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Value of an inhalational model of invasive aspergillosis

WILLIAM J. STEINBACH*, DANIEL K. BENJAMIN JR*$, SCOTT A. TRASI%, JACKIE L. MILLER, WILEY A. SCHELL,

AIMEE K. ZAAS, W. MICHAEL FOSTER & JOHN R. PERFECT

*Division of Pediatric Infectious Diseases, Department of Pediatrics, $Duke Clinical Research Institute, %Division of

Laboratory Animal Medicine and Division of Infectious Diseases and International Health andDivision of PulmonaryMedicine, Department of Medicine, Duke University Medical Center, Durham, NC, USA

Animal models of invasive aspergillosis have been used for virulence studies and

antifungal efficacy evaluations but results have been inconsistent. In an attempt to

reproduce human infection, many Aspergillus animal models have utilized a

pulmonary route for delivery of conidia, largely through intranasal instillation.

However, several radiolabeled particle studies have shown that aerosol delivery is

preferable to intranasal instillation to create a more homogenous delivery to the

lungs. We hypothesized that an inhalational model would be more robust for

studies of invasive aspergillosis pathogenesis and antifungal therapy. We developed

an inhalational model ofAspergillus fumigatus infection using a Hinners inhalation

chamber and demonstrated by quantitative polymerase chain reaction that this

new inhalational model creates a more homogenous murine pneumonia, facilitat-

ing analysis of mutant strains and treatment regimens.

Keywords Inhalation, Hinners, Aspergillus, Pulmonary aspergillosis, Murine

model

Introduction

The primary purpose of an experimental animal model

is to reproduce human disease. Animal models of

Aspergillus infection have been reviewed [1/3] andthere is no universally accepted animal model for

invasive aspergillosis (IA). Variables in each model

include the animal species and strain, Aspergillus

strain, and immunosuppression intensity, frequency

and duration. The method of inoculation has also

varied, including infecting animals by intravenous [4],

intratracheal [5] and intranasal [6] routes. Infection via

the respiratory tract is preferred because it reflects the

initial route of human infection and the vast majority

of IA is manifest as a pulmonary infection. While many

IA animal models have used a pulmonary route for

delivery of the Aspergillus conidia, most have specifi-

cally inoculated by intratracheal, intranasal or selective

intubation routes [1,7].

Intranasal murine models of IA and human disease

differ histopathologically. The key differences noted in

human autopsies have been related to bronchopneu-

monia resulting from both a proliferation of Aspergil-

lus and an exudative response in the alveoli in humans;

while in contrast most Aspergillus lesions in the lungs

of intranasally infected mice are initiated from conidia

that settled on the bronchial mucosa [8]. Conversely,

studies using an inhalational model of Aspergillus

fumigatus have shown pathological changes that more

closely resemble those found at autopsies of humans

with aspergillosis [9].

Numerous other infectious diseases acquired through

aerosols have used inhalational animal models for

experimental study, including Histoplasma capsulatum[10], respiratory syncytial virus [11], influenza [12],

Mycobacterium tuberculosis [13], Burkholderia cepacia

[14], Bordetella pertussis [15], Legionella pneumophila

[16] and Streptococcus pneumoniae [17]. Several pub-

lished studies exist using an inhalational model of

aspergillosis; however, most of these studies examined

antibody formation in immunocompetent animals and

Correspondence: William J. Steinbach, MD, Division of Pediatric

Infectious Diseases, Box 3499, Duke University Medical Center,

Durham, NC 27710, USA. Tel: '/1 919 684 6335; Fax: '/1 919 584

8902; E-mail: [email protected]

Received 4 December 2003; Accepted 30 January 2004

2004 ISHAM DOI: 10.1080/13693780410001712034

Medical Mycology October 2004, 42, 417/425

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these models rarely used immunosuppression in order

to create IA [9,18/27]. Therefore, we have developed a

persistently neutropenic murine IA model using an

inhalational delivery system, and also compared

the development of pneumonia in this model to

the more commonly used intranasal IA model of

infection.

Materials and methods

Immunosuppression

Outbred 6-week-old male CD1 (Charles River) mice

(20/25 g) were housed and fed under aseptic condi-

tions and their sterile water was supplemented with

tetracycline (1 mg/ml) changed once daily. The mice

were immunosuppressed with an intraperitoneal injec-

tion of cyclophosphamide (150 mg/kg) on day (/3, and

a subcutaneous injection of cortisone acetate (250 mg/

kg) on day (/1 of infection. The immunosuppressiveregimen also included additional doses of cyclopho-

sphamide (150 mg/kg or 50 mg/kg) on days '/1, '/4,

and '/7 of infection.

