A vaccine against coccidioidomycosis is justified and attainable.

Coccidioides is a fungal pathogen of humans which can cause a life-threatening respiratory disease in immunocompetent individuals. Recurrent epidemics of coccidioidal infections in Southwestern United States has raised the specter of awareness of this soil-borne microbe, particularly among residents of Arizona and Southern California, and has galvanized research efforts to develop a human vaccine against coccidioidomycosis. In this review, we discuss the rationale for such a vaccine, examine the features of host innate and acquired immune response to Coccidioides infection, describe strategies used to identify and evaluate vaccine candidates, and provide an update on progress toward development of a vaccine against this endemic pathogen.

Quantification of Candida albicans actin mRNA by the LightCycler system as a means of assessing viability in a model of cutaneous candidiasis.

The LightCycler system (two-step reverse transcription-PCR-fluorescent hybridization [LC RT-PCR-FH]) was used to quantify Candida albicans actin mRNA as a means of assessing its viability in a reconstituted skin model of cutaneous candidiasis following the application of an antimycotic. A 192-bp ACT exon fragment was ligated into the pCR2.1 plasmid vector, and dilutions of the cloned insert (pACT; 4.092 kb) were used as the standard reference template. The LC RT-PCR-FH system could detect 1 fg of pACT, equivalent to 2.2 copies of the plasmid. The ACT exon-based PCR primers and FH probes were C. albicans specific, and electrophoretic analysis of the LC RT-PCR-FH assay product showed a 174-bp band in agarose gel. The number of copies of C. albicans ACT mRNA per milligram of tissue decreased with increasing amounts of amorolfine applied to a C. albicans-infected skin model, showing a reduction in viability. Detection and quantification of ACT mRNA in tissue by the LC RT-PCR-FH assay corresponded with cultural isolation of C. albicans from samples. The ACT mRNA-targeted LC RT-PCR-FH assay represents a sensitive, specific, rapid, and quantitative means of assessing the viability of C. albicans in infected tissue. This method may also be useful in evaluating the therapeutic efficacies of antifungal drugs in the treatment of various forms of candidiasis and other fungal diseases.

[Advances in molecular biology of dermatophytes].

During the 44th meeting of The Japanese Society for Medical Mycology in Nagasaki, 2000, a forum was held entitled Advances in Molecular Biology of Dermatophytes. Based on the subject, target molecules and kind of approach, we selected seven presentations from over 100 of the poster abstracts. Six of them concerned identification and one concerned viability. Summaries of the 7 presentations are given in this article. Of presentations on the identification methods, 5 demonstrated their usefulness: 1) A sequence analysis of ITS 1 region in ribosomal DNA of several Microsporum species showed ITS 1 genospecies Arthroderma otae to be composed of A. otae, M. canis, M. equinum and M. audouinii. 2) RAPD may be useful for identifying isolates which are not clearly identifiable by conventional biological techniques. 3) Sequence analysis of CHS 1 was shown to be a rapid tool for species level identification of M. gypseum. 4) PCR-SSCP analysis was also useful for discrimination of dermatophytes with high reproducibility and sensitivity. 5) Strain identification of A. benhamiae isolates may be possible using RFLP analysis of NTS regions in ribosomal DNA. The other presentation concerning identification pointed out some important problems: RFLP of mitochondrial DNA and ITS sequencing of A. benhamiae showed that the results are sometimes in conflict with those obtained from biological techniques, or in some cases, between other molecular techniques. This implies that our concept of fungal species needs to be re-examined and perhaps amended. The presentation on viability introduced quantitative analysis of mRNA of ACT gene, a new application of a molecular technique. Since the mRNA expresses only in living cells, the method is highly useful as an indicator of fungal viability.

Isolation of an intron-containing partial sequence of the gene encoding dermatophyte actin (ACT) and detection of a fragment of the transcript by reverse transcription-nested PCR as a means of assessing the viability of dermatophytes in skin scales.

An internal partial sequence of the gene encoding actin (ACT), 725 to 762 bp in length, was amplified by PCR from the genomic DNA extract of 12 species of dermatophytes and sequenced. An intron that is 56 to 93 bp in length was located along the ACT fragment of all of the dermatophytes at codon position 301 (-3) (a codon number followed by "-3" indicates that the intron directly follows the codon) with reference to the amino acid sequence of human alpha-smooth muscle actin. A primer pair that annealed to exon sequences flanking the ACT-associated intron produced a dermatophyte-specific 171-bp amplicon by reverse transcription-nested PCR (RT-PCR) of dermatophyte ACT mRNA. PCR primer pairs with antisense sequence based on the ACT intron sequence were species specific for dermatophytes, suggesting a potential for use in the identification of dermatophytes. The viability of dermatophytes in skin scales was subsequently assessed by the presence of ACT mRNA in total RNA extracted from a 48-h culture of scale samples in 250 microl of yeast carbon base broth. RT-nested PCR of dermatophyte-infected samples amplified an ACT fragment of the predicted size of 171 bp. The results of viability testing based on ACT mRNA detection by RT-nested PCR correlated with cultural isolation from skin scales. This method is a potential tool for rapidly assessing fungal viability in the therapeutic efficacy testing of antimycotics.

