for intravenous infusion. One drop of water was instilled in the extension tube and connected to the end of the catheter used as an endotracheal tube. When the catheter was inserted correctly into the trachea, the water in the extension tube oscillated in synchrony with the movement of the mouse's thorax, indicating correct placement of the catheter. This method was simple, reliable and use materials that are routinely available. This method is helpful for experimental mouse models that require airway access.

During 1998-2008, there were 31 strains of Gordonia species isolated from clinical specimens in our laboratory. Our identification of the 31 strains of Gordonia species showed that major pathogenic Gordonia species in Japan were classifiable, respectively into 14 and 13 strains of Gordonia sputi and Gordonia bronchialis. The four remaining strains were identified as three Gordonia species: G. aichiensis (2 strains), and G. terrae (1 strain), and G. otitidis (1 strain). Results of drug susceptibility tests for these 31 strains of Gordonia isolates are reported herein.

Objective: Although protein malnutrition impairs immune functions, several studies have recently shown that protein restriction without malnutrition is beneficial to host defenses against invading pathogens and cancer. In an effort to establish the optimum diet for host resistance, we investigated the effect of different dietary protein levels on host resistance to Paracoccidioides brasiliensis. Methods: Mice were fasted for 2 days and then infected with P. brasiliensis. Immediately after challenge with this fungus, mice were refed on diets with three different levels (0%, 1.5%, or 20%) of casein. On days 0-7 after infection, antifungal activity and levels of proinflammatory mediators in the spleen and liver were measured. Results: Mice refed on the 1.5% casein diet showed higher antifungal activity in the spleen and liver compared with mice on the 20% casein diet. The antifungal activity in the spleens of mice refed on the 0% casein diet was intermediate between the antifungal activities of those refed the 1.5% and 20% casein diets. After infection, increases in spleen and liver levels of interleukin-6 and interferon-gamma, liver mRNA levels of antimicrobial proteins (myeloperoxidase, cathepsin-G, and elastase-2), and liver mRNA levels of proinflammatory mediators (interleukin-18, chemokine C-X-C motif ligand 10, nuclear factor-kappaB, inducible nitric oxide synthase, and granulocyte-macrophage colony-stimulating factor) were less profound in mice on the 1.5% or 0% casein diet compared with mice refed the 20% casein diet. Conclusion: The present results suggest that protein restriction without malnutrition could be beneficial to host resistance to P. brasiliensis.

Psychological stress can modulate host defense against invading pathogens. In this study, we investigated the effect of dietary oils on social isolation stress-induced modulation of host resistance to Paracoccidioides brasiliensis. In olive oil-fed mice, 3 weeks of isolation stress resulted in temporarily delayed clearance of this fungus in the liver compared with group-housed mice. By contrast, in soybean oil-fed mice, isolation stress had no significant effect on antifungal activity. The olive oil-fed mice showed greater liver interferon (IFN)-gamma and interleukin (IL)-6 production in response to infection as compared with the soybean oil-fed mice. In the olive oil-fed mice, isolation stress led to greater infection-induced IFN-gamma production in the liver compared with the group-housed animals. These results indicate that the modulatory effects of psychological stress on host resistance to P. brasiliensis can vary depending on dietary fatty acid composition.

症例は75歳女性、胸部CTにて気管支粘液栓と無気肺があり、気管支鏡検査で右B8の粘液栓を確認した。気管支粘液栓からはスエヒロタケが培養され、粘液栓の病理像からMucoid impaction of the bronchi(MIB)と診断した。抗真菌薬であるItraconazoleで改善したが、同薬の中止により、別の部位に粘液栓と無気肺を形成した。Itraconazoleの再投与にて再び改善した。現在Itraconazole投与中であるが、再発はない。本例は喘息の合併はなく、ステロイド剤の投与をせずに抗真菌薬のみで改善している。本菌による呼吸器疾患としては、アレルギー性気管支肺真菌症(ABPM)、菌球、肺化膿症、肺炎の4つの病型が報告されている。ABPMおよびMIBの報告例は本症例を含めて12例である。12例の治療は、抗真菌薬単独6例、ステロイド剤単独3例、併用2例、Bronchial toiletのみ1例である。ABPM/MIB症例でアスペルギルスが検出されない場合や、アスペルギルス抗体が陰性の時は、Schizophyllum communeの可能性を考慮する必要がある。また、治療法の確立には症例の集積が必要である。(著者抄録)