Immunosuppressed but uninfected control mice pre-

viously underwent leukocyte count testing to confirm a

decrease in leukocyte count through tail vein venipunc-

ture using a Unopette capillary tube system (Becton

Dickinson, Sparks, MD). The mice leukocyte counts

were determined before experimentation, at day of

inoculation (day 0), and days '/3, '/7, and '/10 of

infection. A total leukocyte count and manual differ-

ential were determined on each whole-blood sample to

determine the extent of neutropenia. This immunosup-

pression schedule yielded a profound decrease in total

leukocyte count (granulocyte concentration of B/100

cells/ml) 2/3 days after the first cyclophosphamide

injection until approximately day '/10. Manual differ-

entials specifically revealed profound and persistent

neutropenia. Groups of 10/15 animals receiving sev-

eral immunosuppressive regimens were observed for the

natural history of infection over 2 weeks (Fig. 1). Any

animals with the clinical appearance of invasive asper-

gillosis were euthanized.

Aspergillus fumigatus strain

Aspergillus fumigatus strain 293, the strain presently

undergoing sequencing and annotation jointly by The

Institute for Genomic Research (Rockville, MD, USA),

The Sanger Institute (Hinxton, Cambridge, UK) and

the Institut Pasteur (Paris, France), was used in all

experiments. A. fumigatus conidia were grown on

Sabourauds agar for 7 days and harvested in 0.01%

Tween 80 in sterile water on the day prior to inocula-

tion. For the inhalational delivery a conidial suspension

was made to equal 3)/108 conidia per ml, and for the

intranasal delivery a 1)/106/ml conidial suspension

was used.

Inoculation with conidia

A total of 40 mice were used, including 20 mice (two

groups of 10 mice) for each arm of conidial delivery

were immunosuppressed as above. For the inhalational

model, a total of 40 ml of the 3)/108 conidia per ml

suspension was aerosolized in four separate nebulizers

(Aerotech II; CIS-US, Beford, MA) in a Hinners

exposure chamber [28] for 25 min (Fig. 2). The

diamond-shaped acrylic Hinners inhalation chamber

Fig. 1 Survival curves for various immunosup-

pressive regimens. *Listed as cyclophosphamide

(mg/kg) administered on days'/1,'/4,'/7 after

infection after initial dose of 150 mg/kg on day

(/3 and 250 mg/kg of hydrocortisone acetate on

day (/1.

2004 ISHAM, Medical Mycology, 42, 417/425

418 Steinbach et al.

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is approximately 5 cubic liters in size and has been

successfully applied for antigen (ovalbumin) delivery in

a murine model of inflammatory airway disease [29].

The diamond shape allows effective recirculation of

aerosolized spores after delivery into the chamber.

Pressurized air at 30 p.s.i. driving pressure was used

to aerosolize the conidial suspension. Unanesthetized

mice were placed in a wire cage rack subdivided into

individual sections, allowing ample room for move-

ment. Mice freely inhaled the circulating cloud of

conidia yet were unable to huddle with each other

and disrupt adequate and uniform exposure. After

the 25-min inhalation period, a 3-min wash with

pressurized air was performed to clean the chamber

before removing the mice. Surveillance with agar plates

revealed no consistent contamination outside of thebiosafety hood housing the Hinners chamber, but

plates inside the biosafety hood did grow A. fumigatus,

which is postulated to be due to the requirement of

opening the animal cage door to evacuate the last of the

conidia using the biosafety hoods ventilation system.

Mice in the intranasal model were anesthetized with

intraperitoneal pentobarbital (0.75 mg per mouse) and

50 ml of the 1)/106/ml conidial suspension was slowly

pipetted into one of the nares while the mouse was

suspended by the front incisor teeth on a horizontal

string, as previously described [30]. After the full 50 ml

was inoculated, the mice remained suspended for

approximately 10 min in order to allow the conidial

suspension to be aspirated into the lungs.

The 20 mice in each arm of the experiment were

examined by two different methods of analysis,

A. fumigatus quantitative polymerase chain reaction

(PCR) and histopathologic staining. The mice were

killed at 1, 24 and 96 h after inoculation and the entire

lung block removed for examination. We did not

evaluate the lungs immediately after the inoculation

so as to allow the intranasal conidia time for

descension from the nasopharynx into the bronchialtree.