Fluorometric assessment of In vitro antidermatophytic activities of antimycotics based on their keratin-penetrating power.

Keratin particles impregnated with amorolfine or clotrimazole in serial doubling dilutions (64 to 0.125 microg/ml) were used to evaluate the activities of these agents against 20 isolates each of Trichophyton mentagrophytes and Trichophyton rubrum in a yeast carbon broth medium incorporating Alamar Blue dye. The proposed MIC with keratin impregnation (MIC(K)) is defined as the lowest concentration of an agent used to impregnate keratin particles that effects a fluorescence-based fungal growth quotient of 0.05 or less. The conventional colorimetric and visual MICs of amorolfine for the dermatophytes, 64 microg/ml] and 64 microg/ml [range, 16 to >64 microg], respectively) may indicate the strong in vivo antidermatophytic activity of amorolfine as a topical agent. The new antidermatophytic susceptibility testing procedure has potential clinical utility for the in vitro screening of agents for use in the topical treatment of superficial mycoses.

Reverse transcription - 3' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model.

The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue.

Molecular probes for the detection of pathogenic fungi in the presence of human tissue.

Four primer systems, amplifying fragments of the gene coding for the small ribosomal subunit (18S rRNA) were characterised with pure cultures of 65 medically relevant fungal species plus two mushrooms. A primer cocktail (TR1/CA1-TR2/AF2) amplified 59 of 67 fungal species; the universal fungal primer 1 (UF1) in combination with the eukaryotic primers S3 or EU1 amplified 64 and 65 of 67 fungal species, respectively. The design of an additional primer (RZY1) enabled the amplification of the missing members of the zygomycetes. The primer systems amplified all the medically relevant fungi tested. These included eight Candida spp. and seven other yeast species, 13 dermatophytes, 32 moulds (including six zygomycetes and five dimorphic fungi) and two mushrooms. Eleven controls including DNA from Schistosoma mansoni, Escherichia coli, Mycobacterium tuberculosis and man were not amplified. The oligonucleotide CA hybridised with C. albicans, C. tropicalis and C. parapsilosis; the oligonucleotide TR hybridised with the 13 dermatophytes; the oligonucleotide AF hybridised with Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, A. versicolor, A. tamarii, A. clavatus, A. fischeri, but not with A. niger or A. versicolor; and the oligonucleotide HC hybridised with three varieties of Histoplasma capsulatum. These oligonucleotides did not hybridise with the other fungi nor the controls. The specificity of the newly designed primer systems was confirmed by selective amplification of fungal DNA from human lung tissue spiked with fungal biomass and from vitrectomy fluid of a patient with candida endophthalmitis.

Ribosomal genes of Histoplasma capsulatum var. duboisii and var. farciminosum.

A total of 1704 basepairs of the 18S rDNA of Histoplasma capsulatum var. duboisii (HCD, strain CBS175.57) and H. capsulatum var. farciminosum (HCF, strain CBS478.64) were sequenced (EMBL accession no. Z75306 and no. Z75307). The 18S rDNA of HCD was 100% identical to a published sequence of H. capsulatum var. capsulatum (HCC). The 18S rDNA of HCF showed one transversional point mutation at the nucleotide position 114 (ref. Saccharomyces cerevisiae). Hybridization confirmed that, in the 18S rDNA of two out of five strains of HCF, guanine was substituted for cytosine at the nucleotide position 114. Furthermore, identical group 1C1 introns (403 bp) were found to be inserted after position 1165 in four out of five strains of HCF, including the two strains with point mutations in the 18S rDNA, and a slightly different group 1C1 intron (408 bp) was detected in one strain of HCC without this point mutation. Intraspecific sequence variability in the highly conserved 18S rDNA because of occurrence of introns and mutations as a possible source of error in molecular diagnostics is discussed. In addition, internal transcribed spacer regions between the 18S rDNA and the 5.8S rDNA (ITS1) of three strains of HCF, and one strain each of HCC and HCD showed significant sequence variability between varieties and strains of H. capsulatum.

Universal fungus-specific primer systems and group-specific hybridization oligonucleotides for 18S rDNA.

