Comparison of antibody detection with <i>Blastomycesdermatitidis</i> yeast lysate antigens in serum specimens from immunized rabbits and infected dogs

doi:10.4236/oji.2011.13009

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Vol.1, No.3, 74-79 (2011)

doi:10.4236/oji.2011.13009

Open Journal of Immunology

Comparison of antibody detection with

Blastomycesdermatitidis yeast lysate antigens in serum

specimens from immunized rabbits and infected dogs

Will Christenson, Rachel Horton, Kayla Campbell, Kelly Meacham*, A mber Schroeder,

Gene M. Scalarone

Department of Biological Sciences, Idaho State University, Pocatello, USA; *Corresponding Author: [email protected]

Received 9 September 2011; revised 18 October 2011; accepted 8 November 2011.

ABSTRACT

This present study w as designed to evaluate B.

dermatitidis antigens, prepared from two iso-

lates (B5896, 597), when the yeast cells were

allowed to lyse in distilled water for one day or

seven days. The indirect enzyme-linked immu-

nosorbent assay (ELISA) was used to determine

the ability of the lysate reagents to detect anti-

bodies in 30 rabbit and 30 dog serum speci-

mens. Mean absorbance values w ith B5896 lys-

ate antigen ranged from 1.637 (day 1) to 1.461

(day 7) and absorbance values w ith 597 antigen

ranged from 1.579(day 1) to 1.396 (day 7) with

the serum specimens from immunized rabbits.

Serum specimens from infected dogs yielded

absorbance values ranging from 1 .67 2 (day 1) to

1.763 (day 7) w ith the B5896 and values ranging

from 1.909 (day 1) to 1.224 (day 7) with the 597.

Optimal reactivity was obtained with the day 1

lysate using both lysate antigens against the

rabbit sera and with the 597 antigen against the

dog sera. Slightly greater reactivity was evi-

denced with the day 7 B5896 antigen when the

dog sera was tested. Comparative studies are

continuing in order to produce an optimal anti-

genic preparation for antibody detection in

blastomycosis.

Keywords: ELISA; Blastomy cesdermatitidis ;

Antibody Detection; Lysate Antigens

1. INTRODUCTION

Bla stomycesde rmatitidisis a thermally dimorphic fun-

gus that exists in a mycelial mold-like form in nature at

approximately 25˚C, but when incubated at 37˚C, it sur-

vives in the yeast form [1,2]. The organism exists in the

environment as a spore-producing mold and the spores

can be inhaled into the lungs where they develop into

budding yeast cells [3]. In humans, the typical route of

infection is inhalation of aerosolized conidia of B. der-

matitidis. From the respiratory tract, the developing

yeast form may disseminate throughout the body and

affect multiple organ systems, most commonly the lym-

phatic, skeletal, eyes and skin [4]. The disease can be

spread throughout the body in which cutaneous lesions

may occur. Up to 5 to 10 percent of disseminated cases

produce meningitis or epidural/cranial abcesses; if left

untreated, death may result in animals and in humans

[5-7]. Blastomycosis is endemic in North America, Af-

rica, India and parts of Europe. The majority of reported

cases are from North America, primarily in the Great

Lakes area as well as the Mississippi and Ohio River

Valley regions [8,9].

In recent years, there has been an adamant thrust in

understanding the proper diagnosis of this disease be-

cause blastomycosis is often misdiagnosed that can cre-

ate treatment delays [10-14]. As with many other dis-

eases, time is of particular importance in terms of diag-

nosis and treatment [15,16]. Current culture methods

may be effective, but very time consuming. Therefore

researchers have focused on the development of immu-

nodiagnostic assays in attempts to provide improved

methods for the clinical diagnosis of blastomycosis in

humans and animals [2,10,11,14-18]. Our laboratory has

developed various B. dermatitidisyeast phase lysate

preparations and utilized these antigens in the ELISA for

the detection of antibodies in serum specimens from

immunized or infected animals [10,11,13,15]. These

studies have indicated the potential of the yeast lysate

antigens as immunodiagnostic reagents, but additional

studies are required in order to produce an optimal anti-

gen for antibody detection.

