Sequential pathological studies in Japanese quails infected experimentally with Aspergillus fumigatus

Intratracheal inoculation of young quail chicks with Aspergillus fumigatus spores resulted in the development of characteristic gross and microscopic lesions. The lesions were restricted to respiratory tract and there was no dissemination of infection to other tissues of the body.Gross changes in lungs and air sacs were observed within 24 hours and continued up to 20 days while in trachea these were noticed from the 3rd to the 9th day post-infection. The lesions, in general, included congestion and focal haemorrhages in the first 2 days followed by the development of varying-sized greyish-white nodules in the lungs, air sacs and trachea.Microscopic changes consisted of congestion, haemorrhages and a diffuse cellular infiltration in the first 2 days followed by granulomatous reaction with well developed granulomas in lungs, air sacs and trachea. Spores and developing hyphae of Aspergillus could be demonstrated in sections from 24 hours to 20 days of infection.Reisolation of the fungus was consistently achieved from the lungs, air sacs and trachea up to 14 days.     

Phaeohyphomycosis in a free-living eastern box turtle (Terrapene carolina carolina)

A free-ranging eastern box turtle (Terrapene carolina carolina) was referred to the Wildlife Center of Virginia with a three-month history of marked swelling of the right hind limb initially diagnosed as chromomycosis by histopathology. Hematology revealed severe anemia (9%), leukocytosis (12.8 cells x 10(3)/microl), heterophilia (6.14 cells x 10(3)/microl), and monocytosis (0.51 cells x 10(3)/microl). Gross necropsy revealed a firm, encapsulated 3 x 1 cm subcutaneous mass filled with dark brown-black, friable necrotic material of the distal right hind limb. Microscopically, the mass was characterized by a granulomatous inflammatory process with numerous multinucleated histiocytic giant cells. Fungal elements were present within necrotic centers and associated with multinucleated cells. Special stains revealed numerous phaeoid hyphae and yeast; Exophiala jeanselmei was isolated by routine mycologic culture. Phaeohyphomycosis was diagnosed based on the histologic appearance of the fungal elements within the mass and culture results. There was no histopathological evidence of systemic infection. This is the first report of phaeohyphomycosis caused by fungi of the genus Exophiala in free-living reptiles.     

Cutaneous alternariosis: A rare chromohyphomycosis

A 66-year-old woman presented 3 groups of scaly granulomatous lesions on the right forearm and elbow of six months duration. Direct examination of the scales showed hyphae and culture in lactrimel medium grew Alternaria sp. considered a contaminant. No improvement was observed after six months of treatment with oral griseofulvin. A second culture using half of a biopsy grew Alternaria sp. again and in histologic examination short pigmented fragments of hyphae surrounded by a granulomatous reaction were seen in the derma. The patient had marked improvement after treatment with erythromycin per os and miconazole topically; an atrophic scar remained as the only sign of the fungal infection.     

Alternaria fungal dermatitis in a free-ranging javelina (Pecari tajacu)

We report a javelina from Pinal County, Arizona, USA, with severe fungal dermatitis and cellulitis. Extreme emaciation and rostral disfiguration, including left-lateral displacement of the nasal planum, justified euthanasia. A pus-filled tract within the rostrum was observed. Histopathology revealed granulomatous inflammation with hyphae morphologically consistent with Alternaria sp. isolated by culture.     

