Experimental zygomycosis in rabbits: Clinicopathological studies

Zygomycosis was produced experimentally in 20 New Zealand white rabbits (Oryctolagus cuniculus) by intra-nasal administration of spores of Absidia corymbifera. Infected animals showed dullness, depression, coughing and mucopurulent nasal discharge, but no mortality. Haematology revealed no significant change in Hb and PCV, but leukocytosis due to neutrophilia in the initial stages of the experiment. There was a significant increase in serum total proteins, creatinine, AST, ALT, total Igs and CICs. A. corymbifera specific IgM and IgG antibodies were detected in the sera of the infected animals. Gross lesions consisted of pneumonic consolidations of the antero-ventral lobes of the lungs. Microscopically, histology showed formation of pyogranulommas in the lungs. Fungal elements typical of A. corymbifera were demonstrated in the tissues upto 15 days after infection by special stains and confirmed by indirect immunoperoxidase. Re-isolation of the fungus from lungs was also achieved consistently up to 15 days only. It was concluded that intra-nasal instillation of A. corymbifera in rabbits produced significant clinico-pathological alterations with the lesions confined mainly to the lungs. In the present study, neither systemic dissemination of the disease occurred nor were kidneys site of predilection as reported earlier. This revised version was published online in August 2006 with corrections to the Cover Date.     

Bovine fetal cerebral absidiomycosis.

Only 1 case of fetal cerebral mycosis was found in 4015 aborted bovine fetuses. Absidia corymbifera was associated with vasculitis, thrombosis, abscessation and cavitation of the brain. Antibodies against A. corymbifera and other fungal antigens were not detected in fetal serum by immunodiffusion techniques. Immunoglobulin G (IgG) in excess of the normal was detected in the fetal serum by radial-immunodiffusion assay.     

Sequential pathological studies in Asian water buffaloes infected intratracheally with Absidia corymbifera

Zygomycosis was produced experimentally in Asian water buffalo calves (Bubalus bubalis) by intratracheal inoculation of sporangiospores of Absidia corymbifera. Infected animals exhibited dullness, depression, partial anorexia, initial pyrexia, mucopurulent nasal and ocular discharge and coughing during the first week. There was no mortality at any stage of the experiment, which continued for 30 days. The gross and microscopic lesions were restricted to the lungs and there was no dissemination of the fungus to other organs. Gross and microscopic changes in the lungs were observed up to the 20th day post-infection. Gross lesions consisted of pneumonic consolidation of the antero-ventral lobes of the lungs. Microscopic changes consisted of granulomatous reactions with well developed pulmonary granulomas. Distorted hyphae of A. corymbifera were demonstrated in tissue sections up to 15 days post inoculation. Re-isolation of the fungus was achieved consistently for up to 15 days. It is concluded that intratracheal inoculation of A. corymbifera in buffalo calves leads to significant pathological changes in the lungs, but the disease appears to be self limiting 20 days following inoculation.     

Isolation and characterization of a strain of Lichtheimia corymbifera (ex Absidia corymbifera) from a case of bovine abortion

Background  

Lichtheimia corymbifera (previously Absidia corymbifera) is a filamentous zygomycetes belonging to the order Mucorales and to the family Lichtheimiaceae. Members of genus Lichtheimia spp. are cosmopolitan and ubiquitous in nature. Lichtheimia corymbifera is a recognized agent of diseases in man and animals. In cattle it causes abortion and mastitis. Three cases of bovine abortion occurred in a herd located in the Po Valley. Serological examinations were performed on fetal and mother's blood. One of the aborted fetus was referred to our laboratory. The paper describes the isolation and characterization of Lichtheimia corymbifera from a bovine aborted fetus.     

Suspension of non-viable fungal spores as a standard for platelet counting.

In the search among biological particles for a standard for counting human platelets, a strain of Absidia corymbifera was found to have spores that resembled platelets. After fixing in formalin and autoclaving the spores had a similar mean cell volume to that of human platelets. A suspension of these killed spores was tested in 100 laboratories and gave consistent results as a standard for human platelet counting. The Absidia corymbifera standard can be used in electronic counting methods but not in sedimentation methods, as the spores will be removed by centrifugation.     

