Blastomyces dermatitidis 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.

Comparative serological evaluation of 10 Blastomyces dermatitidis yeast phase lysate antigens from different sources.

Yeast phase lysate antigens from 10 isolates of Blastomyces dermatitidis (dog, ERC-2 and T-58; T-27, polar bear; woodpile, ER-3; bat lung, 48938; human, B5929, B5895, B5896, B5931, and CAPP) from different geographical regions, in addition to a Histoplasma capsulatum (G217B) lysate preparation were compared with respect to their reactivity against serum specimens from dogs, rabbits and humans positive for blastomycosis using an indirect enzyme-linked immunosorbent assay. In addition, the lysate antigens were also assayed against histoplasmosis-positive human serum samples to study their cross-reactivity. Variable results were obtained with T-58 and T-27 exhibiting the greatest reactivity. We also noticed that the lysate did not react consistently to serum samples across species with lesser reactivity evidenced when testing dog sera. Finally, T-58 gave the highest cross-reactivity with histoplasmosis-positive sera. The study may prove valuable in the development of antigen candidates for blastomycosis serodiagnosis.

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.

Serological differences in three Blastomyces dermatitidis strains.

Yeast phase lysate antigens, prepared from three isolates of Blastomyces dermatitidis (T-58, Tennessee dog; 48089, Zaire human; ERC-2, Wisconsin dog) were assayed for their ability to detect antibodies in human sera, dog sera and sera from rabbits immunized with each of the lysate antigens. The dog sera were from animals diagnosed with blastomycosis from various endemic regions in North America. T-58 and ERC-2 lysate antigens exhibited a high reactivity with the serum from dogs infected with blastomycosis; however, 48089 lysate showed low reactivity with the same sera. With the immunized rabbit sera, 48089 lysate was the only lysate with a high reactivity with the 48089 serum and it exhibited little reactivity with the heterologous sera. The T-58 and ERC-2 lysate antigens reacted minimally with the 48089 serum but reacted highly with both the T-58 and ERC-2 sera. The human sera were from individuals potentially exposed to B. dermatitidis while working on a prairie dog relocation project in Colorado. Remarkably, all three lysate antigens could detect antibodies in the individuals diagnosed with blastomycosis. This study indicated that there were serological differences in the 48089 Zaire lysate compared with the other lysate antigens and it may be designated serotype 2.

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.

Comparison of seven Blastomyces dermatitidis antigens for the detection of antibodies in humans with occupationally acquired blastomycosis.

Yeast phase lysate antigens, prepared from seven isolates of Blastomyces dermatitidis (dogs, T-58, M-98; human, Le; soil, S; sea lion, SL; polar bear, PB; cat, C) were assayed by enzyme-linked immunosorbent assay (ELISA) to detect antibodies in human sera. The sera were from individuals potentially exposed to B. dermatitidis while working on a prairie dog relocation project in Colorado. All antigens exhibited greatest reactivity with three specimens in two separate assays. Absorbance values ranged from 0.370 (M-98) to 0.427 (LE) (Trial 1) and from 0.579 (M-98) to 0.714 (PB) (Trial 2) with serum 2; from 0.368 (C) to 0.453 (LE) (Trial 1) and from 0.565 (SL) to 0.694 (S) (Trial 2) with serum 3 and from 0.392 (M-98) to 0.506 (LE) (Trial 1) and from 0.557 (SL) to 0.758 (T-58) (Trial 2) with serum 6. The greatest reactivity was observed with sera from two persons (2 and 3 from the same individual and serum 6) who became ill with symptoms of pulmonary disease and sought medical care. Both patients were subsequently diagnosed with blastomycosis by microscopic and culture methods. The study indicated that all of the lysate antigens, regardless of source, could be used to reliably diagnose blastomycosis.

Genetic diversity and transcriptional analysis of the bys1 gene from Blastomyces dermatitidis.