Candida oral flora from 52 Brazilian HIV-infected children was characterized while they received antiviral monotherapy therapy and subsequently, HAART with the use of protease inhibitor. There was a significant increase in non-C. albicans Candida isolates from 9.6-28.8% (P=0.005) after the children were placed on protease inhibitor therapy. Although Candida albicans still remained the most commonly isolated species, relative presence of C. tropicalis (n=9) followed by C. parapsilosis (n=8) markedly increased in association with protease inhibitor therapy. Furthermore, rare Candida species including C. dubliniensis, C. norvegensis, C. humicula and C. rugosa also appeared after the onset of protease inhibitor therapy. Subsequent investigation of the antifungal sensitivity of these diverse isolates, derived during protease inhibitor therapy, demonstrated some variation in antifungal sensitivity. With notable exceptions, the majority were sensitive to amphotericin B while most C. albicans and non-C. albicans Candida isolates were also susceptible to fluconazole, itraconazole and ketoconazole. Amongst exceptions was a single C. tropicalis isolates which was resistant to fluconazole (MIC>64 microl/ml) and one C. albicans-B isolate which showed cross-resistance to all azoles and amphotericin.

The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop-mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMP method had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune-suppressed patients.

2006年本邦において、タイ人AIDS患者の血液培養よりPenicillium marneffeiが分離された。本菌種による感染報告例は本症例が3例目となるが、培養に成功した例はわが国では初めてと考えられる。患者は、41歳、タイ東北部出身の女性で、約10年前に来日。その後もしばしば一時帰国していた。AIDS治療中に発熱のために行った血液培養より、培養初期に白色、やがて暗赤色となる集落が分離され、菌学的および分子生物学的手法によりP.marneffeiと同定され、患者はマルネッフェイ型ペニシリウム症と診断された。アムホテリシンBおよびミカファンギンの投薬により患者は回復し、引き続き通院し経過を観察された。分離株をサブロー・ブドウ糖寒天平板培地にて25℃で培養した集落は、初め白色フェルト状で、次第に黄色から黄緑色となり、さらに培地内に深紅色色素を拡散した。分生子頭は散開性で、その先端に分生子の連鎖を形成していた。ブレイン・ハート・インフュージョン寒天斜面培地にて35℃で培養すると、細かい襞のある灰白色膜様集落を形成し、顕微鏡的には短菌糸より構成されていた。なお、本分離株のリボゾームRNA遺伝子internal transcribed spacer領域の配列は、既知株と100%一致し、DDBJにAB298970として登録されている。臨床検査分野においては、今後HIV感染症の拡散と人々の移動のグローバル化に伴い、病原性輸入真菌症に遭遇する危険性が高まることが予測され、専門機関との連携を含め、初期対応が可能となるような体制作りが必要であると考える。(著者抄録)

A number of cases of Trichophyton tonsurans infection have been reported among sportsmen and women participating in wrestling, judo, and sumo wrestling in Japan, but there have also been sporadic reports of cases with no history of contact with these sports. A molecular method using restriction enzyme analysis of PCR-amplified fragments targeting the non-transcribed spacer region (NTS) of ribosomal RNA gene in fungal nuclei was applied to T. tonsurans strains isolated from sporadic cases in Japan. Five of 6 molecular types recorded in Japan, i.e., NTS types I, II, IV, V, and VI, and two new types, designated NTS VII and NTS VIII, were observed among 10 strains isolated from sporadic cases. The NTS IV strains, considered not to be related to the present epidemic, were found to be the most prevalent molecular type accounting for 4 of the 10 strains isolated. NTS I was the most prevalent type in the current epidemic in Japan, but it was cultured from only one patient who was later noted to be the daughter of a retired judo practitioner. Four subjects had histories of living abroad and were considered to have been infected outside Japan. The strains in these cases were NTS II, V, VI, and VII. The results of this study suggested that the NTS IV strains were originally present in Japan at a low incidence, but that there has been a recent influx of NTS I, II, V, VI, and VII from abroad, which has been accompanied by the secondary spread of strains from wrestlers and practitioners of martial arts to the general community.

