Blastomyces dermatitidis Lysate Antigens: Antibody Detection in Serial Serum Specimens from Dogs with Blastomycosis

Yeast phase lysate antigens prepared from different isolates of Blastomyces dermatitidis (T-58, dog-Tennessee; T-27, polar bear-Tennessee; ERC-2, dog-Wisconsin; ER-3, woodpile-Wisconsin) were compared with respect to the detection of antibodies (indirect enzyme-linked immunosorbent assay-ELISA, peroxidase system) in 126 serial serum specimens (pre-treatment, 30 and 60 days post-treatment with itraconazole) from 42 dogs with diagnosed blastomycosis. Mean absorbance values observed with the four lysate antigens at the three treatment intervals ranged from the most reactive to the least reactive as follows: T-58 (0.270, 0.210, 0.136); T-27 (0.209, 0.156, 0.096); ER-3 (0.189, 0.144, 0.089) and ERC-2 (0.158, 0.129, 0.080). Even though variations in reactivity were evidenced, the lysates prepared from isolates from various geographical regions and sources were all efficacious as antigens for the immunodiagnosis of canine blastomycosis.     

Detection of antibody responses and delayed dermal hypersensitivity with Blastomyces dermatitidis yeast and mycelial lysate antigens

Yeast cell lysate and mycelial lysate antigens prepared from one strain (T-58) of Blastomyces dermatitidis were evaluated with respect to the detection of antibodies and delayed dermal hypersensitivity. Comparable ELISA sensitivity values were evidenced with the two antigens when assayed against serum specimens from dogs with blastomycosis, sera from non-infected dogs residing in endemic and nonendemic areas for blastomycosis and sera from rabbits that were hyperimmunized with B. dermatitidis antigens. Specificity determinations with anti -Histoplasma capsulatum rabbit sera indicated that both reagents exhibited only minimal cross-reactivity; the mycelial antigen was slightly more specific than the yeast phase reagent. Similar sensitivity and specificity results were experienced when the two antigens were used to detect delayed dermal hypersensitivity in guinea pigs previously sensitized with B. dermatitidis or H. capsulatum.     

Secondary Intracerebral Blastomycosis with Giant Yeast Forms

Secondary central nervous system (CNS) blastomycosis is an unusual manifestation of blastomycosis. We report a case of recurrent intracerebral blastomycosis that presented histopathologically with giant yeast-like cells and multinucleation that mimicked Coccidioides immitis. The yeast forms of Blastomyces dermatitidis usually range in size from 8 to 20 μm in diameter. Large or giant yeast forms (20–40 μm) are rare. The four cases previously reported in the literature involving giant yeast cell forms of B. dermatitidis are reviewed here. Intracerebral blastomycosis should be suspected in patients with signs and symptoms of CNS lesions and histories of primary blastomycosis, or treatment with corticosteroids, or comprised immune systems. The diagnosis should be confirmed by culture which presents typical biphasic microbiologic features.     

Serological differences in two Blastomyces dermatitidis isolates from different geographical regions of North America

Yeast phase lysate antigens were prepared from two isolates (T-58 and ERC-2) from different geographic locations, Tennessee and Wisconsin. These lysate were evaluated with respect to their ability to detect antibody in dogs infected with blastomycosis and rabbits immunized with the lysates by an enzyme linked immunosorbent assay (ELISA). Both the dog sera and rabbit sera assays demonstrated that there were serological differences in these two isolates, which implied that there was antigenic variance in geographical populations of B. dermatitidis. These results correlated with a previous molecular study that indicated that there are genetic differences in different geographical populations of the organism. This revised version was published online in June 2006 with corrections to the Cover Date.     

A new atomic absorption spectral assay for the determination of trace IgG using immunonanogold

Nanogold in size of 10 nm was used to label goat anti-human IgG (GIgG) to obtain an immunonanogold probe (AuGIgG) for IgG. In pH 6.8 phosphate buffer solution and in the presence of immunoprecipitator polyethylene glycol 6000 (PEG 6000), IgG reacted with the probe (AuGIgG) to form AuGIgG–IgG–PEG immunocomplex. After the centrifugation to remove the immunocomplex, AuGIgG in the supernatant can be measured by atomic absorption spectrophotometry at gold absorption line 242.8 nm. The results showed that the absorption value decreased as the concentration of IgG increased, and the decreased absorption value was linear to IgG concentration in the range 0.025–0.375 μg/mL, with a detection limit of 0.008 μg/mL. On this base, a new nanogold-labeled atomic absorption spectral assay for IgG was established. The assay was applied to determine IgG in human serum sample with satisfactory results.     