We designed two primer systems that amplify a fragment of the gene coding for the small ribosomal subunit (18S rRNA). A broadly reactive, yet fungus-specific, primer cocktail comprises two previously published primers, TR1 and TR2, which specifically amplify dermatophytes, and two newly designed primers, CA1 and AF2, which specifically amplify Candida and Aspergillus respectively. This primer cocktail amplifies a DNA fragment of approximately 578 basepairs (bp) in length (from position 838 to 1415), which contains variable, possibly species-specific regions (V5, partly V7). Another newly designed primer, UF1 (universal fungal primer 1), along with the eukaryotic primer S3 amplifies a 926-bp fragment (from position 263 to 1188) that includes the variable regions V3, V4 and V5. Both primer systems amplified DNA from Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, Fusarium oxysporum and Trichophyton mentagrophytes, but not the DNA from Prototheca zopfii, Escherichia coli or humans. The previously published oligonucleotides TR and HC, which are specific for dermatophytes and Histoplasma respectively, and the newly designed group-specific oligonucleotides, CA and AF, hybridized with T. mentagrophytes, Histoplasma capsulatum, C. albicans and A. fumigatus respectively, but not with the other six fungi or with the three controls.

A thermostable, alkaline-active, keratinolytic proteinase from chrysosporium keratinophilum.

Thermostable alkaline proteinase was produced by a strain of Chrysosporium keratinophilum when cultured in lactose/mineral salt medium incorporating keratin solubilized with DMSO. The proteinase, partially purified by cold-acetone precipitation followed by gel-filtration on Sephadex G-75, was optimally active at pH 9 and stable from pH 7 to 10 with over 90% relative residual activity after incubation at 25°C for 24 h. The optimum temperature for enzyme activity was 90°C at which the activity half-life was 30 min. Enzyme activity was stimulated by Fe(2+) and inhibited by 1,10 o-phenanthroline. Gel-filtration indicated an M r of 69 kDa.

Production of extracellular collagenolytic proteinases by Histoplasma capsulatum var. duboisii and Histoplasma capsulatum var. capsulatum in the yeast phase.

Yeast cultures of Histoplasma capsulatum var. duboisii and H. capsulatum var. capsulatum in collagen containing defined, semi-defined and complex media produced extracellular collagenolytic proteinases, assayed using 4-phenylazo-benzyloxycarbonyl-L-propyl-L-leucyl- glycyl-L-propyl-D-arginine, a specific collagenase substrate. Significant levels of hydroxyproline were measured in the cultures and clear zones of hydrolysis were produced in collagen buffer agar by the crude enzyme preparations. Hydrolysis of casein and bovine serum albumin at pH 8 suggests the presence, in the crude enzymes, of multiple proteinases rather than a collagenase with broad substrate specificity since collagenolytic activity was not detected at pH 5 and above. Collagenolytic activities in the crude enzymes of both fungi were optimal at pH 4, 40 degrees C and were inhibited by EDTA, phosphoramidion and aprotinin indicating a metallo-serine nature. The molecular weights, estimated by column chromatography, were both 17 kD. The enzymes probably constitute a shared antigen. A probable role in the pathogenesis of histoplasmosis is discussed.

In vitro production of extracellular elastolytic proteinase by Histoplasma capsulatum var. duboisii and Histoplasma capsulatum var. capsulatum in the yeast phase.

Extracellular elastolytic proteinase was produced by yeast cultures of Histoplasma capsulatum var. duboisii and H. capsulatum var. capsulatum in three different induction media, assayed using elastin-orcein as the substrate. Medium-dependent variations in the time-course for enzyme production were observed and no peak was recorded. The proteinases hydrolysed both casein and bovine serum albumin indicating a broad substrate specificity. Both elastolytic proteinases had similar optimum pH (pH 8) and temperature (35 degrees C); over 90% residual elastolytic activity was measured in the crude enzymes of v. duboisii and v. capsulatum after incubation in the pH ranges 4.5-8.0 and 4.0-7.0 respectively, at 4 degrees C for 24 h. The proteinases were not significantly inhibited by any of the tested proteinase inhibitors. The molecular weights, estimated by column chromatography, were 23 kD. A probable role in the pathogenesis of histoplasmosis is discussed.

Potential pathogenicity of Cladosporium tennuisimum, Phaeoisaria clematidis and Ramichloridium subulatum in a mouse model.

The potential pathogenicity of one isolate each of Cladosporium tennuisimum, Phaeoisaria clematidis and Ramichloridium subulatum for mice was investigated by intravenous, intraperitoneal and subcutaneous routes of inoculation with saline and mucin suspensions of the organisms. No mice died during the experimental period. Dark nodular lesions were formed in the liver, spleen, kidneys, intestine, stomach, omentum and diaphragm after inoculation through the intravenous and intraperitoneal routes. Gross lesions were produced in the lungs of one mouse inoculated intravenously with R. subulatum. Localised nodular lesions were formed in the subcutis following the subcutaneous route. Tissue response was characterised by granulomatous inflammatory reaction. Fungal elements were confined within the granulomata. Though the fungi showed limited pathogenic potential, they may represent a hazard under conditions of compromised host immunity.

