(2020) 51:119
Takeshige et al. Vet Res
https://doi.org/10.1186/s13567-020-00844-3

RESEARCH ARTICLE

Open Access

Adiaspore development and morphologicalcharacteristics in a mouse adiaspiromycosis
model
Asuka Takeshige1†, Mie Nakano1†, Daisuke Kondoh1†, Yuma Tanaka1, Akio Sekiya1, Takashi Yaguchi2,
Hidefumi Furuoka1 and Takahito Toyotome1,2,3*

Abstract
Lesions of adiaspiromycosis, a respiratory disease affecting wild animals, have been found mainly in dead mammals
and free-living mammals captured for surveillance. No report has described an investigation of adiaspore formation
progress in the lung. After establishing an experimental mouse model of intratracheal adiaspiromycosis infection withthe causative agent Emmonsia crescens, we observed adiaspore development. The spores grew and reached a plateauof growth at 70 days post-infection. The median adiaspore diameter showed a plateau of around 40 μm. The characteristic three-layer cell-wall structure of adiaspores was observed in the lung at 70 days post-infection. We examined
infection with a few spores, which revealed that adiaspores in the mouse lung progressed from intratracheal infectionof at least 400 spores. Moreover, we developed adiaspores in vitro by culture in fetal bovine serum. Although mostspores broke, some large spores were intact. They reached about 50 μm diameter. Thick cell walls and dense granules
were found as common points between in vitro adiaspores and in vivo adiaspores. These models are expected to be
useful for additional investigations of E. crescens adiaspores and adiaspiromycosis.
Keywords: adiaspiromycosis, adiaspore, Emmonsia crescens
Introduction
Emmonsia crescens (Ajellomyces crescens), a dimorphic
fungus found in soil worldwide [1], is known to be thecausative agent of adiaspiromycosis, a pulmonary disease causing granulomatous lesions in humans [2] andanimals, especially in small mammals [1–7] and rarely inlarge animals [8, 9]. Although the prevalence of adiaspiromycosis in animals remains unknown, Borman reported
adiaspiromycosis in 28.7% of free-living wild mammals
from the southwestern UK [10]. Reported human casesof adiaspiromycosis are few, with disease courses varying*Correspondence: tome@obihiro.ac.jp†
Asuka Takeshige, Mie Nakano and Daisuke Kondoh contributed equallyto this study
1
Department of Veterinary Medicine, Obihiro University of Agriculture
and Veterinary Medicine, Inada‑cho, Obihiro, Hokkaido 080‑8555, Japan
Full list of author information is available at the end of the article

from mild to fatal [2]. Studies have been done of wild
animals captured with traps or found dead incidentally,
then subsequently diagnosed as having adiaspiromycosis
during necropsy. Because no report of the literature has
described a study of the disease course from spore acquisition to mature adiaspore formation in the lung, related
details have remained unknown.
At around 25 °C, E. crescens grows as hyphae. However, the fungus forms adiaspores in mammalian hosts
at around 37 °C [11]. A few infection models have beenreported. An inhalation model has been used with rabbits [12] to analyze humoral immunological responses.
Experimental mouse models of adiaspiromycosis using
intraperitoneal infection routes have been describedin the literature [4, 13, 14]. The models used neither an
intranasal nor an intratracheal route. Adiaspore enlargement dynamics have been examined in mice using an© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material
in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material
is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds thepermitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat​iveco​
mmons​.org/licen​ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Takeshige et al. Vet Res(2020) 51:119

intraperitoneal model, but those circumstances differ
markedly from those in the lung. For the present study,
after we established an experimental mouse model ofadiaspiromycosis via the intratracheal route, we examined adiaspore development in vivo. We also used different inoculation numbers of E. crescens spores from 400
to 4 × 104 to assess their pathogenicity. We examined theadiaspore morphology using optical and electron microscopy. Furthermore, we found new adiaspore development
procedures in vitro and compared associated morphological characteristics of the adiaspores to those of adiaspores formed in vivo.

