Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind. III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 15 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.     

Removal of ABS from solutions by a common fungus of sewage

Conclusions Phialophora jeanselmei (Margarinomyces heteromorphum) is very common in sewage treatment systems, and can remove ABS from solution even in the absence of glucose. Because glucose is present in sewage treatment systems either as a pollutant or as a byproduct of the metabolism of other organisms, it appears that, to some extent, ABS is removed by this organism. Additional work is needed to determine whether this organism can be made a nucleus of an artificial population deliberately established to act upon some special types of wastes; whether a mononucleate-monospore culture capable of more rapid and more complete removal of ABS can be developed; and what pathways of metabolism and decomposition of ABS might be involved with the idea of breaking into the chains of reaction with other organisms.In the presence of added glucose,Phialophora jeanselmei was found to be capable of removing ABS from shaken and still culture solutions containing an ABS concentration of 10 ppm. More adsorption on the mycelium took place in still culture than in aerated culture. In shaken culture a greater loss of ABS from solution resulted; this may be due to metabolic processes since the ABS was neither found in solution nor adsorbed on the mycelium. While wild cultures are capable of a demonstrable amount of activity, culture efficiency could be improved if selected monospore-mononucleate cultures were developed.     

Studies on the relationship between paracoccidioidomycosis in ddY mice and their estrous cycle

The relationship between paracoccidioidomycosis in ddY mouse and its estrous cycle was studied. Adult ddY mice of both sexes were used as experimental animals. Estrous cycle of female mice was examined before inoculation of Paracoccidioides brasiliensis yeast cells and mice were divided into 5 groups such as proestrus, estrus, metestrus-I, metestrus-II and diestrus. Each mouse was inoculated intravenously with 10⁶ P. brasiliensis yeast cell units and sacrificed on day 28 after inoculation. Their internal organs were cultured, and in addition, their histopathologies were studied. As a result, there was no difference in the organ cultures among the male and the female mice of 5 groups. However, histopathologically, the female groups at estrus, metestrus-I and metestrus-II were affected more severely than the male group, and the susceptibility of the female mice to the fungus was closely related to their estrous cycles.Abbreviations BHI-D brain heart infusion agar supplemented with 1.0% of anhydrous dextrose - PAS periodic acid-Schiff techniques - PBS phosphate buffered saline solution - SD standard deviation     

Mitochondrial DNA analysis of Exophiala jeanselmei var. lecanii-corni and Exophiala castellanii

A Japanese clinical isolate (KU-A-0094) which was identified by de Hoog et al. as Exophiala jeanselmei var. lecanii-corni with difficulty, was compared with 5 strains including the type cultures of E. jeanselmei var. lecanii-corni, var. jeanselmei and E. castellanii using RFLP (restriction fragment length polymorphism) patterns of mtDNA (mitochondrial DNA). RFLP patterns of KUA-0094 were identical with those of E. jeanselmei var. lecanii-corni and different from those of E. castellanii with restriction enzymes of HaeIII, MspI and hindIII. Therefore, de Hoog et al.'s identification of KU-A-0094 was confirmed. Additionally, mtDNA-RFLP patterns of E. jeanselmei var. lecanii-corni and E. jeanselmei var. jeanselmei were also different from each other. Consequently E. jeanselmei var. lecanii-corni seem to be a species in its own right rather than a variant of E. jeanselmei. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dematiaceous fungal pathogens isolated from nature

This study was conducted to demonstrate the presence of pathogenic dematiaceous fungi in nature. Using hamster and mouse inoculation techniques, 43 isolates of dematiaceous fungi were recovered from 39 samples of woody plant material and soil from the Virginia environment. Thirteen species were identified and included 4 Phialophora spp., 3 Cladosporium spp., 2 Exophiala spp., Sporothrix sp., Wangiella dermatitidis, Bispora betulina, and Scytalidium lignicola. Evidence is presented for the first isolations of C. trichoides from nature in the United States; these isolates proved to be pathogenic for mice in which they produced disease and death in a course similar to that seen in man. Natural isolates of Phialophora verrucosa, Phialophora repens, Exophiala jeanselmei, and Wangiella dermatitidis were identical to those species isolated from man using the following criteria: morphology, 12% gelatin reaction, and survival in laboratory animals.     