Additional new taxa of Ardisia (Myrsinaceae) from Thailand

Two new taxa of Ardisia from Thailand, A. integra and A. corymbifera var. euryoides , are described and illustrated.     

Experimental cerebral zygomycosis in alloxan-diabetic rabbits: variation in virulence among zygomycetes

We investigated the potential of 33 different zygomycete isolates to cause cerebral disease following the intranasal instillation of their spores into ketotic rabbits with alloxan induced diabetes. The isolates represented six thermotolerant species of Rhizopus (R. arrhizus, R. chinensis, R. microsporus, R. oligosporus, R. oryzae, and R. rhizopodiformis), Absidia corymbifera, Cunninghamella bertholletiae, and Rhizomucor pusillus. All 13 isolates of the thermotolerant Rhizopus species proved to be cerebral pathogens as confirmed by culture and histopathology. One isolate of R. oligosporus and one isolate of R. rhizopodiformis, however, were less pathogenic than isolates of other Rhizopus species tested. Cerebral pathogenicity was noted with 2 of 5 isolates of Rh. pusillus and only 1 of 13 A. corymbifera isolates. Two thermotolerant C. bertholletiae cultures, recovered from human lesions, did not cause either cerebral or pulmonary disease in ketotic rabbits. The incidence of pulmonary zygomycosis caused by the isolates of the species of the four genera under study was as follows: Rhizomucor 24%, Rhizopus 22%, Absidia 9%, and Cunninghamella 0%. This study confirms the pathogenic potential of the thermotolerant species of Rhizopus to cause cerebral zygomycosis in ketotic diabetic rabbits and also revealed the potential of Rh. pusillus and A. corymbifera occasionally to cause the same disease in animals and humans.     

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.     

Factors affecting production of mold mycelium and protein in synthetic media.

The effects of certain cultural conditions on the yield of dry mycelium, protein, and total amino acid content of Rhizopus oligosporus Saito (NRRL 2710), Rhizopus rhizopodiformis (Cohn apud Lichtheim) Zopf (NRRL 6246), and Absidia corymbifera (Cohn) Sacc. et Trotter (NRRL 6247) were studied. The yield of mycelium was found to significantly increase as the spore inoculum was increased from 187,500 to 2,250,000 spores. But the total amino acids (grams/liter) did not change significantly, whereas the percentage of crude protein decreased. An inoculum containing approximately 750,000 spores/ml was used in all of the other experiments. Mycelial production was highest at 37 degrees C for all three molds. However, the best temperature for percentage of crude protein and total amino acids varied with the organism. The mycelial yield and total crude protein of R. oligosporus showed some significant changes as the C/N ratio was increased in 3% glucose medium. In a synthetic medium having a 15:1 C/N ratio, the strains of R. oligosporus, R. rhizopodiformis, and A. corymbifera had better yields from falactose than glucose, not only in dry mycelium but also in total crude protein (grams/liter) and total amino acids (grams/liter). R. oligosporus grew very well on several ammonium salts. but the maximum yield of dry mycelium, total crude protein (grams/liter), and total amino acids (grams/liter) occurred with ammonium sulfate. The optimum pH for both Rhizopus species was 4.0, although R. oligosporus grew equally well at pH 3.0 and slightly less at pH 5.0. The highest yield of mycelium for A. corymbifera was obtained in a medium with an initial pH of 8.0. It was calculated that a fermenter chanrged with an adequate medium and 1,000 lb (about 450 kg) of R. oligosporus or A. corymbifera cells could produce 88 or 90 lb of protein (on a dry-weight basis) per h if the product was removed continuously.     