Materials and methods
Fungal strains and the growth condition

Spores of Emmonsia crescens IFM 46988 (CBS475.77,
UAMG127) used for this study were provided by the
Medical Mycology Research Center, Chiba University.
The strain was cultured routinely on potato dextrose agar
slants at 25 °C for around 4 weeks. A spore suspension
was prepared as described earlier [15].
Intratracheal infection of mice with E. crescens and tissue
section preparation

All animal experiments were approved by the Animal
Care and Use Committee of Obihiro University of Agriculture and Veterinary Medicine (Nos. 27-152, 28-22,
29-170). The intratracheal intubation procedure wasdescribed in an earlier report [16]. We used C57BL/6N
mice (male, 7 weeks) supplied by Charles River Laboratories Japan, Inc. After confirming correct intubation, 25 μL
of spore suspension in saline was introduced through acatheter. We used a larger burden (4 × 106 spores/mouse)and smaller burdens (4 × 102, 4 × 103, 4 × 104 spores/mouse) as spore numbers for intratracheal infection.
After infection with the larger burden, mice were killed
at different time points (three mice each at 1, 2, 3, 4, 6,
8, 10, 30, 50, and 70 days post-infection (dpi) and one
mouse each at 100 and 150 dpi). After tissues including
those of the lung, heart, liver, spleen, and kidney werecollected, they were fixed with 10% formalin and were
subsequently embedded in paraffin. Paraffin blocks weresliced (approximately 3 μm thick) and processed using
usual deparaffinization and hydration procedures. Eachsection was stained with hematoxylin and eosin (HE) or
periodic acid–Schiff (PAS) procedure. For small burden
infections, three mice each were infected and later killed
at 70 dpi. Body weight was measured twice a week after
infection. For comprehensive analyses to detect infectionfoci with smaller burden sizes, six section samples wereprepared for every 50–80 μm thickness of lung paraffinblocks and were examined using HE staining.

Page 2 of 9

TEM analysis

Lungs of mice inoculated 4 × 106 spores were used fortransmission electron microscopy (TEM) analysis. The
lungs were collected at 30, 50, and 70 dpi, fixed with 2.4%
glutaraldehyde (pH 7.4), immersed in 1% phosphate-buffered osmium tetroxide (pH 7.4), dehydrated, substituted
with propylene oxide, and were subsequently embedded
in epoxy resin. After ultrathin sections (approximately
90 nm thick) were cut using a diamond knife and ultramicrotome, they were observed using a transmission
electron microscope (HT7700; Hitachi High Technologies Corp., Tokyo, Japan) without uranyl acetate or leadcitrate staining.
In vitro adiaspore induction

After 1 × 105 spores were inoculated into 2 mL of fetal
bovine serum (FBS) in a 35-mm dish, they were cultured
at 37 °C for 60 days for TEM analysis or 71 days under
5% ­CO2 condition for determination of the adiaspore
diameters. New 2 mL FBS was added every 30 days. To
ascertain the adiaspore diameters, images were taken at
8, 30, 50, and 71 days after inoculation using a fluorescence microscope (BZ-9000; Keyence Co., Osaka, Japan).
For TEM analysis, spores were fixed at 60 days after inoculation with 4% paraformaldehyde in phosphate-buffered saline (Fujifilm Wako Pure Chemical Corp., Osaka,
Japan) followed by 2.4% glutaraldehyde. Then they were
immersed in 1% phosphate-buffered osmium tetroxide (pH 7.4). The precipitated spores were dehydrated,
embedded in LR White resin, and then cut into semi-thinsections (approximately 1 μm thick) for light microscopic
observation with toluidine blue staining, or into ultrathin
sections for analysis using a transmission electron microscope (HT7700; Hitachi High Technologies Corp.).