Blastomyces dermatitidis, a pathogenic fungal organism, is able to exist in two different morphologies, a multicellular mycelium or a unicellular yeast, according to temperature, 25 degrees C and 37 degrees C respectively. The switching between morphologies must be accompanied by a cascade of signaling events in which expression of genes responsible for the change of morphology is increased or decreased. bys1, a gene from B. dermatitidis isolate #58, is expressed at high levels in the unicellular yeast, but gradually diminishes as the temperature is lowered and the organism converts to the mycelial phase where there is no transcription of bys1. We explored if bys1 homologs are found in other B. dermatitidis isolates and if the transcription of the homologs were regulated by temperature. bys1 was identified in all B. dermatitidis isolates tested and could be grouped into two classes by Southern blot, PCR, and DNA sequence. Although the bys1 transcripts of both classes were regulated by temperature, transcription rates varied between the three isolates tested.

Immunological and chemical characterization of glycoproteins in IEF fractions of Blastomyces dermatitidis yeast lysate antigen.

After isoelectric focusing (IEF), fractions of a Blastomyces dermatitidis yeast lysate antigen were analysed for the presence of glycoproteins that may lead to cross-reactivity in immunoassays for the diagnosis of blastomycosis. Five major glycoproteins were apparent, two of which showed cross-reactivity when used in Western blots with sera obtained from dogs with histoplasmosis and coccidioidomycosis. These five glycoproteins were characterized for linkage to the proteins using N-glycosidase F (NGF) and for their lectin binding properties. The cross-reactive 235- and 160-kDa glycoproteins were found to possess mainly O-linked, high-mannose-type carbohydrates, and periodate-mediated oxidation of these molecules eliminated cross-reactivity observed with heterologous sera. Thus, the periodate-treated IEF antigens described here may be useful in solid-phase enzyme immunoassays for the diagnosis of blastomycosis.

Comparative studies on the detection of antibodies and delayed hypersensitivity responses with 10 Blastomyces dermatitidis lysate antigens.

Yeast-phase lysate antigens were prepared from 10 different isolates of Blastomyces dermatitidis. Comparative studies were performed using the lysate antigens in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in sera from dogs with blastomycosis and histoplasmosis. In order to evaluate the ability of the lysate reagents to elicit delayed dermal hypersensitivity (DTH) responses, the lysates were compared as skin-testing antigens in hairless guinea pigs that were previously sensitized with B. dermatitidis or Histoplasma capsulatum killed whole yeast cells. All ten of the lysate reagents were able to detect antibody with the ELISA in the serum specimens from dogs with blastomycosis (absorbance values ranged from 0.184 to 0.272; mean value 0.235). In contrast, when the lysates were assayed against sera from dogs with histoplasmosis, the absorbance values ranged from 0.053 to 0.151, with a mean value of 0.092. All ten lysate antigens were able to elicit a DTH response in the B. dermatitidis-immunized animals (mean axes of induration values ranged from 7.0 to 14.4 mm; mean value 8.6 mm). On the other hand, only minimal reactivity was evidenced in the guinea pigs immunized with H. capsulatum (mean axes of induration values ranged from 0.8 to 2.9 mm; mean value 1.8 mm).

Detection of antibodies and delayed dermal hypersensitivity with different lots of Blastomyces dermatitidis yeast lysate antigen: stability and specificity evaluations.