Histopathologic examination

A board-certified veterinary murine pathologist was

blinded to the source of the sections and evaluated

representative slides from each lung section, grading

according to a five-point pulmonary infarct score

Fig. 2 Hinners inhalational chamber appara-

tus.


Inhalational A. fumigatus animal model 419

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that incorporated necrosis, hemorrhage, edema and

hyphal presence. Scores ranged from 0 to 5 with the

score roughly representing the percentage of tissue

involvement (00/0%, 10/10%, 20/20%, 30/30%,

40/40%, 50/50%). Tissues were fixed in 10% neutral-

buffered formalin and processed routinely for

histopathology. Briefly, tissues were dehydrated withgraded alcohols, lipids removed with xylenes, tissues

infiltrated with paraffin and placed in tissue blocks.

Sections were cut on a rotary microtome at 5-mm

thickness. Adjacent tissue sections were stained with

hematoxylin and eosin as well as Gomoris methenamine

silver.

Quantitative PCR examination

Lung samples were analyzed by Taq Man A. fumigatus

quantitative PCR as previously described [31]. In brief,

lung section tissues were homogenized, DNA extractedand oligonucleotide amplification primers and a dual-

labeled fluorogenic oligonucleotide hybridization probe

complementary to sequence from the A. fumigatus 18S

rRNA gene were used (sense amplification primer

5?-GGCCCTTAAATAGCCCGGT-3?, antisense ampl-

ification primer 5?-TGAGCCGATAGTCCCCCTAA).

The two lungs from each mouse were sectioned into a

total of five sections, divided as three sections of

the right lung and two sections of the left lung,

corresponding to the individual lobes of the respective

lungs.

Statistical analyses

Because this study involved a small number of mice and

multiple samples were taken from each mouse, we used

non-parametric methods and several different methods

of analysis in order to account for the lack of

independence between observations. First, we used

the Fishers exact test to evaluate infection within

individual lung segments. In these analyses, each mouse

contributed no more than one sample to the analysis.

Second, we used Wilcoxons rank sum in order to

evaluate the amount of Aspergillus cells/gram of lung

tissue detected by PCR. In these analyses, each mousecontributed no more than one sample. These data were

also examined using clustered logistic regression. (This

last analysis is not presented in this report, but yielded

similar results to the testing by Fishers exact test and

Wilcoxons rank sum.) Reported P-values are two-

tailed. We used STATA 6.0/7.0 (Stata Corporation,

College Station, TX) for the analyses.

Results

Histopathologic studies

Histopathological examination of a representative sec-

tion of each lung sample with either the hematoxylin

and eosin or Gomoris methenamine silver stain

showed only slight and not clinically significant differ-ences between the inhalational and intranasal models.

Histopathology lung scoring was similar for both

models at 1 h after inoculation, with both models

showing no necrosis, hemorrhage or edema. At 24 h,

only the inhalational model showed any pathologic

changes, consisting of a mean hemorrhage score

equivalent to 10% hemorrhage. However, by 96 h after

inoculation the mean necrosis, hemorrhage, and edema

scores for both models was approximately 10/20% and

not statistically different between the two models. The

Gomoris methenamine silver stain confirmed these

host-response findings, with no statistical difference in

the amount of hyphae observed in the lung sectionsexamined between the two methods of conidial deliv-

ery.

Survival

Like all IA models, the inhalational delivery model still

requires close monitoring of immunosuppression to

maintain appropriate mortality. With an immunosup-

pression strategy using cyclosphophamide at 150 mg/kg

on days '/1 and '/4 we can achieve a 90/100%

mortality, with animals dying between days 6 and 9

after infection. Using another immunosuppressive regi-

men (Fig. 1) with decreased doses of cyclophosphamide(i.e. 50 mg/kg) on days '/1, '/4, and '/7 after infection,

we can modify the acute mortality rate. Therefore, our

inhalational model is reproducible, immunosuppressive

regimen-dependent and induces mortality, but is flex-

ible in its ability to produce death, which is directly

controlled by the immunosuppressive regimens.