The purpose of this present study was to compare the

ability of B. dermatitidisyeast cell lysate antigens, pre-

pared by lysing the cells in water for periods of 1 or 7

W. Christenson et al. / Open Journal of Immunology 1 (2 011) 74-79

7575

days, to detect antibodies in serum specimens from im-

munized rabbits or infected dogs. Most of our previous

comparative studies were performed with antigenic

preparations fo l l owi n g 7 day s of ly si s.

2. MATERIALS AND METHODS

2.1. Antigens

Mycelial phase cultures of two B. dermatitidisisolates

(B5896 and 597) from blastomycosis outbreaks in

Mountain Iron, Minnesota and Eagle River, Wisconsin

were converted to yeast cells by culturing at 37˚C on

BHI agar. Yeast phase lysate reagents were prepared by a

method similar to one that was previously used for the

production of antigen from Histoplasmacapsulatum and

modified in our laboratory for B. dermatitidislysate an-

tigen production [10,11,13,19,20]. The yeast phase cells

were grown for 7 days at 37˚C in a chemically defined

medium in an incubator shaker, harvested by centrifuga-

tion (700 ×g; 5 min), followed by washing with distilled

water, resuspend ed in distilled water and then allowed to

lyse for 1 or 7 days at 37˚C in water with shaking. The

preparations were centrifuged, filter sterilized, merthio-

late added (1:10,000) and stored at 4˚C for further use.

Protein determinations were performed on the lysates

using the BCA Protein Assay Kit (Pierce Chemical Com-

pany, Rockford, IL) and dilutions of the antigenic re-

agents to be used in the assays were based on protein

concentration.

2.2. Serum Specimens

Serum specimens (30) from rabbits that were previ-

ously immunized using lysate preparations of B. derma-

titidis and serum specimens (30) from dogs with blasto-

mycosis, provided by Dr. A.M. Legendre (University of

Tennessee College of Veterinary Medicine, Knoxville,

TN) were assayed. In addition 5 serum specimens from

rabbits immunized with Histoplasmacapsulatum killed

whole yeast cells were used in the assays to compare

cross reactivity with the 4 B. dermatitidis lysate antigens.

2.3. ELISA

The ability of each yeast lysate reagent to detect anti-

bodies in the above serum specimens was determined

using the indirect biotin-streptavidin enzyme-linked

immunosorbent assay (ELISA). Each lysate antigen was

diluted (2000 ng of protein) in a carbonate-bicarbonate

coating buffer (pH 9.6) and then added to triplicate wells

(100 ul) of a Costar 96-well microdilution plate

(Thermo-Fisher Scientific) and incubated overnight at

4˚C in a humid chamber followed by washing three

times with phosphate buffered saline containing 0.15%

between 20 (PBS-T). The serum specimens (1:2500 di-

lution; 100 ul) were added to the microplate wells and

incubated for 30 min at 37˚C. Following this incubation

the wells were washed as above and 100 ul of goat

anti-rabbit or goat anti-dog IgG (H & L) biotin labeled

antibody (Kirkegaard and Perry, Gaithersburg, MD) was

added to each well and incubated for 30 min at 37˚C.

The plates were washed as above and 100 ul of peroxi-

dase labeled streptavidin was added to each well and

incubated for 30 min at 37˚C The plates were again

washed as above and 100 ul of 1-step Ultra TMB-ELISA

peroxidase substrate (Thermo-Fisher Scientific) was

added to each well and incubated for approximately 2

min at room temperature. The reaction was stopped by

the addition of sulfuric acid and the absorbance read at

450 nm using a BIO-RAD 2550 EIA reader.