Comparative evaluations were performed to assess the stability, sensitivity and specificity of eight lots of yeast lysate antigen prepared from a Blastomyces dermatitidis dog isolate (T-58). These antigens were prepared during the period from 1989 to 1995. The lysates were used in an ELISA for the detection of antibodies in serum specimens from dogs with blastomycosis and histoplasmosis. In order to evaluate the ability of the lysates to elicit delayed dermal hypersensitivity (DTH) responses, they were compared as skin-testing antigens in guinea pigs that were previously sensitized with B. dermatitidis or Histoplasma capsulatum killed whole yeast cells. All 8 of the lots of antigen detected antibody in the sera from dogs with blastomycosis (absorbance values ranged from 0.432 to 0.543; mean value of 0.508). The absorbance values ranged from 0.283 to 0.439 (mean value of 0.326) when the lysates were assayed against sera from dogs with histoplasmosis. All of the antigens were able to elicit a DTH response in B. dermatitidis immunized animals (mean axes of induration values ranged from 10.5 mm to 12.5 mm; mean value of 11.6 mm). In contrast, only minimal cross-reactivity was evidenced in the guinea pigs immunized with H. capsulatum (mean axes of induration values ranged from 0 to 4.5 mm; mean value of induration of 1.7 mm).

Induction and detection of cell-mediated reactions with different Blastomyces dermatitidis antigenic preparations.

Blastomyces dermatitidis yeast-phase antigens (killed cell, KWY; lysate, Lys; and filtrate, Fil) from canine isolate T-58 were compared with respect to the induction and detection of cellular immune responses in mice. The antigens exhibited good sensitivity and specificity when used to detect a delayed-type hypersensitivity (DTH) response in mice previously immunized with T-58 or Histoplasma capsulatum antigens. Greater reactivity was observed with the KWY and Lys antigens as DTH-inducing agents (immunogens) than with the Fil antigen. The antigens were also compared with regard to the induction and detection of a lymphoproliferative reaction using splenocytes from normal and sensitized mice, and optimal reactivity was demonstrated with the KWY and Fil antigen preparations both as immunogens (absorbance values 0.22-1.60 and 0.20-0.90, respectively) and as in vitro testing antigens (absorbance values 0.60-1.60 and 0.35-1.00 respectively) (P < 0.01). Peritoneal macrophages from mice sensitized with Fil and KWY antigens showed the greatest in vitro replication inhibition (RI) of B. dermatitidis yeast cells (RI values of 53% and 50% respectively) (P < 0.05). When mitogenic or antigenic lymphocytic supernatants were compared with respect to their ability to enhance the phagocytic activity of unelicited macrophages from normal mice to kill yeast cells, the T-cell mitogens (concanavalin A and phytohaemagglutinin) optimally activated the naive macrophages (45% and 44% RI respectively) compared with the B-cell mitogen (LPS) (23% RI) (P < 0.05). Similar results were obtained with the lymphocytic supernatants from mice immunized with KWY cells when activated using KWY or Fil antigens (46% and 40% RI respectively) (P < 0.05).

Detection of antibodies and delayed hypersensitivity with Rotofor preparative IEF fractions of Blastomyces dermatitidis yeast phase lysate antigen.

Blastomyces dermatitidis (dog isolate T-58) yeast phase lysate antigen was concentrated and separated by Rotofor preparative isoelectric focusing cell (Bio-Rad). The pH values of the fractions were determined and equilibrated to pH 7.2 and then analysed by enzyme-linked immunosorbent assay using horseradish peroxidase enzyme system against serum specimens from dogs with blastomycosis, histoplasmosis, aspergillosis, and coccidioidomycosis. The results showed a peak absorbance at pH 3.89-4.31 (fractions 4 and 5) with the blastomycosis serum specimens. This was a single sharp peak while the rest of the fractions were lower. In contrast the sera from dogs with histoplasmosis showed a peak absorbance at pH 5.54-5.97 (fractions 9 and 10), while the other mycoses showed patterns that did not resemble the blastomycosis or histoplasmosis specimens. Serum specimens from dogs with blastomycosis being treated with itraconazole were also assayed (pre-treatment and 1, 2, 3, and 12 months post-treatment sera). The characteristic peak for blastomycosis was observed and a decrease in the peak was seen as the treatment progressed. Fractions 3-12 were also used to detect delayed dermal hypersensitivity in hyperimmunized hairless guinea-pigs. Fraction 5 (pH 4.31) elicited the optimal response in B. dermatitidis-immunized animals, while no cross-reactivity was observed in guinea-pigs sensitized with Histoplasma capsulatum killed cells.