Quantitative PCR for fungal burden

There were a total of 100 quantitative PCR samples

obtained from 20 mice, each with 5 lung sections. Only

69% (69/100) of those samples yielded fungal burden

values above the lower limit of detection (2.589 log cells

per g lung tissue) for the PCR protocol. Of those

samples detected by PCR, there was not an appreciable

difference in the number of positive samples from the

left lung (72.5%, 29/40) or the right lung (66.7%, 40/60)

(P0/0.66), suggesting that any undetectable sections

were not due to technical issues. There was also no

difference amongst the positive samples within the five



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individual lung sections. On the other hand, a major

difference in distribution of positive samples occurred

between tissues from animals receiving inhalational

conidia versus intranasal conidia; 100% (50/50) of

the samples from animals receiving conidia by inhala-

tion yielded detectable PCR results, compared to

only 38% (19/50) of the intranasally infected animals(P5/0.001). Overall, the inhalational model also did

yield higher Aspergillus burden than the intranasal

model (P0/0.0006).

Fishers exact testing of the five lung sections at all

time-points revealed statistical differences in the pre-

sence of fungus in the lungs between the two delivery

models. While all 10 mice from the inhalational model

had PCR detected Aspergillus in every lung section, in

contrast with the intranasal model only 6/10 (P0/0.09)

and 3/10 (P0/0.003) mice had detectability in the two

left lung sections. On the three right lung lobe sections,

there were only 4/10 (P0/0.01), 3/10 (P0/0.003), and

3/10 (P0/0.003) intranasal sections with detectablefungus compared to the inhalational model.

The mean quantitative PCR values for the inhala-

tional model (Table 1) yielded higher fungal burdens as

well as smaller standard deviations than the intranasal

method. When using the lower limit of PCR detection

as a value for those sections with no detectability of

the fungus, the intranasally infected mice had a

mean fungal burden (log cells per g lung tissue) of

3.3819/1.23. However, the mean levels of the individual

intranasal lung sections were similar throughout the

five lung sections when DNA was detected and

comparable to the inhalational route. Thus, the fungalburden was similar between the two models when

Aspergillus was detected in tissue, but there were

multiple areas of the intranasally infected lungs where

the fungus was not detected.

We also examined the differences between the two

delivery models at the specific time-points of infection.

While all lung samples from inhalationally inoculated

mice revealed Aspergillus at all time-points, at 1 h after

inoculation only 2/15 intranasally infected samples

were detectable by PCR, increasing to 4/15 at 24 h

and 13/20 at 96 h. Using the lower limit of PCR

detectability, 1 h after inoculation the five lung

sections from the intranasal mice had a mean of

2.722 log (cells/g)9/0.183, while the inhaled mice had

a mean of 4.196 log (cells/g)9/0.073. At 24 h after

inoculation the five lung sections of the intranasal mice

had a mean of 2.873 log (cells/g)9/0.18, and the inhaled

mice had a mean of 3.843 log (cells/g)9/0.113. At 96 h

after inoculation the intranasal mice had increased to a

mean of 4.256 log (cells/g)9/0.351, and the five lung

sections of the inhaled mice had a mean of 4.852 log

(cells/g)9/0.272.

Discussion

Although inhalational exposure to aerosolized patho-

gens that create invasive pneumonia intuitively would

seem to be the best model of human disease, one of themost common inoculation methods for IA animal

models is the intranasal pulmonary delivery method.

Several radiolabeled studies have shown the benefits of

inhalational delivery of particles compared to intrana-

sal instillation to create a more uniform distribution

pattern [32/34]. Only one study has directly compared

both an inhalational and an intranasal model of IA [18]

and this study examined only survival and histology.

We compared both the intranasal and our inhalational

method of inoculation and found that lung tissue

quantitative PCR results from the inhalational model

resulted in a more consistent and homogeneous infec-

tion based on lung section sampling, as well as anearlier establishment of infection compared to the

intranasal model.