First human model of in vitro Candida albicans persistence within granuloma for the reliable study of host-fungi interactions

BACKGOUND: The balance between human innate immune system and Candida albicans virulence signaling mechanisms ultimately dictates the outcome of fungal invasiveness and its pathology. To better understand the pathophysiology and to identify fungal virulence-associated factors in the context of persistence in humans, complex models are indispensable. Although fungal virulence factors have been extensively studied in vitro and in vivo using different immune cell subsets and cell lines, it is unclear how C. albicans survives inside complex tissue granulomas. METHODOLOGY/PRINCIPAL FINDING: We developed an original model of in vitro human granuloma, reproducing the natural granulomatous response to C. albicans. Persistent granulomas were obtained when the ratio of phagocytes to fungi was high. This in vitro fungal granuloma mimics natural granulomas, with infected macrophages surrounded by helper and cytotoxic T lymphocytes. A small proportion of granulomas exhibited C. albicans hyphae. Histological and time-lapse analysis showed that C. albicans blastoconidia were located within the granulomas before hyphae formation. Using staining techniques, fungal load calculations, as well as confocal and scanning electron microscopy, we describe the kinetics of fungal granuloma formation. We provide the first direct evidence that C. albicans are not eliminated by immunocompetent cells inside in vitro human granulomas. In fact, after an initial candicidal period, the remaining yeast proliferate and persist under very complex immune responses. CONCLUSIONS/SIGNIFICANCE: Using an original in vitro model of human fungal granuloma, we herein present the evidence that C. albicans persist and grow into immunocompetent granulomatous structures. These results will guide us towards a better understanding of fungal invasiveness and, henceforth, will also help in the development of better strategies for its control in human physiological conditions.     

Allergic fungal sinusitis. A report of two cases with diagnosis by intraoperative aspiration cytology

BACKGROUND: Allergic fungal sinusitis (AFS) is a newly recognized form of sinusitis characterized by opacification of the paranasal sinuses by "allergic mucin" (AM) admixed with scattered fungal organisms. AM consists of necrotic, or partially necrotic, eosinophils and Charcot-Leyden crystals suspended in lakes of laminated, mucinous material. AFS is characterized by the absence of bone or soft tissue invasion, purulent exudate or granulomatous inflammation. Clinically, it is important to differentiate AFS from both acute invasive fungal sinusitis and noninvasive mycetoma because the three diseases are treated with different modalities and have different prognoses. Although the radiologic features of AFS are often characteristic, occasionally it may be difficult to exclude neoplasia. CASES: Two cases of AFS, in which intraoperative diagnosis was made on the basis of the presence of both AM and fungal organisms, are reported. CONCLUSION: Cytologic diagnosis of AFS can be made from intraoperative sinus aspirates from the presence of AM and fungal elements. AM and fungi provide presumptive evidence for a noninvasive, allergic fungal disease and can help reassure clinicians intraoperatively and guide clinical management.     

Ultrastructure of the Host-Pathogen Relationship in Entomosporium Leaf Spot Disease of Photinia

Entomosporium mespili appears to be a hemibiotroph on infected Photinia leaves. This fungal pathogen produced distinctive haustoria in living host cells in young lesions. Each haustorium possessed a long slender neck with a single septum and an enlarged distal body that contained a single nucleus. A collar of host cell wall material was associated with the haustorial neck. Intact haustoria also were found in necrotic cells of older lesions. However, by this stage of disease development, the pathogen also possessed an extensive system of branched, septate hyphae that grew indiscriminately between and through dead and dying host cells. These hyphae eventually gave rise to a subcutaneous layer of sporogenous cells that formed conidia.     

Histomorphological changes in murine fibrosarcoma after hypericin-based photodynamic therapy

Histomorphological changes in murine fibrosarcoma after photodynamic therapy (PDT) based on the natural photosensitizer hypericin were evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80 mm(3) the mice were intraperitoneally injected with hypericin, either in a single dose (5 mg/kg; 1 or 6 h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70 mW/cm(2), 168 J/cm(2)). All groups of PDT-treated animals with single and fractionated hypericin dosing presented primary vascular reactions including vascular dilatation, congestion, thrombosis and oedema. Two hours after PDT there were necrotic changes with small, rather focal appearance. One day after therapy the necrotic areas were enhanced, often affecting a complete superficial layer of tumour tissue. Necrotic areas were accompanied with inflammation and haemorrhages.     