Sensitivity and specificity of an isoelectric focusing fraction of Blastomyces dermatitidis yeast lysate antigen for the detection of canine blastomycosis.

Blastomyces dermatitidis yeast lysate antigen (T-58, dog isolate) fractions prepared using the Rotofor preparative isoelectric focusing (IEF) cell (Bio-Rad) were compared with B. dermatitidis yeast lysate and filtrate reagents with respect to the detection of antibodies in sera from dogs with blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and aspergillosis. A horseradish peroxidase enzyme immunoassay with Turbo TMB substrate was used in the study. One particular IEF fraction (pH 4.3) was optimal in the assay, and it exhibited greater sensitivity (100%) and specificity (93%) than the lysate or filtrate preparations. The highest degree of cross-reactivity was encountered with the histoplasmosis and coccidioidomycosis specimens and considerably less with the cryptococcosis and aspergillosis sera. Studies are in progress to purify further the optimal IEF fraction.

Induction and detection of delayed dermal hypersensitivity in guinea-pig immunized with Blastomyces dermatitidis lysate and filtrate antigens.

Guinea-pigs were immunized using yeast phase antigens (lysate and filtrate preparations) from two strains of Blastomyces dermatitidis (T-58 and Le). Following a sensitization period, the animals were skin tested on days 40 and 216 using T-58 and Le yeast and mycelial phase lysate and filtrate antigen preparations for the detection of delayed dermal hypersensitivity (DTH). Using the Friedman's analysis of variance by rank test, significant differences were found in the efficacy of the immunogens to induce DTH in the animals when skin tested on both occasions (P < 0.05). Optimal reactivity was observed in guinea-pigs immunized with Le yeast lysate (mean axes of induration ranging from 12.0 to 18.8 mm and 7.0 to 18.5 mm on day 40 and 216, respectively) and T-58 yeast filtrate (mean axes of induration ranging from 7.5 to 18.0 mm and 8.0 to 17.0 mm on day 40 and 216, respectively) when the immunogens were administered with adjuvant. When the same data was analysed using the Friedman's test with regard to evaluating the efficacy of the skin test antigens to detect DTH, significant differences were found between them (P < 0.05) with optimal results using the Le mycelial filtrate and yeast lysate antigens (mean axes of induration ranging from 7.0 to 16.5 mm and 7.5 to 14.5 mm, respectively) when skin testing was done on day 40. When skin testing was done on day 216, the T-58 and Le mycelial lysate antigens gave optimal results (mean axes of induration ranging from 9.0 to 18.5 mm and 7.5 to 15.0 mm, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative studies on the detection of delayed dermal hypersensitivity in experimental animals with lysate and filtrate antigens of Blastomyces dermatitidis.

Yeast cell and mycelial lysate and filtrate antigens prepared in our laboratory from two strains of Blastomyces dermatitidis (canine isolate T-58 and human isolate Le) were evaluated with respect to the detection of delayed dermal hypersensitivity in hairless guinea pigs previously immunized with killed whole yeast cells from two strains of Blastomyces dermatitidis (T-58 and Le) and Histoplasma capsulatum (strain G-217A). The optimal potential reactivity (reactivity minus cross-reactivity) with regard to eliciting a dermal response in animals sensitized with B. dermatitidis was achieved with the yeast phase lysate and filtrate antigens prepared from both T-58 and Le isolates (mean axes of induration values ranging from 14.0 to 15.9 mm). In contrast, the mycelial phase reagents exhibited lower potential reactivity (mean axes of induration values ranging from 5.3 to 10.5 mm).

Comparative stability, sensitivity and specificity studies with different lots of Blastomyces dermatitidis yeast and mycelial lysate antigens.