3. RESULTS

3.1. Comparison of Antibody Detection in

Rabbit Sera with the Day 1 and Day 7

L ysate Antigens

Thirty serum specimens from rabbits were immunized

with B. dermatitidis. Two trials were performed using

yeast lysate antigens (B5896 and 597) prepared on day 1

and day 7. Th e results of trial 1 (Figure 1) indicated that

the B5896 day 1 lysate antigen had absorbance values

ranging from 1.122 to 1.759 with a mean value of 1.533,

while the range of the day 7 lysate absorbance values

was 0.914 to 1.672 with a mean value of 1.269. The

second lysate antigen (597) on day 1 had absorbance

values ranging from 1.155 to 1.765 with a mean value of

1.488 and day 7 values ranged from 1.008 to 1.761 with

a mean value of 1.329. In trial 2 the absorbance value

range of day 1 lysate antigen (B5896) was 1.556 to

1.958 with a mean value of 1.740; in contrast the day 7

lysate had a range of 1.498 to 1.859 with a lower mean

value of 1.653. The second trial of the day 1 597 lysate

antigen had absorbance values ranging from 1.487 to

1.863 and a mean value of 1.669. The day 7 absorbance

values of the same antigen ranged from 1.176 to 1.856

with an overall lower mean value of 1.463 (Figure 2).

3.2. Comparison of Antibody Detection in

Dog Sera with the Day 1 and Day 7

L ysate Antigens

Thirty serum specimens from dogs with Blastomyco-

siswere also assayed, as above with the rabbit sera, with

the day 1 and day 7 lysate antigenic preparatio ns. In trial

1 (Figure 3) the B5896 day 1 lysate h ad abso rbance val-

ues ranging from 0.450 to 1.997 with a mean value of

W. Christenson et al. / Open Journal of Immunology 1 (2 011) 74-79

Figure 1. Trial 1 indirect ELISA mean absorbance values using 20 rabbit serum specimens with lysate antigens (B5896 and 597)

prepared on day 1 and day 7

Figure 2. Trial 2 indirect ELISA mean absorbance values using 10 rabbit serum specimens with lysate antigens (B5896 and 597)

prepared on day 1 and day 7.

Figure 3. Trial 1 indirect ELISA mean absorbance values using 20 dog serum specimens with lysate antigens (B5896 and 597) pre-

pared on day 1 and day 7

1.418 and the day 7 antigen values ranged from 0.529 to

2.205 with a greater mean value of 1.714 compared to

the day 1 lysate. Antigen 597 day 1 values ranged from

0.480 to 2.344 with a mean value of 1.765 whereas the

day 7 values ranged from 0.429 to 1.690 with a lower

mean value of 1.124 when compared to 597 (day 1).

Trial 2 data (Figure 4) indicated that the B5896 (day 1)

absorbance values ranged from 1.427 to 2.210 with a

mean value of 1.926 and the day 7 values ranged from

1.639 to 1.994 with a sligh tly lower mean value of 1.812.

Lysate antigen 597 (day 1) had an absorbance range of

1.630 to 2.210 with a mean value of 2.052; in contrast

day 7 lysate values ranged from 0.845 to 1.720 with a

much lower mean val ue o f 1. 32 3.

Openly accessible at

W. Christenson et al. / Open Journal of Immunology 1 (2 011) 74-79

7777

Figure 4. Trial 2 indirect ELISA mean absorbance values using 10 dog serum specimens with lysate antigens (B5896 and 597) pre-

pared on day 1 and day 7

3.3. Antibody Detectio n Mean Abs orbance

Values Obt ained with the Day 1 and Day

7 Lysate Antigens in Sera from Rabbits

and Dogs

The antibody detection resultswith the day 1 and day

7 lysatesfrom the two trials using sera from immunized

rabbits and infected dogs were combined for compara-

tive purposes (Figure 5). All 30 rabbit serum specimens

tested with B5896 lysate antigen showed a day 1 mean

value of 1.637 compared to the day 7 mean value of

1.461. Thirty dog serum specimens tested using the same

antigen indicated a day 1 value of 1.672 and day 7 value

of 1.763. The same 30 rabbit sera were tested using a

different yeast lysate antigen (597) which yielded a

mean value of 1.579 (day 1) while the day 7 lysate re-

sulted in a mean value of 1.396. Lastly, the same 30 dog

sera assayed with antigen 597 resulted in absorbance

values of 1.909 (day 1) and 1.224 (day 7).