Granulomatous Invasive Aspergillosis of Paranasal Sinuses Masquerading as Actinomycosis and Review of Published Literature

Cutaneous aspergillosis is a common systemic mycosis affecting immunosuppressed patients. Here, we describe a novel morphological type of cutaneous aspergillosis in a young immunocompetent woman who presented with a chronic history of multiple nodules and discharging sinuses over left side of the face, mimicking cervicofacial actinomycosis. Skin biopsy showed granulomatous inflammation, and of septate fungal hyphae with acute-angled branching, morphologically resembling Aspergillus. This was confirmed on fungal culture as Aspergillus flavus.     

Abdominal zygomycosis in a bitch due to Absidia corymbifera

We report a case of abdominal zygomicosis in a Doberman bitch. Clinical signs consisted of urinary incontinence and hard abdominal masses detected by palpation. The masses were surgically removed by exploratory laparatomy and had a tumoral-like appearance. A granulomatous reaction containing coarse and non septate hyphae was the main histological finding. Direct microscopic examination revealed the presence of fungal structures. On Sabouraud honey agar the fungus developed fluffy, greyish white colonies that were identified as Absidia corymbifera on the basis of their macro and microscopic morphology.     

Fatal Pulmonary and Cerebellar Zygomycosis due to <Emphasis Type="Italic">Rhizomucor pusillus</Emphasis> in a Ringed Seal (<Emphasis Type="Italic">Pusa hispida</Emphasis>)

A 4-year-old captive ringed seal (Pusa hispida) was treated with subcutaneous antibacterial injections for pus exuding wounds in the skin and associated blubber following a bite attack. Three months after the incident, the animal presented nystagmus and died the following day. At necropsy, there was a 25?×?18?×?25 mm well-delineated, opaque nodular mass in the lung, besides the skin ulcers and localized areas of discoloration in the blubber correlating with the bite wound and injection sites. Histopathology of the pulmonary mass demonstrated severe eosinophilic inflammatory infiltration among numerous intralesional fungal hyphae. The hyphae were irregularly branched, broad and aseptate, consistent of zygomycosis. Magnetic resonance imaging was conducted on the head, which was initially frozen intact, revealing diffuse areas of hyperintensity in the cerebellum. Restricted histopathologic examination of the cerebellum showed severe granulomatous inflammation well spread within the neuroparenchyma, associated with abundant intralesional fungal hyphae similar to those appreciated in the pulmonary mass. Molecular analyses of the fungi in the pulmonary and cerebellar tissue identified the etiologic agent in both sites as Rhizomucor pusillus. The likely route of infection is through inhalation of R. pusillus spores or fragmented hyphae from the environment that developed into an initial pulmonary infection, becoming the source of hematogenous dissemination to the cerebellum. The skin and blubber lesions likely contributed to immunosuppression. Zygomycosis is uncommon in pinnipeds, and the present report emphasizes the importance of considering zygomycete dissemination even when the primary focus is highly confined.     

Overexpression of OgPAE1 from wild rice confers fungal resistance against Botrytis cinerea

A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism.     

Quantification of Fungal Hyphae in Leaves of Deciduous Trees by Automated Image Analysis

An optical method to quantify the fungal hyphae within decomposing leaves of deciduous trees was developed. The plant matrix was partially destroyed under hydrolytic conditions, and fungal hyphae and cellulose residues within the leaves were stained with Calcofluor M2R. Cellulose residues were subsequently depolymerized by cellulase, and fungal hyphae were separated from the remaining plant matrix with a pressurized air-water mixture. An image analysis program to quantify the fungal hyphae was written. The program included the recognition of fungal hyphae, the elimination of stomata from the images, and the measuring of lengths of fungal hyphae. The optical method was verified by a chemical method relying on glucosamine as an indicator of fungal biomass. The fungal biomass in leaves of Fagus silvatica and Quercus petraea at early states of decomposition was 0.2 to 0.4% of the leaf weight. The biomass reached a maximum within 2 to 4 weeks (optical method, 0.5 to 0.7%; chemical method, 1 to 1.4% of the initial leaf weight) and decreased thereafter.     