56歳、男性。胆石膵炎に対し加療受けるも改善せず当院紹介となった。厚生労働省の急性膵炎重症度スコアは転院時13点、CT gradeはVであった。胆石は自然排石していた。PMMA-CHDFやSDDを含む集中治療を開始したが、経過中septic shockに陥り、steroidの使用を余儀なくされた。さらに感染性膵壊死を合併し、第40病日にnecrosectomy+open peritoneal drainageを施行した。当初腹腔内は細菌感染が主であったが、その後真菌が検出されmicafungin sodiumの投与を開始した。しかしその後も感染が制御できず炎症反応高値が遷延し、血液培養からTrichosporon asahiiが検出された。抗真菌薬をfluconazoleへ変更し血液培養は陰性化したが、他の感染を制御できず第138病日に永眠された。近年新しい抗真菌薬の開発・使用に伴い、真菌のbreakthroughが問題となっている。本症例のように著しい免疫不全の状態にある重症患者では特に注意が必要である。(著者抄録)

Objective: The intake of omega-3 polyunsaturated fatty acids and psychological stress can each induce tissue lipid peroxidation. In our present study, we investigated their combined effects on the oxidative status of mouse tissues. Methods: Mice were group-housed (four mice/cage) and fed a diet containing fish oil (as a source of omega-3 polyunsaturated fatty acids), soybean oil, or olive oil for 3 wk. These animals were then 1) housed under the same conditions (four per cage, control group) or 2) individually housed to generate psychological stress conditions (isolation stress). After 2 wk of isolation stress, the levels of thiobarbituric acid-reactive substances (an index of lipid peroxidation) and antioxidants in the liver and kidney and the serum levels of triacylglycerol were measured. Results: Fish oil-fed mice showed increased levels of thiobarbituric acid-reactive substances in their livers and kidneys compared with soybean oil- or olive oil-fed mice. These increases in thiobarbituric acid-reactive substance levels in the fish oil-fed mice were less profound under isolation stress conditions when compared with the group-housed animals on the same diet. In the fish oil-fed mice, isolation stress led to an increase in liver vitamin E levels when compared with their group-housed counterparts. The fish oil-fed mice exhibited lower serum triacylglycerol levels compared with the soybean oil- or olive oil-fed mice, and this decrease was more profound under conditions of isolation stress when compared with group-housing conditions. Conclusion: Dietary fish oil combined with isolation stress results in lower levels of lipid peroxidation in the liver and kidney compared with dietary fish oil alone.

An intra-familial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) housed in a Japanese zoo was studied. The family consisted of an adult couple and two offspring (a male and a female). The porcupettes, born in Japan, showed severe hair loss while the parent animals, imported from the USA. (male) and Canada (female), showed mild symptoms or were asymptomatic. Morphologically identical Tricophyton spp. isolates were recovered within seven days from quills of all animals on chloramphenicol-supplemented potato dextrose agar plates incubated at 37 degrees C. Two representative colonies from each animal were identified as Arthroderma benhamiae Americano-European race based on mating type (+) and the internal transcribed spacer (ITS) 1-5.5S-ITS 2 region of the rRNA gene sequences (AB236404-AB236408). The present cases constituted the second isolation of dermatophytes from porcupines. There were two different ITS types, i.e., the predominant one isolated from all animals and a secondary one recovered from only the mother porcupine. The sequences have never been recorded in Japan or in the GenBank database to the best of our knowledge. In addition, they were located at a cluster involving the type strain and mating strains of A. benhamiae Americano-European race and its F1 progeny. In contrast, 28 rodents (eight species) and three insectivora (1 species) exhibited in the petting zoo were negative for any dermatophytes as determined by culture.

Chaetomium globosum is commonly found in natural environments worldwide and is known to be a causative agent for emerging fungal infections. The present study describes a case of erythematous epilation of a dog caused by C. globosum. A mixed-breed young dog, a 4-months-old male, weighing 7.25 kg, showed depilation, scales, and dermatitis with slightly itchiness on his skin. The main symptom was an erythematous epilation on the left subocular skin 7.5 cm in diameter, accompanied by elephantiasis-like hyperplasia and scales. Similar lesions were observed on the skin on both sides of the ear lobes, the heels, tail, and left angulus oris. The scales from the crusted lesion were cultured on chrolamphenicole-added potato dextrose agar plates at the first visit, as well as followed by ambulatory practices. The isolates at the first visit, 1 and 3 weeks after treatment, were identified as C. globosum by mycological study and the D1/D2 domain of the large subunit rRNA gene sequence. The patient dog was treated by ketoconzole both orally and externally. The lesions were cured, showing new hair growth 9 weeks later. In addition, the susceptibilities to antifungal agents for the present C. globosum isolate were as follows: amphotericin B, 4.0 microg/ml; 5-FC 64.0 microg/ml; itraconazole, 0.5 microg/ml; miconazole, 1.0 microg/ml; fulconazole, 16.0 microg/ml; ketoconazole, 0.25 microg/ml; and micafungin, 16.0 microg/ml.