Experimental infection of chickens with Nocardia asteroides and Nocardia transvalensis.

Ten-day-old cockerels were infected with N. asteroides or N. transvalensis by the oral or intraperitoneal routes. Clinical signs included depression, gasping and emaciation. Grey nodules or foci were observed in the lungs, air sacs, liver and breast muscles. These organs showed necrosis, granulomas, lymphoid follicles and infiltration of lymphocytes, heterophils and macrophages. Intraperitoneal infection resulted in a more severe disease with either organism. N. transvalensis appeared more pathogenic than N. asteroides intraperitoneally and vice versa with the oral route.

Effect of infectious bursal disease virus infection on the severity of Aspergillus flavus aspergillosis of chickens.

Two groups of broiler chickens were infected with infectious bursal disease virus (IBDV) and Aspergillus flavus or A. flavus alone, respectively. Loss in weight was 2.5 times more severe in the IBDV + A flavus birds than in those infected with A. flavus alone. Mottling of liver with grey spots was observed only in the IBDV + A. flavus broilers. Histopathological sections of the lungs showed more necrosis, granulomas and fibrosis in the IBDV + A. flavus group, and the fungus was reisolated for a longer period from this group than from birds infected with A. flavus alone. These observations indicate that IBDV infection can increase the severity of A. flavus aspergillosis.

Pulmonary infections due to Aspergillus flavus in turkey poults and goslings.

Two outbreaks of pulmonary aspergillosis involving a flock of 66 turkey poults and a group of 12 gosling are described. The lungs showed multiple yellowish to greyish nodules. Histology demonstrated hyphae characteristic of Aspergillus and A. flavus was recovered in culture. Sections of liver showed features of aflatoxicosis. Cultures of samples of litter and feed yielded A. flavus in quantity. Representative isolates from lung lesions, litter and feed were found to produce aflatoxin B1.

Lipases of Fonsecaea pedrosoi and Phialophora verrucosa.

Lipase activity was demonstrable titrimetrically in the culture filtrates of Fonsecaea pedrosoi and Phialophora verrucosa on the 6th day of incubation reaching a peak on the 15th and 12th days respectively for the two fungi. Purified lipases of F. pedrosoi and P. verrucosa, with specific activities of 36.0 and 39.4 fold increase respectively, were obtained by cold acetone extraction, gel filtration followed by ion exchange chromatography. The lipases had the same optimum pH (5.5) and temperature (35 degrees C). The molecular weights of the lipases of F. pedrosoi and P. verrucosa, estimated by gel filtration on Sephadex G-100, were 25,000 and 20,000, respectively and the enzymes showed broad substrate specificity. The possible role of lipase in the pathogenesis of infection caused by the fungi is discussed.

Physiological characteristics of environmental isolates of pathogenic dematiaceous fungi.

A total of 39 environmental isolates of pathogenic dematiaceous fungi (Fonsecaea pedrosoi: 14 isolates, Phialophora verrucosa: 6, Cladosporium carrionii: 9, Exophiala jeanselmei: 2, Ramichloridium subulatum: 6, Cladosporium tenuissimum: 1 and Phaeoisaria clematidis: 1) were evaluated for their various physiological characteristics including the ability to produce extracellular enzymes. Significant physiological characteristics included the ability of isolates of all the species except C. tenuissimum to decompose tyrosine, lyse human red blood cells, conversion of R. subulatum from filamentous to yeast form when cultured on Czapek-Dox agar and also on blood agar and the growth of C. tenuissimum on Sabouraud dextrose agar incorporating 3.6 M NaCl. Isolates of all the species except those of C. carrionii produced lipase, while only the isolates of P. verrucosa, E. jeanselmei and Ph. clematidis were positive for phospholipase.

Clinical and pathological features of Aspergillus fumigatus infections in poultry in southern Nigeria.

This study was undertaken to supply information on Aspergillus fumigatus infection of poultry in Nigeria. The disease in broiler chicks was characterized by gasping, droopiness, emaciation and heavy mortality while affected grower chickens showed emaciation, weakness, diarrhoea and 17 per cent mortality. The disease was sporadic in laying flocks. Granulomatous nodules were observed in birds that died in each outbreak. The nodules were numerous and affected mainly the lungs and thoracic air sacs in the broiler chicks while only few large nodules were observed mainly in the abdominal air sacs in the layers.