High turnover of fungal hyphae in incubation experiments

Soil biological studies are often conducted on sieved soils without the presence of plants. However, soil fungi build delicate mycelial networks, often symbiotically associated with plant roots (mycorrhizal fungi). We hypothesized that as a result of sieving and incubating without plants, the total fungal biomass decreases. To test this, we conducted three incubation experiments. We expected total and arbuscular mycorrhizal (AM) fungal biomass to be higher in less fertilized soils than in fertilized soils, and thus to decrease more during incubation. Indeed, we found that fungal biomass decreased rapidly in the less fertilized soils. A shift towards thicker hyphae occurred, and the fraction of septate hyphae increased. However, analyses of phospholipid fatty acids (PLFAs) and neutral lipid fatty acids could not clarify which fungal groups were decreasing. We propose that in our soils, there was a fraction of fungal biomass that was sensitive to fertilization and disturbance (sieving, followed by incubation without plants) with a very high turnover (possibly composed of fine hyphae of AM and saprotrophic fungi), and a fraction that was much less vulnerable with a low turnover (composed of saprotrophic fungi and runner hyphae of AMF). Furthermore, PLFAs might not be as sensitive in detecting changes in fungal biomass as previously thought.     

Outbreaks of phaeohyphomycosis in the chinook salmon (Oncorhynchus tshawytscha) caused by Phoma herbarum

Phoma herbarum has been associated with two outbreaks of systemic mycosis in hatchery-reared chinook salmon (Oncorhynchus tshawytscha) fingerlings. Affected fish exhibited abnormal swimming behavior, exophthalmia, multiple rounded areas of muscle softening, protruded hemorrhagic vents, and abdominal swelling. In all affected fish, swimbladders were filled with whitish creamy viscous fungal mass, surrounded by dark red areas in swimbladder walls, kidneys, and musculature. Clinical and histopathological examinations suggest that the infection may have started primarily in the swimbladder and then spread to the kidneys, gastrointestinal tract, and surrounding musculature. Consistent microscopical findings included broad septate branched fungal hyaline hyphae, 5–12 μm in diameter within the swimbladder, stomach, and often within and adjacent to blood vessels. Profuse growths of woolly brown fungal colonies were obtained from swimbladders and kidneys on Sabouraud medium. On corn meal agar the formation of pycnidia, characteristic of Phoma spp., was detected within 10 days of incubation. Morphological and molecular analyses identified this fungus as Phoma herbarum. This report underscores systemic fungal infections as a threat to raceway-raised salmon.     

Invasive maxilar sinusitis by Rhizopus oryzae

We herein present a diabetic with non Hodgkin lymphoma patient that had been treated with steroids and developed fungal invasive sinusitis. The patient had intensive facial pain that did not respond to antibiotics and on clinical inspection had a necrotic lesion on right nasal area. A smear and biopsy tissue showed broad non septate hyphae and on cultures Rhizopus oryzae was isolated. There was an unfavorable outcome, and the patient died even though liposomal Amphotericin B was administered and surgical treatment was performed.     

Invasive pulmonary pseudallesheriosis in a cross-breed calf

Pulmonary pseudallescheriosis was diagnosed in a two-months old calf. Pneumonic lungs with yellow-white nodules on the surfaces revealed granulomatous lesions microscopically. Septate, pleomorphic hyphae were present in the central caseated core with a bright eosinophilic periphery surrounded by polymorphonuclear cells and macrophages followed by a zone of epithelioid cells admixed with lymphocytes and plasma cells. The fungal agent was demonstrated by Grocott's silver methenamine staining. On isolation, morphologically it was found to be indistinguishable from that ofPseudallescheria boydii. It appears to be first report of fatal mycotic pneumonia in a calf due toP. boydii. The emphasis is given for further detailed investigation on this aspect in veterinary mycopathology.     