Comparative studies were performed to assess the stability and lot-to-lot variation of Blastomyces dermatitidis yeast and mycelial phase lysate antigens. Four lots were prepared from each growth phase of B. dermatitidis strain T-58 (canine isolate) during a 14-month period. Serum specimens from dogs with blastomycosis, histoplasmosis, coccidioidomycosis, cryptococcosis and aspergillosis were assayed for antibody content using an alkaline phosphatase enzyme-linked immunosorbent assay (ELISA). The four lots of the yeast phase reagents were similar with respect to sensitivity and specificity, and the absorbance readings were approximately four times greater with sera from dogs with blastomycosis than with histoplasmosis or coccidioidomycosis. Even less cross-reactivity was evidenced when the sera from dogs with cryptococcosis and aspergillosis were assayed. In contrast, the four lots of the mycelial lysate reagents were considerably less reactive and more cross-reactive than the yeast phase antigens and, as above, the four reagents retained their activity after prolonged storage. Therefore the results indicated that the lysate antigens exhibited a great deal of stability and lot-to-lot variations in activity were not observed.

Comparative studies on the postantifungal effect produced by the synergistic interaction of flucytosine and amphotericin B on Candida albicans.

A turbidometric method was used to measure Candida albicans yeast cell growth and to quantitate the postantifungal effect (PAFE) after exposure to various concentrations of flucytosine and amphotericin B, alone and in combination, for 2 hr at 30 degrees C. The drug concentrations used in the PAFE assays were determined by initial MIC and FIC (fractional inhibitory concentration) evaluations. The PAFE was calculated by the difference in time (hr) required for growth of the control and test cultures to reach the 0.5 absorbance level following removal of the drug by dilution. A synergistic PAFE was evidenced with combinations of the two drugs at concentrations below their individual MICs. Combinations of flucytosine (0.012 to 0.049 micrograms ml-1) and amphotericin B (0.195 to 0.39 micrograms ml-1) produced PAFEs ranging from 6.3 to 21.8 hr. These PAFEs persisted from 0.3 to 14.7 hr longer than those achieved when each of the two agents was assayed separately.

The postantifungal effect of 5-fluorocytosine on Candida albicans.

The in-vitro postantifungal effect (PAFE) of 5-fluorocytosine for Candida albicans for short periods of time was investigated. Yeast cells were exposed for 0.5, 1 or 2 h to a range of concentrations (0.1-3.2 mg/L) of 5-fluorocytosine. The PAFE was quantitated by determinations of the number of colony forming units at hourly intervals (0-10 h) after removal of the drug by dilution. The length of the PAFE was dependent upon the concentration of 5-fluorocytosine and the duration of exposure. An exposure time of 0.5 h resulted in PAFE's ranging from 0 to 4.2 h. Exposure times of 1 and 2 h resulted in longer PAFEs and in many instances suppression of cell growth was seen for the entire evaluation period (up to ten hours).

Synergistic postantifungal effect of flucytosine and fluconazole on Candida albicans.

The in vitro efficacy of flucytosine and fluconazole, separately and in combination, with respect to induction of a postantifungal effect (PAFE) on Candida albicans was studied. PAFE refers to the persistent suppression of fungal cell growth following a short period of exposure to an antifungal agent. A turbidometric method was used to measure cell growth and to quantitate the PAFE following exposure of C. albicans yeast cells to different concentrations of the two agents for 2 h. The PAFE was determined by the difference in time (h) required for growth of the control and test cultures to increase to the 0.5 absorbance level following removal of the drug by dilution. Minimum (MIC) and fractional inhibitory concentration determinations were made and the data used for selecting the concentrations used in the PAFE evaluations. A synergistic interaction of the two drugs at concentrations well below their individual MICs was evidenced. Flucytosine:fluconazole ratios of 1:16-1:32 at concentrations ranging from 0.024-0.098 micrograms ml-1 and from 0.78-1.56 micrograms ml-1, with flucytosine and fluconazole, respectively, induced PAFEs which persisted for 2.5 h longer than those achieved when each of the two agents was assayed separately.