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Evaluation of Plasma (1→3) β-D-glucan Concentrations in Birds Naturally andExperimentally Infected with Aspergillus fumigatusAuthor(s): Julia D. Burco, Michael H. Ziccardi, Karl V. Clemons and Lisa A. TellSource: Avian Diseases, 56(1):183-191. 2012.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/9697-030111-Reg.1URL: http://www.bioone.org/doi/full/10.1637/9697-030111-Reg.1

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Evaluation of Plasma (1R3) b-D-glucan Concentrations in Birds Naturallyand Experimentally Infected with Aspergillus fumigatus

Julia D. Burco,AD Michael H. Ziccardi,A Karl V. Clemons,B and Lisa A. TellC

ASchool of Veterinary Medicine, Wildlife Health Center, University of California, Davis, CA 95616BCalifornia Institute for Medical Research, San Jose, CA 95128

CSchool of Veterinary Medicine, Department of Medicine and Epidemiology, University of California, Davis, CA 95616

Received 2 March 2011; Accepted and published ahead of print 25 October 2011

SUMMARY. Avian aspergillosis, most often caused by Aspergillus fumigatus, is a common and devastating disease affecting arange of bird species. Early diagnosis is difficult and often unreliable. The current study evaluated the utility of measuring (1R3)-b-D-glucan (BG) concentrations in avian plasma samples to aid in the diagnosis of aspergillosis. We evaluated a commerciallyavailable BG assay (FungitellH, Beacon Diagnostics) using 178 plasma samples from naturally infected, experimentally infected, andaspergillosis-free birds. Although there was variation in BG concentration, as reflected by high standard deviations, seabirds withconfirmed aspergillosis had the highest mean BG concentrations (M 5 3098.7 pg/dl, SD 5 5022.6, n 5 22) followed bycompanion avian species and raptors with confirmed aspergillosis (M 51033.8 pg/dl, SD 5 1531.6, n 5 19) and experimentallyinfected Japanese quail (Coturnix japonica; M 5 1066.5 pg/dl, SD 5 1348.2, n 5 17). Variation in severity of disease, differencesamong species of birds with and without disease, and also different levels in environmental exposure likely contribute to thedifferences among avian groups. The overall sensitivity and specificity of the BG test for diagnosis of aspergillosis in birds was 60.0and 92.7%, respectively, with an overall optimized avian cut-off value of $461 pg/dl for positive disease. Our findings suggest that,although BG concentrations are highly variable between and within different avian groups, it could serve as a useful adjunctivediagnostic test for aspergillosis that is applicable to multiple avian species in some settings, particularly as a negative predictor ofinfection.

RESUMEN. Evaluacion de las concentraciones plasmaticas de b-D-glucano (1 R 3) en aves infectadas natural yexperimentalmente con Aspergillus fumigatus.

La aspergilosis aviar, muy a menudo causada por Aspergillus fumigatus, es una enfermedad comun y devastadora que afecta a unagran variedad de especies aviares. El diagnostico temprano es difıcil y poco confiable. El presente estudio evaluo la utilidad de lamedicion de las concentraciones plasmaticas de b-D-glucano (1 R 3) (BG), en aves para ayudar en el diagnostico de la aspergilosis.Se evaluo un ensayo para BG disponible en el mercado (FungitellH, Diagnostico Beacon) con 178 muestras de plasma de avesinfectadas de forma natural, en forma experimental y de aves libres de aspergilosis. Aunque hubo variaciones en la concentracionde BG, como se refleja en las altas desviaciones estandar, las aves marinas con aspergilosis confirmada tenıan las mayoresconcentraciones promedio de BG (M 5 3,098.7 pg/dl, SD 5 5022.6, n 5 22) seguido por las especies rapaces y de companıa concasos confirmados aspergilosis (M 5 1,033.8 pg/dl, SD 5 1531.6, n 5 19) y por codornices japonesas (Coturnix japonica)infectadas experimentalmente, (M 5 1066.5 pg/dl, SD 5 1348.2, n 5 17). Las variaciones en la severidad de la enfermedad, lasdiferencias entre las especies aviares con y sin la enfermedad, y tambien los diferentes niveles de exposicion al medio ambienteprobablemente contribuyen a las diferencias entre los grupos de aves. La sensibilidad y la especificidad totales de la prueba BG parael diagnostico de la aspergilosis en las aves fueron de 60.0 y 92.7%, respectivamente, con un valor de corte optimizado para aves de$ 461 pg/dl como positivo para la enfermedad. Nuestros hallazgos sugieren que, aunque las concentraciones de BG son muyvariables entre y dentro de los diferentes grupos de aves, este metodo podrıa servir como una util prueba complementaria para eldiagnostico de la aspergilosis, que es aplicable a multiples especies aviares en algunas situaciones, particularmente como un predictornegativo de la infeccion.

