Diagnosis of blastomycosis is often done using a combination of clinical signs and cytologic or histopathologic identification of the organism, Blastomyces dermatitidis, from infected tissues. However, these methods are time consuming, invasive, and still lead to misdiagnosis. A competitive enzyme-linked immunosorbent assay (ELISA) can be used for detection of B. dermatitidis antigens, which are present in urine specimens of infected patients. The current study evaluates the use of various antibodies for detection of antigen in dog urine specimens, to provide a better diagnosis of blastomycosis in the future. Our results show that different antibodies against B. dermatitidis produce various sensitivities for antigen detection. The most realistic antibodies for immunodiagnostic tests would be antibodies that can be obtained in larger quantities, i.e. vaccination using a yeast lysate in a laboratory setting. We found that these antibodies produce a comparable and reliable result to that of antibodies obtained from an infected patient.
Blastomycosis is a fungal infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. B. dermatitidis grows in moist, slightly acid areas where rich organic matter is present [1,2]. In this environment it grows in a mycelial form and produces microscopic spores known as conidia. B. dermatitidis is common to areas of North American including the Great Lakes, parts of Canada, and states ranging from Arkansas to North Carolina [3-7]. A mammal may inhale the conidia from the environment, particularly if there is a disturbance of the soil. Inside the warm, moist environment of the lung, the conidia will convert into the yeast phase of B. dermatitidis [
Diagnosis of blastomycosis is often done by a combination of clinical signs and cytologic or histologic identification of the organism [6,9]. Clinical signs are often slow to appear in patients, not revealing a problem until the disease has spread to additional parts of the body. These signs can include fever, anorexia, coughing, dyspnea, ocular and nasal discharge, loss of body condition, cutaneous lesions, and lameness [
Due to the invasiveness, time used for growth and testing, and often misdiagnosis of blastomycosis; there is a continual drive to develop new tests that will provide rapid and reliable results. Antigen testing may be a useful way of diagnosing blastomycosis. Antigen detection has been used to identify histoplasmosis, a fungal disease caused by H. capsulatum [
A competitive enzyme-linked immunosorbent assay (ELISA), shown in
In addition to a competitive ELISA, some assays involve the use of antibody detection from serum specimens of infected patients [13-15]. As the immune system begins to combat the B. dermatitidis infection, cells will produce antibodies that are specific to the yeast antigens. Generally these antibody levels are high enough to be detected by a variety of sensitive and specific assays [13, 14]. With immunocompromised patients, there is commonly a much lower level of antibody specific to B. dermatitidis cells that is difficult to detect using this assay. In these cases a different type of assay is required for the diagnosis of blastomycosis, such as the detection of antigen from urine specimens. Therefore the use of a competitive ELISA antigen detection assay may provide reliable results for the immunodiagnosis of blastomycosis
in humans and animals.
Antibodies against B. dermatitidis can be obtained from serum of infected patients or immunized individuals. Ideally, serum obtained from an immunized animal would be able to provide large quantities for lab testing. This, however, may not be as reliable at detecting B. dermatitidis. This study evaluates the uses of antibodies obtained from infected dog serum specimens as well as antibodies from immunized rabbits in the detection of antigen from dog urine specimens with a competitive ELISA.
The dog urine specimens were provided by A. M. Legendre (University of Tennessee College of Veterinary Medicine, Knoxville, TN). Urine specimens were from several dogs at various stages of blastomycosis from several locations within endemic areas of North America. These urine samples were taken prior to treatment of the dogs. Each urine specimen is designated with a value for each test run. These specimens vary for each experiment, meaning the Specimen A in
Serum containing antibodies against B. dermatitidis were collected previous to this study from New Zealand White rabbits. Rabbits were immunized with a yeast lysate of B. dermatitidis. In addition, a number of serum specimens from dogs with blastomycosis were provided by Dr. A. M. Legendre (University of Tennessee College of Veterinary Medicine, Knoxville, TN). Dog serum specimens were from dogs at different stages of blastomycosis, and were all taken prior to antifungal treatment. Serum was used in various dilutions, the most common
being 1:1000; then when added to the urine specimen reached a final dilution of 1:2000. Dilutions were performed using phosphate buffered saline containing 0.15% Tween 20 (PBS-T).
Serum specimens contain antibody were diluted to various amounts then added to urine specimens. This mixture was allowed to incubate from 30 minutes at 37˚C. For a control and baseline for detection, PBS-T was used in place of urine. Samples were then added in triplicate to a Costar 96-well microplate (Thermo-Fisher Scientific; Waltham, MA); which was coated with B. dermatitidis antigen (2000 ng per ml) diluted in a carbonate-bicarbonate coating buffer (pH 9.6) and incubated overnight at 4˚C in a humid chamber followed by washing three times with phosphate buffered saline containing 0.15% Tween 20 (PBS-T). Following this addition, the plate was incubated at 37˚C for 30 minutes. After incubation, wells were washed as above and 100 μl of goat anti-species (either dog or rabbit) IgG (H&L) peroxidase conjugate was added to each well and incubated for 30 minutes at 37˚C. The plate was washed again as above and 100 μl of peroxidase substrate was added to each well and incubated for approximately 2 minutes at room temperature. The reaction was then stopped by the addition of 2 N sulfuric acid. The absorbance was read at 450 nm using a BIO-RAD 2550 EIA reader (Hercules, CA).
Four different serum specimens were used from rabbits immunized with B. dermatitidis yeast lysates. To identify the quantity of serum needed for adequate antigen detection with the competitive ELISA, serum was first tested using a variety of dilutions. Serum specimens were diluted to 1:250, 1:500, 1:1000, and 1:2000. These were then added to dog urine specimens to make the final serum dilution 1:500, 1:1000, 1:2000, and 1:4000. These results are shown in
As the concentration of the antibody was decreased we see lower absorbance values for the control in addition to the urine specimens. This is due to the amount of antibody binding to the antigens on the plate and the antibody still in solution. As the concentration changes, the amount of antibody that binds to the plate will change based on equilibrium. Based on these results, additional tests were done using a final serum dilution of 1:2000. This showed greater antigen detection than other dilutions used.
Additional immunized rabbit sera were tested to identify an optimal serum for antigen detection, thus allowing for greater sensitivity of the competitive ELISA. Serum was used from rabbits immunized with a variety of yeast lysates made using different B. dermatitidis strains. Comparative analyses of these different serum specimens are shown in