Results
Adiaspores developed in the lung examined using
an experimental mouse model of intratracheal infection

After infection with a higher burden (4 × 106 spores/mouse), mice showed no symptoms. Their body weights
increased gradually along with those of uninfected mice
(Additional file 1). The E. crescens spores and adiasporesassociated with cellular infiltration were found in thelung (Figure 1), but not in the heart, liver, spleen, or kidney. The spores increased in size and showed structures
similar to those of adiaspores (Figure 2), suggesting thatthe model induced adiaspores in mice by instillation
through the intratracheal route.
Maturation of adiaspores from spores in vivo

Spore diameters in the lung were measured at each time
point (Figure 3). At 1 dpi, inflammation mainly from

Takeshige et al. Vet Res(2020) 51:119

Page 3 of 9

1 dpi

6 dpi

10 dpi30 dpi50 dpi70 dpi

100 dpi

150 dpi

Figure 1 Mouse lung lobes infected with E. crescens. Fields of squares are shown in the right panel in each image. Scale bar = 1 mm.

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

70 dpi

100 dpi 150 dpi

PAS

HE

A

4 dpi

6 dpi

8 dpi

10 dpi30 dpi50 dpiFigure 2 Adiaspore development in mouse lung. Specimens were stained with HE (A–I) or PAS procedure (J–R). Scale bar = 50 µm. All images
are of the same scale.

accumulated neutrophils was observed in the infectedlungs (Figure 4A, B). Inoculated spores were found in
and around the bronchi and the alveoli (Figure 4C). Some
spores were phagocytosed by macrophages (Figure 4D).
The median spore sizes at 1 dpi (3.6 μm) was similar tothat of recovered spores from potato dextrose agar slant
in vitro (3.2 μm mean diameter). Enlarged spores were
found in the lung at 4 dpi (median 7.2 μm, Figure 3). The
thick cell walls of some spores were observed clearly, as
portrayed in Figure 2A, J. Infiltration of lymphocytesaround bronchi and blood vessels were observed mainly
at 8 dpi. Some spores were surrounded by multinucleated giant cells (arrows in Additional file 2). The medianspore size reached 13.5 μm at 8 dpi (Figure 3). The average spore size reached 41.1 ± 7.5 μm at 70 dpi. The average size did not exceed 50 μm until 150 dpi in the strain(Figure 3).
The three-layered cell wall structure was observed
clearly in most adiaspores at 70 dpi (Figure 2G). At 100
and 150 dpi, some adiaspores showed a multilayered cellwall structure (Figure 2H, I). The outermost layer of thecell wall showed a bleb-like structure on some adiaspores(Figure 2F, G). As early as 4 dpi, an internal space with
unstructured materials was observed in some adiaspores(Additional file 3). The area expanded gradually during
infection. In contrast to HE images, granular materials

Takeshige et al. Vet Res(2020) 51:119

Page 4 of 9

80

Diameter (μm)

60

40

20

0

0

50

100
Days post infecon150

Figure 3 Spore and adiaspore diameters observed in mouse
lung. White bars show the median at each time point.

in the space were strongly stained with PAS stain (Additional file 4). At 30 dpi and later, some adiaspores were
found with debris-like materials or were empty (Additional file 5). Some showed that the cell wall structure
had partly ruptured (left image in Additional file 5). We
conducted TEM analysis for mouse lungs recoveredat 10, 30, 50, and 70 dpi. Adiaspores observed at 10 dpi

retained intracellular contents and a membrane structure of organelles (Figure 5A, A′), which are similar to theendoplasmic reticula surrounding the nucleus. Althoughseveral nucleus-like structures were observed in an adiaspore, cellular membranes to septate these nuclei were
not found in the adiaspore (Figure 5A, A′). Similarly tothe results observed under an optical microscope, adiaspores showed internal space with unstructured materials(arrows in Figure 5B, C). A bleb-like structure on the outermost layer of the cell wall and a three-layered cell wall
structure were confirmed using ultrastructural analysis(Figure 5D′). Most adiaspores were found to have readily
visible electron-dense granules at 30 dpi and higher resolution (Figure 5B–D). We identified characteristics of E.
crescens development from spore to adiaspore using thismouse model with infection via the intratracheal route.
Adiaspores induced by fetal bovine serum in vitro