Histological and ultrastructural features of gastrointestinal basidiobolomycosis

Basidiobolus ranarum is a fungus found in the dung of amphibians, reptiles, and insectivorous bats. Its structural elements include both hyphae and zygospores. Patients with B. ranarum infection may present with subcutaneous, gastrointestinal, or systemic lesions. Here we report a case of gastrointesinal badidiomycosis in a 13-year-old male child who presented with acute abdomen. Exploration revealed a mass in the ascending colon. On histology, transmural granulomatous inflammation composed of abundant eosinophils, lymphocytes, histiocytes and giant cells was seen. Histochemical stains revealed broad, non-septate, hyphae-like structures surrounded by an eosinophilic sheath. On an ultrastructural level, fungal hyphae, spores, and macrophage-laden crystalloids were observed. The diagnosis of gastrointestinal basidiobolomycosis was established and the patient received antifungal treatment. This paper reviews the relevant literature regarding basidiomycosis, and discusses its diverse clinicopathological features, as well as distinguishing it from other diseases.     

Cytodifferentiation of the Seeds (Protocorms) and Vegetative Propagation Corms Colonized by Mycorrhizal Fungi

The stipecell, subepidermal parenchyma cells and inner cortical parenchyma cells were differentiated from Gastrodia elata Bl. seed and protocorm cells when they were colonized by the fungal hyphae of Mycena osmundicola Lange and M. orchidicola Fan et Guo. The hyphae aggregated in the suspensor remnant surrounding stipecell, primarily penetrated the stipecell, and then colonized the embryo of seed. Stipecell is the unique invading site of the hyphae. Subepidermal parenchyma cells containing pelotons of hyphae is also a kind of passage cells of hyphae, but, when primarily colonized by hyphae, they can degenerate a little of hyphae. The hyphae colonizing inner cortical parenchyma cells were totally degenerated, and the function of inner ocrtical parenchyma cells is digestive. The vegetative propagation corms, which differentiated from protocorms, were recolonized by Armilariella mellea (Vahl:Fr.) Karst., and the hyphae of A. mellea and M. osmundicola were found in the same cell, but there is a layer of cells uncolonized by mycorrhizal fungal hyphae. This means the two fungal species can not crisscross colonize the cell of G. elata.     

Relationship between fluorescein diacetate-stained hyphae and oxygen utilization, glucose utilization, and biomass of submerged fungal batch cultures.

The relationship between fungal activity and staining with fluorescein diacetate (FDA) was investigated by growing Penicillium citrinum and Rhizoctonia solani in submerged batch cultures at different initial glucose concentrations and aeration rates. A modified FDA staining method, similar to the Jones and Mollison technique (P. Jones and J. Mollison, J. Gen. Microbiol. 2:54-69, 1948), was developed to assess both total and FDA-stained hyphae. In previous studies, soil hyphae stained with FDA were considered viable. However, determination of a quantitative relationship between FDA staining and fungal activity is necessary before such an assumption can be made. Growth rates and the rate of change in the percentage of FDA-stained hyphae were significantly correlated. The regression equation calculated for the relationship was: growth rate (mg . ml-1 . h-1) = 0.34 + 1.1 (rate of change in the percentage of FDA-stained hyphae [. ml-1 . h-1]). Changes in activity as measured by O2 utilization, glucose utilization, and biomass correlated significantly with changes in the percentage of FDA-stained hyphae, although the relationships among these parameters were different for each fungal species. Fungal growth stage was also correlated with the percentage of FDA-stained hyphae. Staining was 10% or greater during fungal growth and less than 10% during the late growth, stationary, and death phases. Thus, the rate of change in the percentage of FDA-stained hyphae can be used to predict fungal activity rate changes for single fungal cultures and growth rates for mixed fungal cultures, and the growth stage can be assessed by the percentage of FDA-stained hyphae.