A novel highly boron tolerant bacterium, <Emphasis Type="Italic">Bacillus boroniphilus</Emphasis> sp. nov., isolated from soil,that requires boron for its growth

Three strains of gram-positive, motile, rod-shaped and boron (B)-tolerant bacterium were isolated from naturally B containing soil of Hisarcik area in the Kutahya Province, Turkey. The strains, designated as T-14A, T-15Z^T and T-17s, produced spherical or ellipsoidal endospores in a terminal bulging sporangium. The strains required B for the growth and can tolerate more than 450 mM B. These also tolerated up to 7.0% (w/v) NaCl in the presence of 50 mM B in agar medium but grew optimally without NaCl. The temperature range for growth was 16–37°C (optimal of 30°C), whereas the pH range was 6.5–9.0 (optimal of 7.5–8.5). The DNA G + C content was 41.1–42.2 mol% and the predominant cellular fatty acid was iso-C_15:0. The major respiratory quinone system was detected as MK-7 and the diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis of 16S rRNA gene sequences data and DNA–DNA re-association values, we concluded that the three strains belong to a novel species of the genus Bacillus, the type strain of which is T-15Z^T and for which we proposed the name, B. boroniphilus sp. nov. (DSM 17376^T = IAM 15287^T = ATCC BAA-1204^T).     

Isoelectric focusing and ELISA evaluation of a <Emphasis Type="Italic">Blastomyces dermatitidis</Emphasis> human isolate

Blastomyces dermatitidis is a dimorphic fungal organism and the causative agent of blastomycosis. This organism is endemic east of the Mississippi river as is the fungal organism Histoplasma capsulatum. This study was performed to determine if sensitive and specific antigens from the B. dermatitidis yeast phase lysate (human isolate 592) could be separated using isoelectric focusing (IEF) to eliminate antigens that are cross-reactive with H. capsulatum. Indirect enzyme linked immunosorbent assays were performed to test for reactivity and cross-reactivity and indicate that certain fractions (4–6) were highly reactive. Fraction 16 exhibited a high degree of cross-reactivity with H. capsulatum. This study indicates that IEF may be a useful method for the separation of B. dermatitidis proteins.     

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev)

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66–117 μg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75–91% of total carotenoids under various regimes of growth. β-Carotene varied in the range 5–23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and β-carotene. The anticipated metabolic precursor γ-carotene (β,ψ-carotene) constituted a minor fraction (≤8%) under all conditions of growth.     

T-2 and HT-2 toxin analysis in cereals and cereal products following IAC cleanup and determination via GC-ECD after derivatization

The authors present a new and sensitive method for the determination of T-2- und HT-2 Toxin in cereals and cereal products in the low ppb level. A representative part of the cereal sample is extracted with a mixture of methanol-water (90:10) and the extract is cleaned on the commercially available immunoaffinity column T-2test™ (IAC), eluted with methanol, derivatized by pentafluorpropionic anhydride (PFPA) and measured on a GC-ECD. The method has been successfully validated on wheat, rye and oats. The recovery rates with wheat and rye endowed on a level of 50 ppb and with 85 ppb naturally contaminated oats were 71–115% with a coefficient of variation of 5.7–19.5%. The detection limits of the method with a signal to noise level of 3:1 were 1.5–2.3 μg/kg for HT-2 and 1.1–1.7 μg/kg for T-2 toxin. Financial support: Federal Ministry of Food and Agriculture (part of the project 05HS 001 — Improvement and validation of type A trichothecene (T-2 toxin and HT-2 toxin) analysis and occurrence of these mycotoxins in food marketed in Germany)     

Occurrence ofAlternaria andFusarium mycotoxins in winter wheat from domestic crop in year 2003