Four strains of Ochroconis gallopava from 3 out of 15 Japanese hot springs were isolated. Colonies of the hot spring isolates were uniformly floccose and dark olive green on the surface and dark brown on their reverse side on potato dextrose agar (PDA) plates, however, they became felty, flat, and brownish-black, and produced a reddish-brown pigment after several times of subculture at room temperature. Shapes and sizes of conidia of the four strains were individual, while the D1/D2 domain of the large subunit ribosomal RNA gene sequences showed 99.7% identity in the GenBank database. The DNA pattern of the hot spring isolates amplified by species specific loop mediated isothermal amplification method were as the same pattern as that of a clinical isolate. The minimum inhibitory concentrations of antifungal agents to O. gallopava isolated from the hot springs were ranged from 0.5 to 1 microg/ml in amphotericin B, 1 to 16 microg/ml in flucytosine, 0.125 to 0.25 microg/ml in itraconazole, 1 to 4 microg/ml in miconazole, 16 to 64 microg/ml in flconazole and 0.03 to 0.5 microg/ml in micafungin. The isolates had fatal outcome in experimentally infected mice intravenously with severe invasiveness to brains and kidneys. These findings suggested that O. gallopava habitats in hot springs could be one of sources for infection.

Pathogens of the Aspergillus species are frequently seen in deep-seated mycoses. We previously demonstrated that the culture filtrate of Aspergillus fumigatus (CF) has immunosuppressive effects on polymorphonuclear leukocytes (PMNs), which act as the main phagocytes to hyphae of Aspergillus fumigatus (A. fumigatus). But little is known about the gene expression profiles involved in it. Therefore we investigated the changes in gene expression in human PMNs treated with CF or gliotoxin at two time points, using microarray analysis. CF and gliotoxin changed the expression of 548 and 381 genes, respectively. Only 51 genes showed the same expression patterns with the two stimulants, and CF-induced changes in gene expression occurred comparatively earlier than those induced by gliotoxin. Among 31 genes encoding apoptosis, which were up- or down-regulated in this assay, only 3 genes were similarly changed by both kinds of stimulation. Apoptosis was detected and quantified using two apoptosis assays. CF and gliotoxin changed the expessions of only 3 out of 19 regulated genes related to inflammatory mediators and receptors similarly. The up-regulation of the gene encoding annexin 1 (ANXA1), which is known to be involved in extravasation and apoptosis of neutrophils, may play a role in the immunosuppressive effect of A. fumigatus. The difference in expression changes between CF and gliotoxin is presumed to be caused by the interaction among the components of CF and therefore the interaction is an area of interest for further investigation.

Psychological stress has been shown to modulate immune functions. In this study, we investigated the effect of dietary oils (olive oil, soybean oil, and fish oil) on the social isolation stress-induced modulation of lymphocyte immunological activities in mice. In olive oil-fed, but not soybean oil- or fish oil-fed, mice, a 2-week isolation stress decreased the lymphocyte proliferative response, reduced the interferon-gamma and interleukin (IL)-10 secretions and increased the IL-4 secretion by lymphocytes. The isolation stress reduced the arachidonic acid content of lymphocytes markedly, moderately, and not at all in the olive oil-, soybean oil-, and fish oil-fed mice, respectively. In the olive oil-fed, but not soybean oil- or fish oil-fed, mice, the isolation stress up-regulated the expression level of mRNA for splenic heat-shock protein 70 and increased lymphocyte sensitivity to the antiproliferative effect of corticosterone. This is the first demonstration that effect of psychological stress on lymphocyte immunological activities can vary depending upon the dietary fatty acid composition.