Key words: b-D-glucan, aspergillosis, avian, Fungitell assay

Abbreviations: AUC 5 area under the curve; BG 5 b-D-glucan; CAPE 5 Companion Animal Pet and Exotic Service;CFU 5 colony-forming units; ELISA 5 enzyme-linked immunosorbent assay; GMS 5 Gomori’s methenamine silver; PO 5 orally;ROC 5 receiver operator characteristic; Se 5 sensitivity; SID 5 once daily; VMTH 5 Veterinary Medicine Teaching Hospital

Aspergillosis is a devastating fungal disease affecting a varietyof avian species. It is caused by infection with one of the ubiqui-tous saprophytic fungi of the genus Aspergillus, most commonlyAspergillus fumigatus. Although mammals can acquire aspergillosis,birds are more commonly affected by this organism, likely due totheir unique anatomy, especially in the face of external stressors suchas captivity (23,40). Some birds, including production, companion,and captive wild birds, are highly susceptible to infection. Inproduction birds such as young turkeys, chickens, and rheas,aspergillosis causes syndromes ranging from acute to chronic

respiratory infection to mycotic pododermatitis and neurologicimpairment (1,13,39,43). Prevalence in excess of 8.5%, which isassociated with high mortality, has been reported in commercialturkey flocks (29). Frequently, the infected companion pet birdsinclude African grey and Amazon parrots (Psittacus erithacus andAmazona spp.) (9,26). Among captive wild birds, raptors are onegroup that is highly susceptible to infection with Aspergillus spp. withsome species having specific predispositions; those include goshawks(Accipiter spp.), gyrfalcons (Falco rusticolus), red-tailed hawks (Buteojamaicensis), and golden eagles (Aquila chrysaetos) (11,15,35).Captive seabirds housed in zoologic institutions and aquaria, aswell as seabirds undergoing rehabilitation (especially those recoveredin mass numbers during oil spills), comprise another subgroup that

DCorresponding author. Oregon Department of Fish and Wildlife, 7118NE Vandenberg Ave., Corvallis, OR 97330. E-mail: julia.d.burco@state.or.us

AVIAN DISEASES 56:183–191, 2012

183

is extremely susceptible to this devastating disease (10,14,16,19,21,25,37,38).

Due to the prevalence of Aspergillus spp. infection in birds, thepoor prognosis of infected individuals, and the difficulty in detectingearly infection, it is critical to identify diagnostic tests that aid in theearly diagnosis of this disease. Historically, to diagnose aspergillosisin birds, avian practitioners have relied heavily on nonspecificmarkers of inflammation such as plasma protein electrophoresis,white blood cell count, complete blood cell count, and total protein,combined with clinical impressions (6,19,20).