The aim of this study was a monitoring of the occurrence ofAlternaria andFusarium mycotoxins in winter wheat from domestic crop in the year 2003. Altenuene was determined in 56 (100%) samples of winter wheat, range 14.5–41 μg/kg, mean 25 μg/kg. Alternariol was determined in 16 (28.6%) samples of winter wheat, range 6.3–22.1 μg/kg, mean 5.7 μ/kg. DON was determined in 42 (100%) samples of winter wheat, range 250–3500 μg/kg, mean 330 μg/kg. T2-toxin was determined in 42 (100%) samples of winter wheat, range 25–337 μg/kg, mean 99 μg/kg. ZEA was not determined in samples of winter wheat. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germary, May 17–19, 2004 Financial support. Supported (one part of experiments, the determination of Fusarium mycotoxins) by the Ministry of Agricu ture of the Czech Rebublic (Propect No QF3121)     

<Emphasis Type="Italic">Blastomyces Dermatitidis</Emphasis> Antigen Detection in Urine Specimens from Dogs with Blastomycosis Using a Competitive Binding Inhibition ELISA

A competitive binding inhibition enzyme linked immunosorbent assay (ELISA) was used to detect Blastomyces dermatitidis antigens in urine specimens from dogs with blastomycosis. Sera from rabbits immunized with B. dermatitidis killed whole yeast cells were used as the primary antibody in the competitive ELISA. This initial study was performed to determine if B. dermatitidis antigen detection was possible and to test the efficacy of the rabbit sera as a primary antibody. An indirect ELISA was also performed to compare antigen detection in urine to antibody detection in the sera of the infected dogs. The results indicate 100% (36/36 specimens) detection of both antigen and antibody. Cross reactivity with Histoplasma capsulatum, as well as non-specific binding with the normal urine specimens, was observed with the competitive binding inhibition ELISA.     

Experimental design for the production of tensio-active agent by <Emphasis Type="Italic">Candida lipolytica</Emphasis>

The strategy of optimization using sequential factorial design was employed to enhance the tensio-active emulsifying agent produced by Candida lipolytica using soybean oil refinery residue as substrate. A full factorial design was used to evaluate the impact of three fermentation factors—amounts of refinery residue, glutamic acid and yeast extract. This allowed exclusion of the yeast extract. Full factorials designs were then sequentially used to optimize the levels of the residue and glutamic acid. The surface tension value was finally reduced to 25.29 mN/m. The maximum emulsifier activity using different substrates was within 40 h of cultivation. The surface tension of the cell-free broth containing the biosurfactant remained very stable during exposure to a wide range of pH (2–12), temperatures (0–120°C) and salinity (2–10% NaCl). The combination of an industrial waste and a cheap substrate therefore seems to be very promising for the low-cost production of potent biosurfactant.     

Study of sugarcane pieces as yeast supports for ethanol production from sugarcane juice and molasses

Due to the environmental concerns and the increasing price of oil, bioethanol was already produced in large amount in Brazil and China from sugarcane juice and molasses. In order to make this process competitive, we have investigated the suitability of immobilized Saccharomyces cerevisiae strain AS2.1190 on sugarcane pieces for production of ethanol. Electron microscopy clearly showed that cell immobilization resulted in firm adsorption of the yeast cells within subsurface cavities, capillary flow through the vessels of the vascular bundle structure, and attachment of the yeast to the surface of the sugarcane pieces. Repeated batch fermentations using sugarcane supported-biocatalyst were successfully carried out for at least ten times without any significant loss in ethanol production from sugarcane juice and molasses. The number of cells attached to the support increased during the fermentation process, and fewer yeast cells leaked into fermentation broth. Ethanol concentrations (about 89.73–77.13 g/l in average value), and ethanol productivities (about 59.53–62.79 g/l d in average value) were high and stable, and residual sugar concentrations were low in all fermentations (0.34–3.60 g/l) with conversions ranging from 97.67–99.80%, showing efficiency (90.11–94.28%) and operational stability of the biocatalyst for ethanol fermentation. The results of this study concerning the use of sugarcane as yeast supports could be promising for industrial fermentations. L. Liang and Y. Zhang have contributed equally to this work.     