3.4. Specificity Determinations with the B.

dermatitidislysate Antigens

Five serum specimens from rabbits that had been im-

munized with H. capsulatumwhole yeast cells were as-

sayed with both B. dermatitidis and H. capsulatum lysate

reagents in order to obtain initial evidence with regard to

specificity of the day 1 and day 7 lysate antigens. As

shown in Table 1, two of the B. dermatitidis prepara-

tions (Antigen 2: B5896, day 7 and Antigen 3: 597, day

1 exhibited a lower level of cros s reactivity than the An-

tigen 1: B5896 day 1 and Antigen 4: 597 day 7 when

compared to the reactivity evidenced with the homolo-

gous H. capsulatum antigens. As indicated above this

data was obtained with a limited number of H. capsula-

tumsera and further studies are required in order to as-

sess the specificity of the different lysate preparations.

4. DISCUSSION

The production and evaluation of B. d ermatitidis yeast

cell lysate antigenic reagents has been a major thrust of

our research endeavors for several years. Our initial re-

search work on the preparation of B. dermatitidis lysates

was based on previous investigations on the preparation

of lysate antigens for either the detection of antibod ies or

delayed dermal hypersensitivity in coccidioidomycosis

and histoplasmosis. In studies on the development of

skin-testing and serological antigens for coccidioido-

mycosis, Coccidioidesimmitis spherules were lysed in

distilled water for a period of 40 days [19,21,22]. In

contrast a one-day period of lysis of H. capsulatum yeast

cells was sufficient to produce a reagent that detected

both delayed dermal hypersensitivity as well as being

useful for the detection of antibodies in the ELISA

[19,20]. With regard to our work with B. dermatitidis

yeast phase lysate antigens many of our comparative

studies have been done with reagents following growth,

harvesting and allowing the cells to lyse for a p eriod of 7

days [10,11,13,15,23,24].

The primary objective of this present comparative

study was to compare yeast phase lysate reagents that

had been prepared following either 1 day or 7 days of

lysis in water. The lysate antigens were prepared from

human isolates of B. dermatitidis from outbreaks in

Mountain Iron, Minnesota (B5896) and Eagle River,

Wisconsin (597) and assayed using the ELISA to detect

antibodies in serum specimens from immunized rabbits

or infected dogs. The evidence indicated that the day 1

lysates from both isolates reacted optimally with regard

to detecting antibodies in the rabbit sera and the 597 day

1 lysate showed a greater degree of sensitivity than the

day 7 lysate when the dog sera were assayed. In contrast

the B5896 day 7 lysate exhi bited sli ght ly great e r reacti vit y

than the day 1 preparation with th e dog se rum specimens.

W. Christenson et al. / Open Journal of Immunology 1 (2 011) 74-79

Figure 5. Combined (trial 1 and trial 2) mean absorbance values using rabbit and dog serum specimens with lysate antigens (B5896

and 597) prepared on day 1 and day 7.

Table 1. H.capsulatumserum specimens were teste d with 4 B. dermatitidislysate antigens [B* = (1) B5896 (day 1); (2) B5896 (day 7);

(3) 597 (day 1); (4) 597 (day 7)] and with 4 H. capsulatum lysate antigens [H* = (1) G217B; (2) G217B; (3) G217A; (4) G217B].

Cross-reactivity study Antigen 1 Antigen 2 Antigen 3 Antigen4

B* 1.626 1.597 1.44 1.535

H* 1.713 1.942 1.83 1.785

Therefore when the data from the above sensitivity

comparisons and the specificity determinations against

the limited number of H. capsulatum serum specimens it

appeared that the day 1 597 lysate antigen was an opti-

mal reagent for antibody detection. These results indi-

cate the potential of the day 1 lysates and also provide

evidence for continuing comparative studies on the

preparation of such lysates from additional B. dermatiti-

dis isolates in an effort to develop lysate antigens with a

high degree of sensitivity and specificity for use in the

immunodiagnosis of Blastomycosis in humans and ani-

mals.

5. ACKNOWLEDGEMENTS

This research was supported by the Department of Biological Sci-

ences, Idaho State University.

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