The FDA has approved two commercial enzyme-linked immu-nosorbent assays (ELISA) that are utilized extensively in diagnosinghuman invasive aspergillosis via detection of fungal cell wallcomponents. One is the PlateliaH test (Bio-Rad Laboratories,Hercules, CA), a sandwich ELISA that detects circulating fungalgalactomannan (a component of the fungal cell wall) in serum,plasma, urine, and bronchoalveolar lavage fluid (5,22,32). ThePlatelia test has been evaluated in some avian species and showspromise as a diagnostic tool (3,7,8). The second FDA-approved testis the FungitellH assay (Associates of Cape Cod, Falmouth, MA),which is an antigen test that detects circulating concentrations of(1R3)-b-D-glucan (BG); BG is a major cell wall component ofmost pathogenic fungi except for Cryptococcus neoformans andzygomycetes (24). This assay has been used widely as an aide indetecting invasive aspergillosis in humans (24,33,34). We are awareof only a single study evaluating the test in avian species (36); thestudy involved 18- to 35-day-old broiler chickens with or withoutaspergillosis. Although severity and duration of disease was notmentioned, those authors reported no difference in BG concentra-tion between the infected chickens and the controls (36). Therefore,the objective of this study was to evaluate the potential diagnosticutility of the Fungitell BG assay in detecting aspergillosis in multipleavian species.

MATERIALS AND METHODS

Description of study groups. Aspergillosis-positive birds evaluatedin this study were comprised of three separate groups. The first groupwas represented by birds presented to the Companion Animal Pet andExotic Service (CAPE) at the University of California, Davis VeterinaryMedical Teaching Hospital (‘‘VMTH cases-CAPE’’) between 1995 and2005 (n 5 19). Species represented included raptors, psittacines, andwaterfowl. BG data were obtained using retrospective sampling ofbanked patient plasma samples. Only samples that had been collected4 days or less prior to confirmation of disease (via necropsy or biopsywith culture and histopathology) were included to ensure these samplesaccurately reflected the current disease process. The second aspergillosis-positive avian group included seabirds (‘‘Positive Seabirds’’) representedby 15 individuals (some of which were sampled multiple times; totalsamples n 5 22). For the purposes of this study, seabirds include speciesat historic risk of developing aspergillosis in captivity and are representedby the following families: Gaviidae (loons), Podicipedidae (grebes),Alcidae (common murres and puffins) and Phalacrocoracidae (cormo-rants). Aspergillosis-positive seabirds included birds undergoing reha-bilitation at a facility in northern California; the birds had died or wereeuthanatized due to poor prognosis. These birds were housed in acombination of indoor, net-bottom pens and warm and cold water poolslocated in indoor or outdoor environments. All seabirds had been placedon prophylactic itraconazole upon intake at 20 mg/kg orally (PO) oncedaily (SID) as outlined by Oiled Wildlife Care Network protocols (31).Blood samples were collected upon admittance to the rehabilitationfacility and repeated every 2–4 days until release, death, or euthanasia forhumane reasons. Late disease testing was defined as the last serial sampleprior to death. Only those seabirds that had necropsies performed todefinitively confirm the presence or absence of disease were included in

the study. Any evidence of fungal pathology upon gross necropsyresulted in culturing of tissue samples.

The third aspergillosis-positive group consisted of experimentallyinoculated Japanese quail (Coturnix japonica; ‘‘Infected Quail’’; n 5 17).Ten-day-old Japanese quail were experimentally infected (via trachealinoculation) with A. fumigatus conidia (41). These quail were hatchedand raised at the University of California, Davis Avian Science facility,where they were housed indoors in individual cages with paper towelbelow the cages to absorb organic matter. For purposes of apharmacokinetic study, the birds were divided into three groups; notreatment, voriconazole 20 mg/kg PO SID, and voriconazole 40 mg/kgPO SID (Table 1) (41). Plasma was collected from these birds prior toeuthanasia and stored frozen at 280 C for BG testing.