Growth kinetics and Pho84 phosphate transporter activity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under phosphate-limited conditions

The effect of phosphate (P _i ) concentration on the growth behavior of Saccharomyces cerevisiae strain CEN.PK113-5D in phosphate-limited batch and chemostat cultures was studied. The range of dilution rates used in the present study was 0.08–0.45 h⁻¹. The batch growth of yeast cells followed Monod relationship, but growth of the cells in phosphate-limited chemostat showed change in growth kinetics with increasing dilution rates. The difference in growth kinetics of the yeast cells in phosphate-limited chemostat for dilution rates below and above approximately 0.2 h⁻¹ has been discussed in terms of the batch growth kinetic data and the change in the metabolic activity of the yeast cells. Immunological detection of a C-terminally myc epitope-tagged Pho84 fusion protein indicated derepressive expression of the Pho84 high-affinity P _i transporter in the entire range of dilution rates employed in this study. Phosphate transport activity mediated by Pho84 transporter was highest at very low dilution rates, i.e. 0.08–0.1 h⁻¹, corresponding to conditions in which the amount of synthesized Pho84 was at its maximum.     

Geographic Distribution of Human Blastomycosis Cases in Milwaukee, Wisconsin, USA: Association with Urban Watersheds

Most studies of endemic blastomycosis and outbreaks have involved rural areas. Case homesites in rural Northern Wisconsin have been associated with waterways and sand soils. ARC-GIS was used to geocode addresses and to observe geographic features of homesites from 45 State-mandated reports of human blastomycosis in urban Milwaukee County, Southeastern Wisconsin 2000–2004. Each case property was directly observed, and houses and duplexes (N = 38) were compared with 151 same-street control homesites. Categorical data was analyzed using a chi-square or Fisher’s exact test; continuous variables by Kruskal–Wallis test. One case cluster was seen on Milwaukee’s North side where the estimated annual incidence was 2.8/100,000 compared to 0.96/100,000 for the entire county. Cases were less common in the most urbanized watersheds (0.49/100,000/yr) versus Lake Michigan shores (0.85) versus remaining three open watersheds (1.4) [P<0.01]. Case homesites averaged 1067 m to waterways and none were on sand soils. (Comparison is made to a Northern Wisconsin community where case homesites averaged 354 m to waterways, 24/25 were on sand soils and annual incidence was 74/100,000.) No unique features of case homesites were identified in Milwaukee County. In this urban area of Wisconsin, relatively low incidence rates may be explained, in part, by lower density of inland waterways and lack of sand soils, however, blastomycosis cases appear to be associated with open watersheds.     

Clinical Characteristics and Outcomes in Patients with Pulmonary Blastomycosis

Background  Blastomycosis is an uncommon granulomatous infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. The most frequent clinical infections involve the lung, skin, and bone. Pulmonary manifestations range from asymptomatic self-limited infection to severe diffuse pneumonia causing respiratory failure. Objectives  To establish the clinical characteristics and outcomes of patients with pulmonary blastomycosis diagnosed at hospitals in Manitoba and northwestern Ontario, Canada. Methods  A retrospective review of medical records was done for 318 patients with blastomycosis in these regions. Results  The majority of patients were Caucasian (198 (62.5%) patients), male (193 (61%) patients), and residents of Ontario (209 (65.7%) patients). Most patients were treated in an inpatient hospital ward (266 (84%) patients) and survived (294 (92%) patients). Pulmonary involvement, either alone or associated with other sites, was present in 296 (93%) of the 318 patients; 22 (7%) patients had no evidence of pulmonary blastomycosis. The majority of patients had localized lung disease (1–3 quadrants on chest radiograph involved; 225 (82%) patients). Of 294 (92%) patients requiring hospitalization, 266 (90%) patients received all inpatient care on a general medical ward; 28 (10%) patients received some care in the intensive care unit (ICU). Factors associated with ICU admission included diffuse pulmonary disease (four quadrants involved on chest radiograph), diabetes, and prior use of antimicrobial therapy. Twenty-four (8%) patients died, and multivariate analysis showed that older age and Aboriginal ethnicity were the significant risk factors for death from blastomycosis. Conclusion  Blastomycosis is a cause of serious, potentially life-threatening pulmonary infection in this geographic region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A survivor of breast cancer with immunity to MUC-1 mucin, and lactational mastitis