A recent case of canine histoplasmosis, the first confirmed case of disseminated infection accompanied by carcinoma in Japan, was diagnosed by clinical characteristics, histopathological examination, chest radiographs, ocular fundoscopy and molecular biological data. The clinical manifestations were not limited to cutaneous symptoms but were referable to disseminated infection, similar to human autochthonous cases. The partial sequences of the internal transcribed spacer (ITS1/2) regions of the ribosomal DNA genes of this and other Japanese canine histoplasmosis strains were 99-100% identical to the sequence AB211551 derived from a human isolate in Thailand, and showed a close relationship to the sequences derived from Japanese autochthonous systemic and cutaneous human cases. The phylogenetic analysis of 97 sequences of the ITS1/2 region disclosed six genotypes. The genotypes derived from Japanese autochthonous human and dog cases belonged to the cluster consisting of Histoplasma capsulatum var. capsulatum and H. capsulatum var. farciminosum sequences, indicating that these varieties might cause not only cutaneous but also systemic histoplasmosis, regardless of their host species. The current status of the 3 varieties of Histoplasma capsulatum according to the host species remains a subject of further investigation.

A 2-year-old spayed female mongrel dog showed claudication with abnormal ossification containing fungal cells detected by biopsy. The dog was treated with ketoconazole and itraconazole perorally for 5 months; however, the osteomyelitis became aggravated, and an amputation from the scapula was performed. The right superficial cervical lymph node became swollen 5 months after the operation. The lymph node contained PAS positive fungal elements and a portion of tissue produced mycelial fungal growth on potato dextrose agar supplemented with chloramphenicol. The culture was identified as Lecythophora hoffmannii based on morphology, physiology and 100% identity in the sequence of the D1/D2 domain of the large subunit ribosomal RNA gene of the fungal species in the GenBank database (accession number AB100627). In addition, the sequence from the present isolate was submitted as AB189164. The isolate showed resistance to antifungal agents, i.e., amphotericin B, 5-FC, fluconazole, itraconazole, miconazole and micafungin. The dog developed cachexia 2 months after the onset of lymphadenopathy, and was euthanized on the 459th day after onset of clinical symptoms. This was the first disseminated case of L. hoffmannii infection in Japan.

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.

Ochroconis gallopava is a species of dematiaceous fungi recognized as a causative agent of zoonotic and emerging fungal infections. It affects the central nervous system and respiratory tracts of humans, birds and cats. We designed O. gallopava species-specific primer sets to aid in its identification by a loop-mediated isothermal amplification (LAMP) method based on the D1/D2 domain of the LSU rDNA sequence. The LAMP method successfully detected the gene from both fungal DNA and experimentally infected brains and spleens of mice and will be helpful in the diagnosis of O. gallopava infection.

<title>ABSTRACT</title> We developed a pair of primers that specifically identifies <italic>Coccidioides</italic> species, etiologic agents of the human fungal disease coccidioidomycosis. These primers could be used for distinguishing <italic>Coccidioides immitis</italic> and <italic>Coccidioides posadasii</italic> by simply comparing the amplicon sizes on an agarose gel.

The Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University is the only organization in Japan to possess a series isolates of Coccidioides spp., which are the most virulent pathogenic fungi and which are treated as biosafety level 3 microorganisms. Recently, the genus Coccidioides has been classified into two species, C. immitis and C. posadasii, based on their endemic areas and genotyping; the former species is endemic to the state of California, and the latter is endemic to other parts of North and South America. We reevaluated 19 isolates of Coccidioides immitis stored in our center using a multiple gene analysis. Five isolates were identified as C. immitis and 14 as C. posadasii. Their sequence information in GenBank will help to identify the two genospecies of Coccidioides spp.

A 5-year-old male Siberian husky bred outdoor in Tokyo had a swollen paw with interdigital granulomatous lesions in the left hindlimb. The dog had no apparent pulmonary or gastrointestinal involvement. Histopathological analysis of the skin lesions demonstrated yeast-like organisms predominantly within macrophages. Sequence analysis of fungal ribosome RNA gene isolated from a paraffin sample revealed a 100% homology with the teleomorph of Histoplasma capsulatum. The present case may support the concept of primary cutaneous canine histoplasmosis as an endemic phenotype recognized in Japan.