We examined adiaspore formation in vitro, observed at
8, 30, 50, and 71 days post-inoculation (Figures 6 and
7). As the culturing time increased, the spores became
larger. They reached a plateau in terms of size at 50 days
post-inoculation (Figure 7). At 8 days post-inoculation, some spores formed a germ tube-like protrusion (white arrows in Figure 6). At 50 days and 71 days

Figure 4 Infection foci in mouse lung at 1 day after E. crescens infection. These specimens were stained with HE (A, B) and PAS procedure (C,D). The field of a square in A is magnified in B.

Takeshige et al. Vet Res(2020) 51:119

Page 5 of 9

10 dpi
A

A’

5 µm

D

30 dpi50 dpi

B

1 µm

C

10 µm

10 µm

D’

10 µm

5 µm

70 dpiFigure 5 TEM images of adiaspores in mouse lung at 10 (A, A′), 30 (B), 50 (C), and 70 (D, D′) days post-infection. Fields surrounded with awhite square in A–D are magnified respectively in A′–D′. White arrowheads in A′ indicate the endoplasmic reticulum-like structure surrounding
the nucleus-like structure (N in A′). White arrows in B and C and a black arrow in D′ respectively point to unstructured materials and a bleb-likestructure.

Figure 6 Adiaspore development in FBS in vitro. Culture durations were shown in each image. White arrows indicate germtubes. Arrowheads
indicate remarkably large adiaspores. Scale bar = 50 µm.

Takeshige et al. Vet Res(2020) 51:119

Page 6 of 9

Infection focus formation with small burdens of E. crescens
spores in mouse lung

Diameter (µm)

60

40

20

0

0

20

40
Days aer inoculaon60

80

Figure 7 Diameters of adiaspores cultured in vitro. White bars
show the median at each time point.

post-inoculation, although most of the adiaspores wereof ca. 30 µm in diameter, some adiaspores had grown
remarkably large under the culture (arrowheads in Figure 6). Among our observations, the largest adiaspore
at 71 days post-inoculation had diameter of greaterthan 120 µm in. The cell wall indicated by a white arrow
in Figure 6 was ruptured, thereby resembling the cellshown in the left image of Additional file 5. Adiasporescultured for 60 days are shown in Figure 8 (TEM analysis) and in Additional file 6 (stained with toluidineblue). As shown in Figure 6, some adiaspores inducedin vitro showed a three-layer structure of the cell walland electron-dense granules in the spores. They exhibited similar structural features to those of adiaspores
formed in the lung on the experimental mouse model.

Next, we examined E. crescens infections with a smallburden size (4 × 102, 4 × 103, or 4 × 104 spores/mouse)through the intratracheal route to elucidate how a few
spores establish infection in mouse lung tissues. Body
weights increased as they did for an uninfected mouse(Additional file 7); no symptom was apparent. We used
three mice for each inoculation burden and observed theinfection foci in mouse lungs at 70 dpi after infection.
Adiaspores were found in the lung of each mouse that
had been infected with 4 × 102 and 4 × 103 spores. Under
the condition of infection with 4 × 104 spores, althoughadiaspores were not found in every mouse, the infiltration of lymphocytes (10 foci) was found. These results
suggest that E. crescens can form infection foci and adiaspores in mouse lung under a small burden: even at 400
spores/mouse.