Experimental Induction of Lyme Arthritis in Outbred Mice

Outbred ddY mice inoculated with live cells of Borrelia burgdorferi strain 297 into hind footpad displayed swelling of the footpad at days 7 to 11 after inoculation. Marked neutrophilic infiltration was observed in the subcutaneous tissue and the part of bone tissue which was partially destroyed, and synovial layer of articular capsule was thickened and protruded into the joint space in the histopathological examination of footpad inoculated with live Borrelia cells. The inflammation peaked at day 7 and B. burgdorferi was cultured from bladder and heart of the mice at day 14 after inoculation. The mice inoculated with heat-inactivated cells at 56 C for 30 min did not show any significant histopathological change. In this mice model, nontreated littermates were not infected in contact with infected littermates for 14 days of experimental period. The outbred ddY mice model is useful for evaluating the effectiveness of vaccination against Lyme disease.     

Infectivity and Early Antibody Response to Borrelia burgdorferi Sensu Lato Isolated in Japan in Outbred Mice

Borrelia burgdorferi sensu lato isolated from Ixodes ovatus (B. japonica), I. persulcatus and patients with erythema migrans (EM) in Japan were determined on infectivity and arthritis induction-activity in outbred mice. Infectivity of B. japonica was weak and did not induce the development of footpad swelling by subcutaneous (s.c.) inoculation into the footpad. Challenged strain, NO129-M of B. japonica, to ddY mice were reinoculated to the mice at various cell numbers (1 × 10-1 × 10⁶ cells/mouse). The strain isolated from the mouse did not reinfect ddY mice and did not induce the production of specific antibody to the homologous strain. On the other hand, strains from I. persulcatus and patients with EM in Japan infected the mice and induced a serious inflammatory response in Borrelia-inoculated footpad as well as strains belonging to the three genospecies, B. burgdorferi sensu stricto, B. garinii, and B. afzelii, related to Lyme disease, from North America and Europe. The mice were infected with 10 cells of strain HP1 isolated from I. persulcatus in Hokkaido and of strain 297 isolated from a patient in the U.S.A. by subcutaneous inoculation into the hind footpad, or by intradermal inoculation into the back. Antigens of ca. 20, 23–24 (Osp C), 29, 39, 41 (flagellin) and 45 kDa reacted with the pooled sera from mice inoculated with strains HP1 and 297, but Osp A and Osp B did not.     

Evaluation of skin test for chromoblastomycosis using antigens prepared from culture filtrates ofFonsecaea pedrosoi,Phialophora verrucosa,Wangiella dermatitidis andExophiala jeanselmei

Antigenic substances were prepared from culture filtrates ofFonsecaea pedrosoi, Phialophora verrucosa, Wangiella dermatitidis andExophiala jeanselmei. These antigenic substances were evaluated for detecting cutaneous delayed hypersensitivity in rats experimentally-infected withF. pedrosoi, P. verrucosa, W. dermatitidis, E. jeanselmei, Cladosporium carrionii andFonsecaea compactum and in patients with chromoblastomycosis caused byF. pedrosoi. TheF. pedrosoi antigen elicited positive reactions in all of the animals infected withF. pedrosoi and in 5 of 6 patients. TheP. verrucosa, W. dermatitidis andE. jeanselmei antigens elicited positive reactions in all of the animals infected with the homologous species. These antigens displayed cross-reactivity in some of the animals and patients, whereas more than half of them exhibited positive reactions only to the antigens prepared from the homologous species. These results suggest that a delayed-type skin test using the antigens prepared by the authors may be useful not only for the diagnosis of chromoblastomycosis but also for the identification of species of the causative agent.     

Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo Dasypus novemcinctus )

We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo ( Dasypus novemcinctus ).Pulmonal inflammatory area was evaluated by intravenous inoculation of 10⁶ yeast cells of each isolates in young, male, ddY mice. Moreover, the partial sequence of GP43kDa gene of P. brasiliensis was analyzed. The lung inflammatory area was greater in animals inoculated with isolate D3S1. The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1. This study suggested that the same armadillo might be susceptible to multiple P. brasiliensis isolates simultaneously. This revised version was published online in August 2006 with corrections to the Cover Date.     

Acute oral intragastric pathogenicity and toxicity in mice of <Emphasis Type="Italic">Paecilomyces fumosoroseus</Emphasis> isolated from whiteflies

Paecilomyces fumosoroseus, monospore culture EH-506/3, isolated in Mexico from Bemisia tabaci whitefly was tested for acute oral intragastric pathogenicity and toxicity in CD-1 mice. Animals were inoculated by gavage with only one dose (10⁸ conidia/animal) of viable (72 mice), heat-killed (24 mice) fungus and compared to 18 control mice. Clinical observations were done daily; mycological and histological tests were performed during necropsies at days 3, 10, 17, and 21 after the inoculation. No mice were clinically ill or died. At the end of the study, their mean weight corresponded to healthy adults. Positive fungal cultures of feces were obtained only 24 h after inoculation. Positive cultures were found in 15 out of 360 organs (liver, spleen, kidney, brain, lung) in 12 of 72 mice inoculated with viable conidia. Gross pathology exhibited splenomegaly and liver paleness in mice inoculated with viable and heat-killed fungus. Non-germinated conidia were observed in studied organs, without any pathological tissue reaction, suggesting no mycological or histopathological evidence of fungal multiplication. The fungus was able to persist, but did not cause permanent damage to the host. This study supports the non-pathogenic/toxic status of P. fumosoroseus EH-506/3 when administered intragastrically in mice.     