<title>ABSTRACT</title> Yeasts from caves have rarely been examined. We examined yeasts collected from bat guano samples from 20 bat-inhabited limestone and volcanic caves located in 11 prefectures in Japan. Of ∼700 yeast-like colonies, nine <italic>Trichosporon</italic> species were recovered from 15 caves. Two of these were known species, and the remaining seven are potentially novel species, based on molecular phylogenetic analyses. In addition to <italic>Trichosporon</italic> species, identifiable strains of eight ascomycetous yeasts and one basidiomycetous yeast were recovered at frequencies of 5 to 35%. Our findings suggest that <italic>Trichosporon</italic> spp. are the major yeast species in bat guano in Japan and that bat guano is a potentially rich source of previously undescribed yeast species.

Stachybotrys chartarum is a dematiaceous fungus that is ubiquitous in our living environment. This fungus has long been regarded as non-pathogenic and its inhalation effect on humans has been scarcely studied. Recently, however, epidemiologic studies on acute idiopathic pulmonary hemorrhage in infants suggested that the fungus might be potentially pathogenic to humans. To determine the pathogenicity of this fungus, its interaction with the host defense system was studied using polymorphonuclear leukocytes (PMNs) and macrophages. Histopathological analysis of mice intratracheally injected with this fungus was also performed. The results disclosed that the conidia of S. chartarum were resistant to the antifungal activities of alveolar macrophages in terms of phagocytosis, killing and inhibition of germination. However, the conidia could not survive in the lungs of mice when injected intratracheally. Lavage fluid of mycelia that contained the dark slimy material coating the surface of conidia showed cytotoxic activity against macrophages and PMNs. Intratracheal injection of conidia in mice resulted in intraalveolar infiltration of PMNs. When using multiple injections during a 3-week period, strong eosinophilic infiltration into the proximal alveoli and perivascular tissues was observed. Our results suggest that inhalation of conidia may cause serious damage to the human lung, particularly when repeated.

Coccidioidomycosis, caused by inhaling Coccidioides immitis, is a mycosis imported from endemic regions including the southwestern United States. C. immitis is so virulent that even a short-term stay in the endemic area can provide a chance for infection. Here, we report a 33-year-old Japanese man with formation of a fungus ball inside the pulmonary cavity secondary to coccidioidomycosis with a duration of 8 years, which is considered rare. He was infected with C. immitis in the United States in 1996. A nodule remained in the lung, which later cavitated with fungus ball formation. We identified Coccidioides immitis in the cultured specimen from the cavity and serum antibodies against it. We performed a lobectomy in 2003 since anti-fungal treatment was only temporarily effective. He is still free of disease 6 months later.

Schizophyllum commune has recently emerged as a causative agent of human mycosis, but the details of its virulence are not yet known. To elucidate the pathogenicity of S. commune, a murine model of invasive pulmonary infection was established. ICR mice, not immunosuppressed or immunosuppressed by cortisone acetate, were infected with S. commune by intratracheal inoculation with agar beads containing the basidiospores. All immunosuppressed mice died within 2 weeks. Pathology examination revealed massive mycelial invasion into the lungs, penetration into adjacent vessels, and systemic dissemination, suggesting much higher virulence of this fungus than was previously estimated. This is the first murine model of pulmonary infection by S. commune, which we believe can be of assistance during subsequent investigations of this infection.

74歳,女性のTrichophyton tonsuransによるblack dot ringwormを報告した.患者は20年来,頭皮に痒みと落屑があり,1999年に直接鏡検で真菌陽性が判明した.臨床所見は前頭部から後頭部に及ぶ大型の脱毛斑で,その中に多数の黒点と,鱗屑を伴った紅色丘疹が散在していた.病毛の直接鏡検で毛内性大胞子菌性寄生を認め,培養所見では集落は中央が綿状に隆起し辺縁が白色粉状で,裏面は赤褐色を呈した.スライドカルチャーでは,棍棒形～球形,極端には風船様に膨らんだ大小種々の小分生子と,少数の大分生子,及びラセン体を認めた.毛髪穿孔試験陰性.ウレアーゼ試験陽性.ITS1-5.8S-ITS2領域のリボソームRNA遺伝子の解析で,新潟県の2例の高齢女性の頭部白癬から分離されたT.tonsurans 2株と100%一致した.以上より,本菌株をT.tonsuransと同定した.一方,最近流行している格闘技選手や最近の単発例とはITS1領域で3塩基相違があったことからT.tonsuransには種内多型があり,輸入株とはITS1領域で鑑別可能な日本の在来株が存在することが示唆された.在来型のT.tonsuransが千葉県で分離されたのは今回初めてであるが,菌学的に酷似するT.coccineumが,第二次大戦前に多数分離されていたことを考えると,本県には古くからT.tonsuransが存在していた可能性がある.