Four groups of aspergillosis-negative birds were available forcomparison and to aid in developing BG cut-off values to use fordisease detection. The first group included ‘‘Specific-Pathogen-Free(SPF) chickens’’ sampled at 16 days of age (n 5 9). This group alsoprovided information about how the environment may influence BGlevels; these birds were compared with healthy chickens raised in a more-traditional setting. The second group, referred to as ‘‘Hopkinschickens,’’ included normal, healthy 3.5-mo-old chickens housed in anon-SPF environment on a substrate composed of pine shavings (n 554). The third group is referred to as ‘‘control quail’’ and was comprisedof healthy adult Japanese quail (n 5 23) also raised at the Avian Sciencefacility at the University of California, Davis. Samples from theseclinically healthy quail were compared to those from the Aspergillus-infected quail described above. The fourth aspergillosis-negative aviangroup, ‘‘Negative Seabirds,’’ was represented by 38 samples from a totalof 29 individuals. These birds shared the conditions of the aspergillosis-positive seabirds listed above but were not diagnosed as having thedisease based on culture and histopathology at necropsy. Samples fromthese aspergillosis-negative seabirds were compared to aspergillosis-positive seabird samples for validation of the Fungitell assay in anaturally infected avian group.

All sampling, treatment, and care of these animals was approved bythe University of California, Davis Animal Care and Use Committee.

Confirmation and classification of infection status. Histopathol-ogy, using a combination of periodic acid Schiff and Gomori’smethenamine silver (GMS) stains and culture, were the gold standardsfor diagnosis of aspergillosis. Live birds had an initial presumptivediagnosis based on clinical signs or other diagnostics, but only those thatdied on their own, or were euthanatized, were included in the final dataset to confirm disease status via culture and histopathology. Positiveseabird cases were further classified into disseminated (systemic) orlocalized forms based on necropsy findings. Those cases having two orfewer walled-off granulomas were classified as local infections, and thosewith more than two granulomas, diffuse pneumonia, or both wereclassified as having disseminated disease. Only a subset of positiveseabirds, 10 of 15, had sufficient necropsy descriptions to appropriatelyclassify them in this manner. Fungal burden was estimated in inoculatedJapanese quail via histopathology categorization based on the burden offungal hyphae present in GMS-stained tissues and via colony-formingunit (CFU) counts per gram of lung tissue (wet weight). For definingaspergillosis-negative avian groups, only seabirds were confirmednegative via necropsy findings. Other negative birds were clinicallyhealthy and were not experimentally inoculated with A. fumigatus.

b-glucan assay. Plasma BG concentrations were measured using theFungitell BG assay during natural (seabirds, companion birds, andraptors) or experimental infections (quail) and compared to confirmedaspergillosis-negative and clinically healthy avian subgroups. Confirma-tion of positive and negative cases in seabirds and Japanese quail, using agold standard of histopathology and culture, also allowed for furtherevaluation of the Fungitell BG assay to establish cut-off valuerecommendations for categorizing positive and negative cases. Allplasma samples were collected, frozen at 280 C, and sent to BeaconDiagnostics, Associates of Cape Cod Inc., Falmouth, MA where theFungitell BG assay was performed.

Data analysis. BG concentrations were log10 transformed to convertdata to a normal distribution prior to group comparisons. To maintain

184 J. D. Burco et al.

independence of samples, only final plasma samples (i.e., the last plasmasample taken prior to death or euthanasia) were used from individualsthat had repeat sample measurements, over time, for the independentt-test and one-way ANOVA. A one-sided Student’s t-test for groupswith unequal variances was performed to evaluate the difference in BGvalues between overall aspergillosis-positive and -negative birds’ plasma.A one-way ANOVA was then performed to evaluate differences betweengroups. Post hoc multiple comparisons between groups were evaluatedusing a Bonferroni adjustment. Means (M ), standard deviation (SD),and sample size were presented for the various avian groups and includedall data points. Differences between localized and systemic infectionwere evaluated using a Mann-Whitney U-test. Recommended diagnosticdetection cut-offs were based on optimizing specificity and sensitivityusing receiver operator characteristic (ROC) analysis as reportedpreviously (12). For overall evaluation of BG in all avian species,negative samples included SPF chickens, Hopkins chickens, uninfectedquail, and aspergillosis-negative seabirds; positive samples includedinoculated Japanese quail, aspergillosis-positive seabirds, and clinicalraptor or companion avian species seen at VMTH-CAPE. All analysisand diagnostic detection cut-off recommendations were based on BGlevels reported by Associates of Cape Cod Inc. using the Fungitell BGAssay. Statistical analyses were done using appropriate software (STATAv. 11.0, College Station, TX), and an alpha level of 0.05 was used todetermine statistical significance for all tests.