Antigenic characterization of some potentially pathogenic mucoraceous fungi

The antigenic profiles of 10 mucoraceous fungi--Absidia corymbifera, Mortierella wolfii, Mucor miehei, M. pusillus, M. racemosus, Rhizopus arrhizus, R. microsporus, R. oryzae, R. rhizopodiformis, R. stolonifer,--Candida albicans and Aspergillus fumigatus were compared by crossed immunoelectrophoresis (XIE). Antigen-rich material was obtained from homogenized hyphae (or yeasts in the case of C. albicans), and antisera by multiple subcutaneous innoculation of rabbits with macerated but viable hyphal fragments of Ab. corymbifera, M. pusillus, R. oryzae or Asp. fumigatus. Unique and common antigens were demonstrable amongst the mucoraceous species although Mort. wolfii revealed little antigenic similarity with the others. Considerable sharing of antigens between Ab. corymbifera and M. pusillus was evident. Little or no cross reactivity was seen between extracts of C. albicans and Asp. fumigatus and the mucoraeceous antisera. R. oryzae and R. arrhizus, now regarded as synonymous, revealed close antigenic similarity. On the other hand, the distinction between both M. pusillus and M. miehei--which are regarded by some as belonging to a separate genus Rhizomucor--and less thermotolerant M. racemosus was reflected in their antigenic dissimilarity. Partial separation and characterization of antigens from the crude Absidia extract was achieved by concanavalin A-Sepharose chromatography. Antigens with and without affinity for concanavalin A could be demonstrated. Cross reactivity between Absidia antigens and M. pusillus antiserum appeared to be contained predominantly in material (possibly carbohydrate) which bound to concanavalin A and could be eluted with alpha-methyl-D-mannoside.     

Five cases of zygomycosis.

Combined histological and mycological study of tissue specimens established a proven diagnosis of cutaneous zygomycosis in four patients. All patients had been treated with wide spectrum antibiotics and one patient (liver transplantation) was in addition also treated with cyclosporine. All had acidosis and cutaneous breaks and four had also been treated with systemic corticosteroids. The infecting organisms were Absidia corymbifera (n=2), Rhizopus stolonifer (n=1) and Mucor circinelloides (n=1). Combined treatment with i.v. conventional and liposomal formulations of amphotericin B and surgical treatment lead to a favourable clinical and mycological cure in three patients (A. corymbifera and R. stolonifer infections). One lymphoma patient with suspected Rhizopus pusillus infection of the lungs (presence of hyphae in sputum and positive culture) had an unfavourable outcome. The patient had been treated with wide spectrum antibiotics, corticosteroids and showed severe neutropenia and acidosis. The clinical presentations are outlined, including the outcomes and predisposing factors and focus on the diagnostic procedures, treatment and preventive measures.     

一株产生淀粉分解酶犁头霉的分离鉴定及其酶学性质

从保宁麸醋醋曲中定向筛选得到一株产生淀粉分解酶的菌株CQB43,其酶活力为105.2 U/mL,RDA值达到27.9%。通过形态观察和分子生物学鉴定确定该菌株为伞枝犁头霉Absidia corymbifera。对该菌株分泌生淀粉酶酶学性质的研究结果表明,该酶在pH为4.0-5.6的范围相对稳定,最适pH是5.0;在60°C以下的范围内具有较好的热稳定性,最适作用温度为40°C。研究金属离子对其活力影响的结果表明,Co2+对该生淀粉糖化酶有激活作用,Fe3+和Ca2+对该酶有抑制作用。     

Ecology of fungi associated with moist stored barley grain

A study of the ecology of fungi associated with moist stored barley grain was carried out on samples from a partially sealed silo in Cumberland. Samples were collected during harvest and after storage at high moisture content in a partially sealed silo. At harvest, the field fungi Alternaria, Fusarium, Cladosporium and Mucor spp. were dominant both in the husks and in the dehusked, surface-sterilized grain but they were rapidly succeeded by the storage fungi: Aspergillus, Penicillium and Absidia spp. then constituted about 80 % of the initial invasion. At this stage internal grain infection rarely exceeded 5 % compared to 65–80 % infection in the husks. In subsequent months, there was a remarkable increase in invasion, mainly by Penicillium, Aspergillus and Absidia spp. and by yeasts, which appeared both in the husks and in dehusked grain. In the third month of storage, Penicillium cyclopium, P. roqueforti, Absidia corymbifera, Aspergillus candidus, A. terreus and yeasts became dominant. The thermophilous species Aspergillus fumigatus, A. terreus, Absidia corymbifera, Mucorpusillus and Dactylomyces crustaceus were isolated from husks and in dehusked grain incubated at 37 and 45 °C. Most grains were infected significantly by single genera.     