In order to confirm the phylogenetic relationships of Histoplasma capsulatum, the partial sequences of large subunit (28S) ribosomal gene (D1/D2 region) of 49 isolates were studied. The similarity values of the 49 isolates were more than 99.0% across 617 base pairs, however, the 49 isolates were divided into 9 groups. These 9 groups were independent of 3 varieties, var. capsulatum, var. farciminosum and var. duboisii. These results showed that analysis of the nucleotide sequence of the 28S rRNA gene was very effective for identification of H. capsulatum and that three varieties of H. capsulatum should be reclassified according to the phylogenetic relationship established from analysis of the D1/D2 region sequences.

74歳男.骨髄異形成症候群(MDS)のためプレドニゾロンを内服中であった.今回,右足底の腫瘤を主訴とした.右足底内側には径22mmの表面肉芽様腫瘤がドーム状に隆起していた.また,アキレス腱部,土踏まず部に弾性軟,多結節性の皮下腫瘤を認めた.病理組織は,真皮から皮下組織にかけて炎症細胞浸潤と肉芽腫様病変を示し,その中に小円形で好塩基性の莢膜を持ち,内腔が明調な菌要素が多数存在していた.PCR法による遺伝子検索では,クリプトコックスに特異的なバンドを検出した.フルコナゾールの投与により腫瘤は徐々に縮小し,色素沈着を残して平坦化した.しかし,その後,アキレス腱部からの生検で皮下に少数の菌体を認め,イトラコナゾールの内服を併用した.症例はMDS自体とステロイド内服による細胞性免疫の低下により皮膚クリプトコックス症を発症したと考えられた.診断にはPCR法が有用だと思われた

Paracoccichoidomycosis is a deep mycosis Caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Gliotoxin, one of the mycotoxins produced by Aspergillus fumigatus, has various, potent bioactivities. However, it has not been considered to be a toxic (or virulence) factor because of its slow production. The aim of the present study was to investigate the effects of aeration on the cytotoxicity of A. fumigatus culture filtrate, and to determine the optimal condition for the rapid production of gliotoxin from this fungus. Fungal culture filtrates were made in three different containers under various conditions of aeration and O-2 concentration. These filtrates were compared in terms of their cytotoxicity on murine macrophages and analyzed by gas chromatography. The culture filtrate showed high cytotoxicity when it was made under highly aerated conditions, but it was significantly less cytotoxic when prepared under non-aerated conditions. The cytotoxic activity became evident within 15 h of culture at 20% O-2, when the fungus had already started producing gliotoxin. The culture filtrates also contained some other as yet unidentified substances that might also to some extent contribute to the cytotoxicity. In light of these results, the authors propose that a highly aerated condition is responsible for the rapid production of gliotoxin, and that gliotoxin might play an important role in the respiratory infection by A. fumigatus, with other toxic substances acting additively or synergistically.

The lesions of histoplasmosis in dogs in Japan differ from those in dogs in North America. Affected dogs in Japan have had multiple granulomatous or ulcerated foci in skin or gingiva and have not had pulmonary or gastrointestinal lesions. The present report introduces a polymerase chain reaction (PCR) diagnosis of canine histoplasmosis and the characteristic of disease in Japan. The surgically removed skin ulcerate samples from a 5-years-old female Shiba-inu native to Japan without traveling out of the country were evaluated. Tissue samples had many yeast-like organisms in the macrophages. DNA was extracted from paraffin-embedded tissue samples. A nested PCR technique was applied. The detected sequence of the internal transcribed spacer of ribosomal RNA gene had 99.7% in homology with Ajellomyces capsulatus (the teleomorph of Histoplasma capsulatum). Clinical manifestations, historical background of equine epizootic lymphangitis in Japan, and a human autochthonous case of histoplasmosis farciminosi indicated that this dog might have been infected with H. capsulatum var. farciminosum as a heteroecism.