 The human mucin, MUC-1, is a transmembrane glycoprotein that is produced by both normal an malignant epithelium. The MUC-1 produced by malignant epithelium is underglycosylated, which leads to the expression by tumors of novel T and B cell epitopes on the mucin polypeptide core. Similar underglycosylation occurs in the lactating breast. In this report, we describe a long-term survivor of breast cancer whose tumor strongly expressed the T- and B-cell-stimulatory epitopes. Five years after presenting with the tumor, the patient had her first pregnancy, at which time she developed fulminant lymphocytic mastitis. We demonstrate that the lactating breast produced mucin expressing the same “tumor-specific” epitopes as the original cancer. The patient had circulating anti-mucin antibodies of both the IgM and IgG isotypes (these are not found in normal controls), and mucin-specific cytotoxic T lymphocytes in the peripheral blood. Limiting  –  dilution analysis for mucin  –  specific cytotoxic T lymphocytes in three different experiments yielded frequencies of 1 in 3086, 1 in 673, and 1 in 583, compared to approximately 1 in 10⁶ in normal controls. The patient remains clinically free of carcinoma after 5 additional years of follow-up. We propose that the original tumor primed the patient’s immune response against the mucin epitopes, and that the re-expression of these epitopes on the lactating breast evoked a secondary immune response. It is tempting to speculate that the vigor of her anti-mucin immunity may have helped protect this patient against recurrent tumor. Received: 12 February 1996 / Accepted: 5 November 1996     

Mycotoxins in horse feed

A total of 62 samples of commercial horse feed preparations (complementary feeds) containing cereal mixtures (“muesli” or mash, n = 39; pelleted feeds, n = 12), and plain horse feed grains (maize, n = 5; oats, n = 4; barley, n = 2) were purchased from 21 different producers/distributors from the German market. All samples were analysed by competitive enzyme immunoassays (EIA) for six different mycotoxins (mycotoxin groups). Analytes (detection limit, mean recovery) were: deoxynivalenol (DON, 10 μg/kg, 84%), zearalenone (ZEA, 5 μg/kg, 93%), fumonisin B₁ (FB₁, 2 μg/kg, 113%), T-2 toxin (T-2, 0.1 μg/kg, 71%), sum of T-2 + HT-2 toxin (T-2/HT2, 0.2 μg/kg, 97%), ochratoxin A (OTA, 0.2 μg/kg, 67%), and total ergot alkaloids (Generic Ergot Alkaloids “GEA”, 30 μg/kg, 132%). All samples contained DON (16–4,900 μg/kg, median 220 μg/kg), T-2/HT-2 (0.8–230 μg/kg, median 24 μg/kg), and T-2 (0.3–91 μg/kg, median 7 μg/kg). ZEA was detected in 98% of the samples (7–310 μg/kg, median 61 μg/kg). Most samples (94%) were positive for FB₁ (2–2,200 μg/kg, median 27 μg/kg). Ergot alkaloids were detected in 61% of samples (28–1,200 μg/kg, median 97 μg/kg), OTA was found in 42% of samples (0.2–4 μg/kg, median 0.35 μg/kg). The results demonstrate that a co-contamination with several mycotoxins is very common in commercial horse feed from the German market. The toxin concentrations were in most cases well below the levels which are usually considered as critical or even toxic. The highest mycotoxin concentrations were mostly found in single-grain cereal feed: the maximum values for DON and FB₁ were found in maize, the highest T-2/HT-2 toxin concentrations were found in oats, and the highest concentration of ergot alkaloids was found in barley. In composed feeds, no correlation between cereal composition and mycotoxin levels could be found.     

Imaging and determining friction forces of specific interactions between human IgG and rat anti-human IgG

Covalently immobilized rat anti-human immunoglobulin (IgG) monolayers on thiol-modified gold substrates and human IgG linked with the tips were fabricated using the self-assembled monolayer method, and interactions between these systems were studied by friction force microscopy (FFM). In addition to observation of distinct nanostructures of protein monolayers due to recognition events, FFM also quantified the friction force due to protein–protein-specific interactions. The average friction force due to interactions between the antigen functionalized tip and the antibody monolayer was determined as 200–250 pN, significantly greater than that between either the bare tip and the antibody monolayer (0–50 pN), or the blocked antigen tip and the antibody monolayer (50–100 pN), indicative of antigen/antibody-specific interactions. These results, taken together, suggest that FFM is not only capable of tracking recognition events, but also quantifying the friction force due to specific interactions between biological molecules, such as antigen and antibody.