Experimental mucormycosis in congenitally athymic (nude) mice

Athymic nude (nu/nu) mice and their phenotypically normal (nu/+) littermates displayed a similar susceptibility to acute lethal infection withAbsidia corymbifera. Although the clinical manifestations of acute infection were also similar in both groups, the nude mice tended to develop more extensive lesions and were less effective in eliminating viableA. corymbifera spores than their heterozygous littermates. The results suggested that thymus-dependent processes did not play an essential role in primary resistance to mucormycosis.     

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.     

Microbial oxidation of terfenadine and ebastine into fexofenadine and carebastine

The oxidation of tert-butyl-phenyl group of title compounds by some microorganisms was studied. We have optimized the conditions of culture to increase the formation of acid metabolites and to avoid the formation of side products. We showed that an oxidative activity is induced by soybean peptones in Streptomyces platensis. The biologically active compounds, fexofenadine and carebastine, are produced in good yield (86-95%) by Absidia corymbifera.     

Evaluation of<Emphasis Type="Italic">in Vitro</Emphasis> antifungal activity of<Emphasis Type="Italic">N</Emphasis>-Benzylsalicylamide derivatives

A series of 65 derivatives of N-benzylsalicylamide was tested against eight potentially human pathogenic fungi by microdilution broth method modified according to M27-A standard. The majority of these compounds showed only weak in vitro antifungal activity. The most significant effect was observed against filamentous fungi Trichophyton mentagrophytes, Absidia corymbifera, and Aspergillus fumigatus while yeasts, in general, were less susceptible. N-(4'-Chlorobenzyl) salicylamides, N-(3',4'-dichlorobenzyl)-salicylamides, and partially N-benzylsalicylamides exhibited relatively high in vitro antifungal activity. The most efficient derivatives had MIC < or = 7.8 mumol/L against T. mentagrophytes. Regression analysis suggested an indirect relationship between MIC values and lipophilicity (log P).     

Detection of 3-nitropropionic acid and cytotoxicity inMucor circinelloides

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the generaPenicillium, Aspergillus, orFusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro-and micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides. Presented at the 30^th Mykotoxin Workshop Utrecht, Netherlands, April 28–30, 2008.     

3-Phenyl-5-methyl-2H,5H-furan-2-ones: tuning antifungal activity by varying substituents on the phenyl ring

A series of racemic 3-phenyl-5-methyl-2H,5H-furan-2-ones related to a natural product, (-)incrustoporine, was synthesized, and their antifungal activity evaluated. The key structural feature, furanone ring, was closed via H2SO4-mediated cyclization of 2-phenylpent-4-enoic acids. The compounds displayed antifungal activity, especially against filamentous fungi. Expressed as the minimum inhibition concentration (MIC) in micromol/L, the activity of the most promising derivative against Absidia corymbifera matched that of ketoconazole (31.25 micromol/L). In terms of microg/mL, the substance was more active (7.6 microg/mL) than this standard antifungal drug (16.6 microg/mL).     

Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments

About 34 wild fungal species associated with edible oil mill wastes were isolated by the serial dilution technique. Methods for rapid screening of fungal species against production of extracellular enzymes such as amylase, protease, cellulase, and lipase are reported. Among all the species, Aspergillus versicolor exhibited high amlylolytic and gelatinolytic activity, whereas Penicillium citrinum showed only high amylolytic activity. Maximum cellulolytic activity was recorded for Absidia corymbifera, As. niger, Cunninghamella echinulata, Curvularia lunata, Fusarium solani, Mucor racemosus, Paecilomyces variotii, and Syncephalastrum racemosum. The fungal species Ab. corymbifera, As. fumigatus, As. japonicus, As. nidulans, As. terreus, Cun. verticillata, Cur. pallescens, F. oxysporum, Geotrichum candidum, M. racemosus, Pe. citrinum, Pe. frequentans, Rhizopus stolonifer, and Trichoderma viride exhibited maximum lipase activity. This study confirms the isolated fungi present on a wide range of substrates in the ambient environment, and these results could provide basic data for further investigations on fungal extracellular enzymes.First two authors equally contributed to this work