Loss ofHindIII cleavage sites in thed-amino acid oxidase gene in some inbred strains of mice

Summary d-Amino acid oxidase cDNA was amplified by a polymerase chain reaction using RNA extracted from the mouse kidney. When digested withHindIII, the cDNAs of the BALB/c and ddY/DAO^– mice were cleaved into two fragments whereas the cDNA of the ddY/DAO⁺ mice was not. Sequencing revealed that nucleotide-471 of the cDNAs was G in the BALB/c and ddY/DAO^– mice whereas it was substituted for C in the ddY/DAO⁺ mice. This base substitution was the cause of the failure of the cleavage of the cDNA of the ddY/DAO⁺ mice.Examination of other strains of inbred mice showed thatd-amino-acid oxidase cDNAs of A/J, AKR, C57BL/6, CD-1, CF#1, ICR, DBA/2, NZB and NZW mice were cleaved withHindIII into two fragments whereas those of C3H/He, CBA/J and NC mice were not. Genomic DNAs extracted from the mice of these 15 strains were digested withHindIII and hybridized withd-amino-acid oxidase cDNA. A 18.2-kb fragment hybridized with the probe in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice whereas two fragments of 12 kb and 6.2 kb hybridized in the other mice. These results are consistent with those of the cDNAs, confirming the loss of theHindIII cleavage site in the C3H/He, CBA/J, ddY/DAO⁺ and NC mice. The Southern hybridization revealed a loss of a differentHindIII cleavage site in the A/J, AKR, C57BL/6, DBA/2, ICR and NZB mice.The substitution at nucleotide-471 should cause a substitution of an amino acid residue. However, this substitution did not affect catalytic activity ofd-amino acid oxidase.     

Application of flow cytometry to differentiating Exophiala dermatitidis E. moniliae and E. jeanselmei from each other

We applied a flow cytometry apparatus (FCM) to differenciating Exophiala dermatitidis, E. moniliae and E. jeanselmei from each other. The wavelength of the argon laser emitted from the FCM was 488 nm and the aperture of nozzle from which the stream of fluid containing single cells was blown out was 100 m. By irradiating the stream with laser by either the forward light scatter (FLS) or by the perpendicular light scattr (PLS), we were able to get two pieces of informations. Histograms displayed by the FLS indicate the cell size, while dot displays by the PLS reflect the cell structure. As a result, E. dermatitidis was clearly differenciated from either E. moniliae or E. jeanselmei by their histograms by FLS. In addition, dot displays by the PLS differenciated E. moniliae from E. jeanselmei.In conclusion, flow cytometry is available for differenciating E. dermatitidis, E. moniliae and E. jeanselmei from each other.     

Granuloma formation and killing functions of granuloma in congenitally athymic nude mice infected with Blastomyces dermatitidis and Paracoccidioides brasiliensis

We did this experiment to clarify the mechanism of granuloma formation and the killing functions of granuloma in nude mice against Blastomyces dermatitidis and Paracoccidioides brasiliensis infections. B. dermatitidis A-295 and P. brasiliensis B-1183 were the cultures used. Congenitally athymic nude (nu/ nu) mice and their heterozygous (nu/ +) littermates of BALB/ c background were the test animals. From culture A-295, 0.1% and 1% cell suspensions (wet weight) were prepared and from culture B-1183 0.2% and 2% cells suspensions were prepared. Ten nu/ + and 10 nu/ nu mice were allotted to each of four cell suspensions. For experimental blastomycosis each mouse was inoculated intravenously with 0.2 ml of the cell suspension of A-295 and for experimental paracoccidioidomycosis, with 0.15 ml of the cell suspension of B-1183. Two mice from each of the four groups were killed at 5, 8, 12, 18 and 25 days after inoculation, and histopathologic sections, stained with H&E or by PAS, were prepared from various internal organs.In the nu/ nu mice inoculated with B. dermatitidis A-295 granuloma was formed in the brain tissue after the 12th day. However, mononuclear cells, which formed the granuloma, did not kill the fungal cells, and the fungal cells continued to multiply in the granuloma. On the other hand, in the heart, kidney and fat tissue, their histopathological findings after the 18th day were clumps of fungal cells with slight cell reactions. In these organs the exertion of cell-mediated immunity was necessary for granuloma formation against the fungal infection.In the nu/ nu mice infected with P. brasiliensis B-1183, granuloma appeared in the brain and kidney after the 18th day and fungal cells continued to multiply within the granuloma as well as in those inoculated with culture A-295.These results show that the exertion of cell-mediated immunity plays an important role as the defense mechanisms of hosts against these fungal infections. However, PMNs also play an important role in the mouse's defense mechanisms against these fungal infections.We assume that the defense mechanisms of immunocompetent mice against B. dermatitidis or P. brasiliensis infection consist chiefly of two steps: in the first step phagocytosis by PMNs occurs and in the second step cell-mediated immunity enters into play.     