Only the inhalational method of delivery resulted in

quantitative PCR detectability of Aspergillus DNA in

all lung sections in every mouse. In addition, even

among the intranasally infected lung sections that did

have PCR detectability, there was a large standard

deviation of fungal burden. This indicates that the

inhalational model produced not only a more homo-

genous infection among all the lung sections, but also

within the individual lung parenchyma samples. These

results also indicate a general slower presence or growth

of the fungus within the lung parenchyma in the

intranasal method of inoculation with the inocula

used, as shown by a much lower fungal burden at the

earlier time points of infection. In fact, the quantitative

results are probably even more disparate as the fungal

burden in the intranasal model is overestimated be-

cause when the quantitative PCR yielded an undetect-

able level, we considered the level in tissue to be at the

Table 1 Quantitative polymerase chain reaction (PCR) (log cells per

g lung tissue) values with inhalational delivery

Lung

Section

Mean

(log cells per g tissue)

Standard

Deviation

Range

Total 4.35 9/0.66 3.15/5.74

Left 1 4.31 9/0.83 3.15/5.55

Left 2 4.44 9/0.73 3.34/5.56

Right 1 4.28 9/0.56 3.59/5.32

Right 2 4.50 9/0.68 3.73/5.74

Right 3 4.23 9/0.54 3.51/5.18

Limit of PCR detection is 2.589 log cells/gram tissue.



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PCRs lowest limit of detection in the analyses. There-

fore, the true fungal burden in the intranasal model

is probably less and with a wider range in fungal

burden.

Homogeneity of infection through an inhalational

model offers many potential advantages for further

study of lung tissue, including quantifying fungal

burden on one harvested lung lobe while utilizing other

lobes for other purposes (e.g. histology, RNA isola-

tion). A heterogeneous pattern of infection such as that

observed in the intranasal infection route could pro-

duce inconsistent and lobe-dependent results in each

animal, particularly altering any diagnostic testing

results. The ability to create a uniform infection is

attractive for both antifungal agent strategies as well as

virulence studies with different strains. Consistency and

rapidity of infection also allows testing of earlier

infection time points as well as potentially using less

numbers of animals. Finally, the inhalational exposure

technique is performed on awake, anaesthetized mice,so there is no recovery time and no risk of anesthesia.

Our histopathology staining analyzed only two 5-mm

sections of each lung sample, whereas PCR analyzed a

homogenized portion of an entire sample, therefore

increasing the sensitivity of the PCR assay. Given the

uniformity of fungal burden as assessed by quantitative

PCR, histopathology of the entire lung via serial

sections would be expected to support PCR results.

Additionally, the act of inflating the lungs for histo-

pathology sample processing may have diminished the

visible fungal burden. This should not affect the

identification of established lesions, but may wash

away conidia which are not deeply embedded in thetissue or remove conidia present in the alveoli as well as

alveolar macrophages that may have been attempting

to clear them.

It has been proposed that fungal burden, not

survival, should become the primary outcome measure

for antifungal animal model experimentation in Asper-

gillus infections [35]. It is clear that our model does

have a high mortality rate depending on the immuno-

suppressive regimen, as shown in Fig. 1. On the other

hand, the difficulties in accurately quantifying an

Aspergillus tissue burden of entwined hyphae using

colony forming units (c.f.u.) in tissue are well known

[36], and previous studies have shown the increased

sensitivity and precision of A. fumigatus quantitative

PCR over c.f.u. measurement [31]. Specifically, the

PCR-based quantification of A. fumigatus tissue bur-

den can detect every cell in a filamentous fungal mass.

For example, in one study the PCR assay detected a

10 000-fold increase in fungal burden, while the same

tissue displayed less than a 10-fold increase in the

number of c.f.u. [31]. If quantitative PCR for detection

of fungal burden becomes the new standard, we will

need to utilize an animal model strategy with a

consistent and homogeneous infection at the target

organ.

The concept of an inhalational model of IA is not

entirely new, and we found 13 other reports ofinhalational invasive aspergillosis models (Table 2).

However, the majority of reports follow the experi-

mental methods of two earlier reports [19,37]. In 1959,

Sidransky and Friedman [19] first described a closed

bell jar containing a cylindrical wire mesh and the use

of a powder atomizer for dispersal of vacuum suction

dried spores. In 1960, Piggott and Emmons [37]

described a general inhalation exposure device using a

1-l Erlenmeyer flask with a layer of agar seeded with

spores at the bottom. Mice were placed in horizontal

side arms until their noses extended beyond the open

end of the tube. At the top of the chamber was a rubber

stopper with an angled plastic tube attached to asyringe and the aerosol exposure was created when one

investigator pumped air into the chamber by strokes

with the plunger and a second investigator rotated the

tube to direct the jet of air over the agar and spores. In

prior experiments we recreated the Piggott and Em-

mons model and discovered several potential problems

with this model system. First, we were unable to

consistently reproduce the inoculum on the poured

agar bottom. In addition, when using a hand atomizer

or balloon as the air delivery device we found an

inconsistent air flow each time, as the exact dispersal is

highly dependent on force, consistency, duration andangle of delivery. Finally, another potential limitation

of using a hand atomizer lies in testing mutant

Aspergillus strains, where an unknown conidiation

defect could affect dispersal of the spores from an

agar plate.