Discussion
For this study, we established an experimental mousemodel for intratracheal infection with E. crescens. Subsequently, we observed adiaspore development in micelungs. Adiaspores in mice were observed in the lung only,
which is consistent with cases of wild animals reported
earlier [5, 6, 8].
Additionally, we established a novel in vitro culturingmodel for adiaspore formation. The model uses FBS as
the medium. Adiaspores were maintained for more than
70 days, until 150 days in the model (data not shown). As
described earlier, solid media such as brain–heart infusion agar have been used frequently for in vitro culture[10, 11]. Solid media present long-term culture difficulty

Figure 8 Adiaspores cultured in FBS for 60 days in vitro. The magnified image in a black frame of A is shown in B.

Takeshige et al. Vet Res(2020) 51:119

because of drying. For a model using a liquid medium(FBS), medium addition or culture medium replacement
can be done for long-term culturing. Moreover, the collection of adiaspore from FBS is easier than collection
from solid media. Therefore, in vitro culture is useful for
additional analyses of adiaspores, such as gene expression analysis. For the present study, we compared theadiaspore morphologies of those grown through infection of the lung and those grown through in vitro culture.
Both models are complementary, but not perfect: in vitro
culture model entails no host immunological response;in vivo infection presents difficulty when analyzing thereactions and changes of E. crescens. Parallel analyses
using both models are expected to be beneficial for additional understanding of E. crescens and the diseases theycause.
It is particularly interesting that E. crescens infection with a small burden size, even as few as 400 spores,
formed an infection focus. Although E. crescens is notdispersed in the bloodstream or other organs, the species
can form infection foci with small burdens in immunocompetent hosts. In a natural environment with E. crescens, small mammals including rodents are incidentally
exposed to E. crescens spores. The inhaled spores form
adiaspores in their lung.
Infected mice in the model showed no symptoms,
which is consistent with most reported cases, showing alack of readily apparent symptoms from infection. Schaffer-White and colleagues reported two cases of Northern hairy-nosed wombats (Lasiorhinus krefftii) [17], anddescribed that the cause of death of one wombat was
pulmonary adiaspiromycosis. Northern hairy-nosed
wombats might be less resistant to the causative agents.
Susceptibility to E. crescens and infection severity arethought to vary among animals, suggesting that further
investigations should be conducted to ascertain the pathogenicity to wild animals, including endangered animals.
Our experimental model induces adiaspores with average diameter of around 50 μm. Adiaspores of E. crescens
in wild animal cases are around 300 μm (50–500 μm)
diameter [18]. The reason for the small maximum adiaspore size in our model remains unknown. The bodyor lung size of the host might limit the adiaspore size,
but the mean diameter of adiaspores was found to begreater than 300 μm in small mammals such as field voles(Microtus agrestis) and bank voles (Clethrionomys glareolus) [19]. Additional study using multiple strains is warranted because only one strain was used for this study.
Most adiaspores described in earlier reports were
empty or with residual eosinophilic components[6, 7, 20, 21]. In contrast to them, most adiasporesobserved in our study remained filled with basophiliccomponents. Empty adiaspores and adiaspores with

Page 7 of 9

residual eosinophilic components were observed more
frequently in the lungs at 150 dpi compared with otherearlier time points, suggesting that most cases arefound in wild animals after lengthy infection.
Hughes and Borman reported adiaspiromycosis in awild European rabbit (Oryctolagus cuniculus), whichshowed a bleb-like structure of the outermost layer on
the surface. Bleb-like structures similar to those were
found during our observations [22]. Multinucleate
giant cells surrounded adiaspores shown in Additional
file 2 resemble those of an earlier report of a study conducted by Hughes and Borman [22].
We were able to detect membrane structures of organelles in adiaspores at 10 dpi. At 30 dpi and higher resolution, instead of membrane structures, electron-dense
granules were observed in the center of adiaspores.
Those lipofuscin-like structures were also observedin adiaspores that had been formed in vitro. Similar
structures were found in adiaspores in a wild Franklin’s ground squirrel [23]. Electron microscopic observation of the granules revealed similar characteristics
to those found for lipofuscin: dense particles observed
as a brown-yellow material under optical microscopy(Additional file 6). Lipofuscin is well known to be an
age-related material. It is composed mainly of lipids
and proteins that are highly oxidized. These data suggest that adiaspore aging produces a lipofuscin-likestructure in vivo and in vitro.
Results and a model of the adiaspore fate from thetime-course analysis in vivo are presented in Figure 9.
Animals acquire E. crescens spores through inhalation.
A few spores form infection foci. After infection, spores
expand in size and form adiaspore structures with athree-layer cell wall including an outermost bleb-likelayer. During and after formation, some adiaspore cell
walls rupture, thereby releasing the spore contents.
Empty adiaspores are only found in the lung of animals
with adiaspiromycosis after long-term infection.