Fungi in bathwater and sludge of bathroom drainpipes

Samples of bathwater from 14 homes and 22 public bathhouses and sludge in drainpipes from 19 house-hold bathrooms were plated out onto potato dextrose agar supplemented with chloramphenicol. Several media were used to study colony morphology of the isolates and the thermotolerance and alkaline tolerance of each isolate were examined.Eleven sludge samples produced 12 isolates of Exophiala jeanselmei, 2 of E. dermatitidis and 1 of E. moniliae. Five household bathwater samples produced 2 isolates of E. jeanselmei, 4 of E. dermatitidis and 1 of E. alcalophila. One isolate of E. jeanselmei, 2 of E. dermatitidis, 3 of E. moniliae and 2 of unidentified Exophiala species were recovered from 6 samples of the bathwater dissolving Chinese medicine in the bathtubs of public bathhouses. One isolate of E. jeanselmei was recovered from the 15 samples of bathwater from public bathhouses. Bathwater and sludge in bathroom drainpipes may be an important habitat of Exophiala species.     

Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀ = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹ CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.     

The effects of insect pathogenic soil fungi and ectomycorrhizal inoculation of birch seedlings on the survival of Otiorhynchus larvae

• 1 Weevil larvae of the genus Otiorhynchus are a serious problem in agriculture and forestry, causing damage to a wide range of plant species, primarily by larval feeding on roots. Otiorhynchus larvae are a serious pest in forest plantations in Iceland, causing 10–20% mortality of newly‐planted seedlings.
• 2 We studied the effects of soil fungi on the survival of Otiorhynchus sulcatus larvae. The larvae were introduced into pots with birch seedlings grown in: (i) nursery peat; (ii) nursery peat inoculated with three different species of ectomycorrhizal fungi; (iii) nursery peat inoculated with insect pathogenic fungi; (iv) nursery peat inoculated with ectomycorrhizal fungi and insect pathogenic fungi; and (v) nursery peat inoculated with natural forest soil from Icelandic birch woodland.
• 3 Larval survival was negatively affected by inoculation of: (i) the ectomycorrhizal fungus Laccaria laccata; (ii) the ectomycorrhizal fungus Cenococcum geophylum; (iii) the insect pathogenic fungus Metarhizium anisopliae; and (iv) forest soil. Inoculation with the ectomycorrhizal fungus Phialophora finlandia did not have any significant effect on larval survival. No significant synergistic effect was found between insect pathogenic and ectomycorrhizal fungi.
• 4 It is concluded that ectomycorrhizal and insect pathogenic fungi have a significant potential in biological control of Otiorhynchus larvae in afforestation areas in Iceland. Further studies are needed to establish the effect of these fungi in the field and to analyse how mycorrhizal fungi affect root‐feeding larvae.

     

Tissue responses against Cladosporium trichoides and its parasitic forms in congenitally athymic nude mice and their heterozygous littermates