Finally, a group recently developed an inhalational

model of IA [38] using a rectangular-shaped aerosol

chamber for 1 h with an aerosolized inoculum of

5)/109 conidia. While this model used a similar

immunosupression protocol to our model, we used a

diamond-shaped Hinners inhalation chamber (Fig. 2)

to maximize the recirculation of spores instead of a

rectangular chamber and utilized quantitative PCRtechnology to determine the extent of spore deposition

in the various lung sections following inhalation. We

also validated our model compared to the more

commonly used intranasal model using similar immu-

nosuppression and found that infection with our

inhalational delivery in the Hinners chamber was

more homogenous.



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Table 2 Previous immunosuppressed inhalational models of invasive aspergillosis

Murine

strain

Immunosuppression Chamber Method Aspergillus

isolate

Conidia

concentration

Inhalati

CF1 Cortisone acetate (5 mg)

on day (/2

Closed bell jar with a

cylindrical wire mesh

Powder atomizer A. flavus NS 10/30 m


on day (/2, or on days

0, '/1, '/4, '/6

Modified Piggott and

Emmons

Powder atomizer,

six or eight sprays

A. flavus NS 5/12 m


on day (/2

Sidransky, 1959 Powder atomizer,

five sprays total

A. flavus NS 5 min


on day (/2

Sidransky, 1959 Powder atomizer A. fumigatus 400 mg dry

spores

20 min

Swiss white Cortisone acetate (4 mg)

on day (/2,

(2.5 mg) on day 0, (2.5 mg)

on day '/2

Sidransky, 1959 Powder atomizer A. fumigatus,

and six other

Aspergillus

spp.

1)/105/

4)/105NS


on day (/2

Sidransky, 1959 Powder atomizer A. flavus NS NS


on day (/2

Piggott and Emmons Pump 100 ml air

over 15 s

A. fumigatus Inoculate 107,

grow 5 days

3/4 min

CF1 Cortisone acetate (100 mg/kg)

once daily for 3 days


Emmons

Atomizer A. flavus NS N/A

OF1 Cortisone acetate (125 mg/kg)

on days (/3, (/1, 0

Anesthetized mouse

held by nose at neck

of flask

Air through syringe A. fumigatus NS 1 min

CD1 Cortisone acetate (2 mg)

on days (/2, 0

Piggott and Emmons Pump 100 ml air A. fumigatus 3)/107

conidia

2 min

CF1 Cortisone acetate (100 mg/kg)

on days (/1, 0, '/1


Emmons

Atomizer A. fumigatus

and A. flavus

NS 0.5/1 m

NS Corticosteroids Inhalation flask Atomizer A. fumigatus

and A. flavus

NS NS

Balb/c Cortisone acetate (250 mg/kg)

and cyclophosphamide

(200 mg/kg) on days (/2, '/2

Plexiglass rectangular

chamber

Nebulizer A. fumigatus 5)/109

conidia

1 h

NS, not specified; c.f.u., colony-forming units.

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Conclusion

While the intranasal delivery of conidia has shown over

the years to lead to disease and animal death, an

inhalational model of IA might be a better model as it

reproduces the physiologic acquisition of human dis-

ease and allows for confident sampling of any lung

section. In this study we demonstrate by quantitativePCR that an inhalational delivery system creates a

more efficient and homogenous pneumonia in mice

compared to the more commonly used intranasal

delivery method. We propose the use of an inhalational

delivery system to better reproduce human acquisition

and pathology of IA and yield consistent and uniform

quantitative counts and histopathology to study viru-

lence of A. fumigatus strains and analyze antifungal

treatment regimens.

Acknowledgements

W.J.S. is an NICHD Fellow of the Pediatric Scientist

Development Program (NICHD K12-HD00850).

W.M.F. received support from R01-NIH-HL62641;

D.K.B. received support from NICHD 1 R03

HD42940-02; and J.R.P. received support from P01-

AI-449175 (Duke University Mycology Research

Unit). We acknowledge the support of Merck and the

assistance of Cellular and Molecular Technologies with

the quantitative PCR.