In the lung

IntactBurst

Spore
Growing
spore

Adiaspore

Intact but empty

Figure 9 Graphical summary of adiaspore development in vivo.

Takeshige et al. Vet Res(2020) 51:119

Supplementary information

Page 8 of 9

Received: 4 July 2020 Accepted: 7 September 2020

Supplementary information accompanies this paper at https​://doi.
org/10.1186/s1356​7-020-00844​-3.
Additional file 1. Body weights of mice infected with 4 × 106 sporesor uninfected. Error bars represent standard deviations of the means.
Additional file 2. Adiaspores surrounded by multinucleate giant cells
at 8 dpi. Arrows and arrowheads respectively indicate multinucleate giant
cells and surrounded adiaspores.
Additional file 3. Adiaspores in mouse lungs at 4, 10, 30, and 50 dpi.
Additional file 4. Representative adiaspores in which granular materials (arrow) were observed. The adiaspores are those of mouse lungs at
70 dpi, stained using PAS procedure.
Additional file 5. Adiaspores at 30 dpi (left) and 70 dpi (right) inmouse lungs. The adiaspore found at 30 dpi was not intact, but it
retained debris-like materials. The adiaspore (arrow) found at 70 dpi wasempty.
Additional file 6. Adiaspore development in FBS in vitro. Adiaspores
were stained with toluidine blue.
Additional file 7. Body weights of mice infected with 4 × 102,
4 × 103, or 4 × 104 spores, or uninfected. The error bars represent
standard deviations of the means.
Abbreviations
dpi: days post infection; FBS: fetal bovine serum; HE: hematoxylin and eosin;
PAS: periodic acid–Schiff; TEM: transmission electron microscopy.
Acknowledgements
This study was supported by intramural funding of Obihiro University ofAgriculture and Veterinary Medicine and by the National BioResource Project,
Japan (http://www.nbrp.jp/). We appreciate DBCLS TogoTV for providing animage used in this study.
Authors’ contributions
AT, MN, and DK contributed equally to this study. DK, TY, HF, and TT participated in the study design. AT, MN, DK, YT, AS, and TT conducted the experiments. AT, MN, DK, and TT analyzed the data. TT wrote the manuscript. All
authors read and approved the final manuscript.
Funding
This study was supported by intramural funding of Obihiro University ofAgriculture and Veterinary Medicine.
Availability of data and materials
Data supporting the conclusions of this article are included within the article
and its additional files.
Ethics approval and consent to participate
All animal experiments were approved by Animal Care and Use Committee of
Obihiro University of Agriculture and Veterinary Medicine (No. 27-152, 28-22,
29-170).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
Department of Veterinary Medicine, Obihiro University of Agriculture
and Veterinary Medicine, Inada‑cho, Obihiro, Hokkaido 080‑8555, Japan.
2
Medical Mycology Research Center, Chiba University, 1‑8‑1 Inohana,Chuo‑ku, Chiba 260‑8673, Japan. 3 Diagnostic Center for Animal Healthand Food Safety, Obihiro University of Agriculture and Veterinary Medicine,
Inada‑cho, Obihiro, Hokkaido 080‑8555, Japan.
1

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