RESULTS

Overall, BG concentrations were measured from 182 avian plasmasamples (124 aspergillosis-negative and 58 aspergillosis-positivesamples) using the Fungitell BG assay. All naturally infected aviancultures were identified as A. fumigatus except for one, Aspergillusniger, in a red-throated loon. No other fungal organisms were isolatedusing routine culture techniques. Despite the variability in results,overall, BG concentrations in samples from aspergillosis-positive birds(M 5 2098.5 pg/dl, SD 5 535.4) were significantly higher than thosein plasma samples from aspergillosis-negative birds (M 5 155.7 pg/dl,SD 5 19.6), t 5 27.96 (Welch’s df, 78), P , 0.001.

There was a significant difference in BG concentrations amongall avian subgroups, F (6,158) 5 18.49, P , 0.001 (Fig. 1).

Aspergillosis-positive seabirds, represented by 15 individuals with atotal of 22 samples, had the highest average BG concentrations(M 5 3098.7 pg/dl, SD 5 5022.6; Table 1). Companion avianspecies and raptors with confirmed aspergillosis seen at theveterinary school (VMTH-CAPE) had a mean BG concentrationof 1033.8 pg/dl (SD 5 1531.6, n 5 19). Experimentally infectedquail had the next highest BG concentration (M 5 1066.5 pg/dl,SD 5 1348.2, n 5 17).

Post hoc comparisons between individual groups demonstratedsignificant differences in BG concentrations between negative con-trol quail and experimentally inoculated quail (P 5 0.01) as well asbetween aspergillosis-negative and -positive seabirds (P , 0.001).Among the disease-negative groups, there was also a significantdifference in BG concentration between SPF chickens and Hopkinschickens (P 5 0.019; Fig. 1).

Dissemination of infection (i.e., multifocal lesions often withvascular invasion) may impact circulating plasma BG concentrationin seabirds. As shown in Table 1, seabirds with known systemicinfection had higher circulating BG concentrations than did thosewith known localized infection; larger controlled studies areneeded to determine whether this is a significant finding. All fiveaspergillosis-positive seabirds that had repeated testing demonstratedan increase in BG over time, with two of the seabirds demonstratingan exponential increase as the disease progressed (Fig. 2a, b). Incontrast, most aspergillosis-negative seabirds demonstrated stableBG concentrations with subsequent sampling (Table 1). Of theexperimentally inoculated quail, two of the three that received notreatment had high BG concentrations (Table 2). However, BGconcentrations were not significantly different amongst treatmentgroups (F (2,14) 5 0.51, P 5 0.64) and duration of infection(days to euthanasia [P 5 0.41]). Individual sample values of theinoculated quail also varied widely (range [R]5 51–4839 pg/dl;Table 2). Nevertheless, many of the high fungal counts and increasedfungal invasion of tissues corresponded to elevated BG levels(Table 2).

ROC curves were generated for seabird, quail, and overall aviansample groups due to sufficient sample size and adequate positiveand negative control groups (Fig. 3). Seabirds were further classified

Fig. 1. Comparison of b-D-glucan concentrations (pg/dl) between multiple aspergillosis-positive (VMTH cases-CAPE, infected quail, andpositive seabirds; represented by dark bars) and aspergillosis-negative (SPF chickens, Hopkins chickens, Control quail, and negative seabirds;represented by light grey bars) avian groups (mean 6 1 SE). Avian groups with different italicized letters were significantly different from each other(P , 0.05, Bonferroni post hoc tests).

b-D-glucan and avian aspergillosis 185

Tab

le1.