References

1 Shibuya K, Naoe S, Yamaguchi H. Animal models of A.fumigatus infections. Contrib Microbiol 1999; 2: 130/138.

2 Yamaguchi Y. Opportunistic fungal infections. In: Miyaji M (ed.).

Animal Models in Medical Mycology. Boca Raton, FL: CRC

Press, 1987: 101/158.

3 Schmidt A. Animal models of aspergillosis / also useful for

vaccination strategies? Mycoses 2002; 45: 38/40.

4 Sabetta JR, Miniter P, Andriole VT. The diagnosis of invasive

aspergillosis by an enzyme-linked immunosorbent assay for

circulating antigen. J Infect Dis 1985; 152: 946/953.

5 Spreadbury CL, Krausz T, Pervez S, Cohen J. Invasive aspergil-

losis: clinical and pathological features of a new model. J Med Vet

Mycol 1989; 27: 5/15.

6 Matsumoto S, Wakai Y, Nakai T, et al. Efficacy of FK463, a new

lipopeptide antifungal agent, in mouse models of pulmonary

aspergillosis. Antimicrob Agents Chemother 2000; 44: 619/621.7 Shibuya K, Ando T, Wakayama M, et al. Pathological spectrum

of invasive pulmonary aspergillosis: study of pulmonary lesions of

54 autopsies and the relationship between neutrophilic response

and histologic features of lesions in experimental aspergillosis. Jap

J Med Mycol 1997; 38: 175/181.

8 Shibuya K, Takaoka M, Uchida K, et al. Histopathology of

experimental invasive pulmonary aspergillosis in rats: pathologi-

cal comparison of pulmonary lesions induced by specific virulent

factor deficient mutants. Microb Pathol 1999; 27: 123/131.

9 Merkow LP, Epstein SM, Sidransky H, et al. The pathogenesis of

experimental pulmonary aspergillosis. An ultrastructural study of

alveolar macrophages after phagocytosis of A. flavus spores in

vivo. Am J Pathol 1971; 62: 57/74.

10 Schlitzer RL, Chandler FW, Larsh HW. Primary acute histoplas-

mosis in guinea pigs exposed to aerosolized Histoplasma capsu-

latum . Infect Immun 1981; 33: 575/582.

11 Woolums AR, Anderson ML, Gunther RA, et al. Evaluation of

severe disease induced by aerosol inoculation of calves with

bovine respiratory syncytial virus. Am J Vet Res 1999; 60: 473/

480.

12 Heinen PP, van Nieuwstadt AP, Pol JM, et al. Systemic and

mucosal isotype-specific antibody responses in pigs to experi-

mental influenza virus infection. Viral Immunol 2000; 13: 237/

247.

13 Brooks JV, Orme IM. Evaluation of once-weekly therapy for

tuberculosis using isoniazid plus rifamycins in the mouse aerosol

infection model. Antimicrob Agents Chemother 1998; 42: 3047/

3048.

14 Yamagishi Y, Fujita J, Takigawa K, et al. Experimental pneumo-

nia produced by clinical isolates of Pseudomonas cepacia in mice.

Kansenshogaku Zasshi 1993; 67: 816/822.

15 Canthaboo C, Xing D, Douglas A, Corbel M. Investigation of an

aerosol challenge model as alternative to the intracerebral mouse

protection test for potency assay of whole cell pertussis vaccines.

Biologicals 2000; 28: 241/246.

16 Twisk-Meijssen MJ, Meenhorst PL, van Cronenburg BJ, et al.

The course of Legionella pneumonia in guinea pigs after

inhalation of various quantities of L. pneumophila. Immunobiol-

ogy 1987; 176: 108/124.

17 Coil JA, Dickerman JD, Boulton E. Increased susceptibility of

splenectomized mice to infection after exposure to an aerosolized

suspension of type III Streptococcus pneumoniae. Infect Immun

1978; 21: 412/416.

18 Tang CM, Cohen J, Krausz T, et al. The alkaline protease of

Aspergillus fumigatus is not a virulence determinant in two murine

models of invasive pulmonary aspergillosis. Infect Immun 1993;

61: 1650/

1656.19 Sidransky H, Friedman L. The effect of cortisone and antibiotic

agents on experimental pulmonary aspergillosis. Am J Pathol

1959; 35: 169/184.

20 Sidransky H, Verney E. Experimental aspergillosis. Lab Invest

1962; 11: 1172/1183.