The tissue responses against Cladosporium trichoides and its parasitic forms were studied using nude (nu/nu) mice and their heterozygous (nu/+) littermates of BALB/c background.1.0,0.1 and 0.01% cell suspensions were prepared from a culture broth which had been inoculated with the C. trichoides and cultured with reciprocal shaking at 27 ° C for 7 days. Sixty nu/nu or 60 nu/+ mice were divided into three groups consisting of 20 each which was allotted to one of the three cell suspensions. Each mouse was inoculated intravenously with 0.1 ml of either the cell suspensions. Two mice from each of the six groups were sacrificed at adequate intervals until 30 days after inoculation and histopathologic sections stained with H & E or by PAS were prepared from their visceral organs.There were no characteristic findings in the nu/nu and nu/+ mice inoculated with the 0.01% cell suspension. When inoculated with the 1.0% cell suspension, the brain was the favorite target organ in both groups of mice and the kidney was the second. When inoculated with the 0.1% cell suspension, brain lesions were observed only in the nu/nu mice. The susceptibility of the nu/nu mice was higher than that of the nu/+ mice.The parasitic forms in the brain of the nu/nu and nu/+ mice were slender septate true hyphae with or without polymorphonuclear leucocyte infiltrate, while in the liver, spleen and lung of both groups of mice the parasitic forms were short thick hyphae, moniliform hyphae, chlamydospores or round cells (sclerotic cells). Many giant cells containing fungal elements appeared in the liver of the nu/nu mice.     

Chemical and Biological Properties of Lipopolysaccharide from a Marine Bacterium,Photobacterium phosphoreum PJ-1

The chemical and biological properties of the lipopolysaccharide (LPS) isolated from a marine bacterium, Photobacterium phosphoreum PJ-1, were studied. This LPS consists of 40.6% carbohydrate, 27.3% fatty acid, 0.2% 2-keto-3-deoxyoctonate (KDO) and other components. One characteristic of this LPS is its small amount of KDO, the basic component of the usual LPS. Electrophoresis in sodium dodecylsulfate polyacrylamide gel revealed at least two staining bands for carbohydrates. These bands were continuous and broad, and showed rapid electrophoretic mobility which corresponded closely to the fastest moving band of LPS from Salmonella typhimurium. This LPS preparation had adjuvant activity, lethality for ddY mice, and the ability to gel Limulus amebocyte lysate, and the strength of these activities corresponded closely to those of LPS preparations from Escherichia coli 0111:B4 and S. typhimurium. In the test for lethality of the LPS for ddY mice, the lethal action appeared in two phases depending on the dose used for intravenous (i.v.) injection : the early lethal action appeared within 30 min after injection of 250 μg or less, and the late lethal action occurred gradually after 16 hr at doses of 500 μg or more. The total (both phases) LD50 of this LPS (i.v.) for ddY mice was 265 μg per mouse and in only the late phase it was 500 μg. These results show that in spite of structual differences in regard to KDO content, LPS from P. phosphoreum PJ-1 has some biological properties similar to those of LPS from E. coli 0111:B4 and S. typhimurium but it shows no immunological cross-reaction with other LPS.     

Mitochondrial DNA analysis ofExophiala moniliae

Mitochondrial DNA(mtDNA) analysis with restriction enzymes, Hae III, Hind III and Msp I was performed in 17Exophiala moniliae strains. The results were as follows: (1)E. moniliae could be classified into 10 types based on restriction patterns, (2)E. moniliae is suggested to be a complex organism because of extensive mtDNA polymorphism among strains likeE. jeanselmei and (3) two types ofE. moniliae are identical with two types ofE. jeanselmei. These results suggest thatE. moniliae is not genetically defined fromE. jeanselmei and the taxonomical status ofE. moniliae requires reevaluation     

Investigation on the potential application of biological agents in the extension of shelf life of fresh beef in Nigeria

The objective of this study was to evaluate the ability of lactic acid bacteria (LAB) cultures to preserve fresh beef at room temperature, with a view to promoting safety and availability of the product in Nigeria. Two LAB strains, Pediococcus pentosaceus LIV 01 and P. acidilactici FLE 01, were applied as starters (10⁶ cfu/g) on sliced fresh beef samples, and were stored for 7 days at 30°C. Analyses of microbiological, thiobarbituric acid (TBA) and free fatty acids (FFA) were carried out during storage. Results indicated reduction in the Enterobacteriaceae, Staphylococcus and coliforms in starter inoculated samples. TBA and FFA were lower in starter culture inoculated samples compared to controls during storage. In a challenge experiment against the LAB cultures during a 7-day storage, two sets of meat were inoculated separately with 10⁶ cfu/g each of pathogenic organisms Listeria monocytogenes and Salmonella Typhimurium. There was about 1 log reduction in the L. monocytogenes on day 1 while counts were below detection limit (<2 log) on day 2 in meat samples inoculated with P. pentosaceus alone and in combination with P. acidilactici. Counts of S. Typhimurium showed about 2 log reduction in starter inoculated samples during storage while an increase by about 3 log was observed in control samples. The protective ability of the LAB strains could be exploited in shelf life extension and control of foodborne pathogens in fresh beef; their use as biological preservatives may help in promoting public health, safety and availability of the product in Nigeria.