21 Epstein SM, Verney E, Miale TD, Sidransky H. Studies on the

pathogenesis of experimental pulmonary aspergillosis. Am J

Pathol 1967; 51: 769/788.

22 Ford S, Friedman L. Experimental study of the pathogenicity of

Aspergilli for mice. J Bacteriol 1967; 94: 928/933.

23 Sandhu DK, Sandhu RS, Damodaran VN, Randhawa HS. Effect

of cortisone on bronchopulmonary aspergillosis in mice exposed

to spores of various Aspergillus species. Sabouraudia 1970; 8: 32/

38.

24 Kothary MH, Chase T Jr, MacMillan JD. Correlation of elastaseproduction by some strains ofAspergillus fumigatus with ability to

cause pulmonary invasive aspergillosis in mice. Infect Immun

1984; 43: 320/325.

25 Le Conte P, Joly V, Saint-Julien L, et al. Tissue distribution and

antifungal effect of liposomal itraconazole in experimental

cryptococcosis and pulmonary aspergillosis. Am Rev Resp Dis

1992; 145: 424/429.

26 Cacciapuoti A, Loebenberg D, Corcoran E, et al. In vitro and in

vivo activities of SCH 56592 (Posaconazole), a new triazole



7/30/2019 13693780410001712034

9/9

antifungal agent, against Aspergillus and Candida . Antimicrob

Agents Chemother 2000; 44: 2017/2022.

27 Menzel F Jr, Jackson C, Patera A, et al. Combination treatment

of posaconazole and granulocyte colony-stimulating factor

against Aspergillus in a mouse pulmonary infection model. In:

Program and Abstracts of the 42nd Interscience Conference on

Antimicrobial Agents and Chemotherapy, San Diego. Washing-

ton, 2002: abstract M-858.

28 Hinners RG, Burkart JK, Contner GL. Animal exposure cham-bers in air pollution studies. Arch Environ Health 1966; 13: 609/

615.

29 Whitehead GS, Walker JK, Berman KG, et al. Allergen-induced

airway disease is mouse strain dependent. Am J Physiol Lung Cell

Mol Physiol 2003; 285: L32/L42.

30 Cox GM, Harrison TS, McDade HC, et al. Superoxide dismutase

influences the virulence of Cryptococcus neoformans by affecting

growth within macrophages. Infect Immun 2003; 71: 173/180.

31 Bowman JC, Abruzzo GK, Anderson JW, et al. Quantitative

PCR assay to measure Aspergillus fumigatus burden in a murine

model of disseminated aspergillosis: demonstration of efficacy of

caspofungin acetate. Antimicrob Agents Chemother 2001; 45:

3474/3481.

32 Brain JD, Knudson DE, Sorokin SP, Davis MA. Pulmonary

distribution of particles given by intratracheal instillation or byaerosol inhalation. Environ Res 1976; 11: 13/33.

33 Pritchard JN, Holmes A, Evans JC, et al. The distribution of

dust in the rat lung following administration by inhalation and

by single intratracheal instillation. Environ Res 1985; 36: 268/

297.

34 Oberdorster G, Cox C, Gelein R. Intratracheal instillation versus

intratracheal inhalation of tracer particles for measuring lung

clearance function. Exp Lung Res 1997; 23: 17/34.

35 Wheat LJ. Combination therapy for aspergillosis: is it needed, and

which combination? J Infect Dis 2003; 187: 1831/1833.

36 Eisenstein DJ, Biddinger PW, Rhodes JC. Experimental murine

invasive pulmonary aspergillosis. Am J Pathol 1989; 93: 510/515.

37 Piggott WR, Emmons CW. Device for inhalation exposure

of animals to spores. Proc Soc Exp Bio Med 1960; 103:

805/806.

38 Sheppard DC, Filler SG, Rieg G, et al . Development

of a clinically relevant, reproducible inhalational model of

invasive aspergillosis. In: Program and Abstracts of the 43rd

Annual Interscience Conference on Antimicrobial Agents and

Chemotherapy , Chicago, IL. Washington, DC, 2003: abstract M-

372.

39 Loebenberg D, Cacciapuoti A, Parmegiani R, et al. In vitro and

in vivo activities of SCH 4247, the active enantiomer of the

antifungal agent SCH 39304. Antimicrob Agents Chemother 1992;36: 498/501.



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