Det

aile

dd

escr

ipti

onof

avia

nsu

bgro

up

sin

clu

ded

for

pla

smab

-D-g

luca

nte

stin

g.A

B

Avi

ansp

ecie

sSa

mp

les

Sam

ple

dat

e(s)

Dat

eof

euth

anas

iaor

dea

thD

iagn

osis

Dis

ease

stat

usC

BG

(pg/

dl)

MR

SD

VM

TH

-CA

PE

case

s(n

519

)

Red

-tai

led

haw

k1

2/2/

922/

4/92

Nec

rop

syP

osit

ive

818

Red

-tai

led

haw

k1

12/2

5/92

12/2

6/92

Nec

rop

syP

osit

ive

1814

Gre

ath

orn

edow

l(B

ubo

virg

inia

nus)

14/

21/9

34/

23/9

3N

ecro

psy

Pos

itiv

e70

0R

ed-t

aile

dh

awk

112

/9/9

212

/9/9

2N

ecro

psy

Pos

itiv

e81

5B

lue-

fron

ted

Am

azon

(Am

azon

aae

stiva

)1

8/1/

968/

1/96

Nec

rop

syP

osit

ive

155

Red

-tai

led

haw

k1

12/1

4/95

12/1

5/95

Nec

rop

syP

osit

ive

226

‘‘Par

rot’

’1

10/4

/97

10/8

/97

Nec

rop

syP

osit

ive

372

Red

-tai

led

haw

k1

11/1

4/98

11/1

5/98

Nec

rop

syP

osit

ive

591

Red

-tai

led

haw

k1

11/3

/99

NA

En

dos

cop

y;cu

ltu

reP

osit

ive

1766

Red

-tai

led

haw

k1

9/7/

009/

8/00

Nec

rop

syP

osit

ive

593

Red

-tai

led

haw

k1

6/23

/97

6/25

/97

Nec

rop

syP

osit

ive

765

Blu

ean

dgo

ldm

acaw

(Ara

arar

auna

)1

10/1

7/96

NA

Air

sac

cult

ure

Pos

itiv

e49

1T

ouca

n(R

amph

asto

ssu

lfura

tus)

17/

14/0

77/

14/0

7N

ecro

psy

Pos

itiv

e11

16G

reat

hor

ned

owl

110

/15/

9610

/17/

96N

ecro

psy

Pos

itiv

e10

7G

old

enea

gle

19/

20/9

69/

26/9

6N

ecro

psy

Pos

itiv

e33

7E

clec

tus

par

rot

(Ele

ctus

rora

tus)

111

/30/

9512

/5/9

5N

ecro

psy

Pos

itiv

e86

5O

rin

oco

goos

e(N

eoch

enju

bata

)1

1/19

/99

2/1/

99N

ecro

psy

Pos

itiv

e35

9R

ed-t

aile

dh

awk

18/

23/9

58/

31/9

5N

ecro

psy

Pos

itiv

e69

4B

arn

owl

(Tyt

oal

ba)

110

/19/

0210

/21/

02N

ecro

psy

Pos

itiv

e70

59A

llV

MT

H-C

AP

Eca

ses

1033

107–

7059

1532

SPF

chic

ken

s9

5/6/

2001

5/6/

2001

Nec

rop

syN

egat

ive

5320

–78

19.4

Hop

kin

sch

icke

ns

544/

10/2

001

N/A

Nec

rop

syN

egat

ive

274

23–1

197

257.

4C

ontr

olqu

ail

232/

28/2

001

2/28

/200

1C

lin

ical

lyh

ealt

hy

Neg

ativ

e19

428

–415

113.

1In

ocu

late

dqu

ail

175/

5/01

–5/1

5/01

5/5/

01–5

/15/

01N

ecro

psy

Pos

itiv

e10

6651

–483

913

48

Asp

ergi

llosi

s-n

egat

ive

seab

ird

s(n

538

)

Red

-th

roat

edlo

on(G

avia

stel

lata

)2

7/3/

07,

7/10

/07

7/13

/07

Nec

rop

syN

egat

ive

145,

88C

omm

onm

urr

e(U

ria

aalg

e)2

7/9/

07,

7/10

/07

7/11

/07

Nec

rop

syN

egat

ive

336,

316

Com

mon

mu

rre

26/

29/0

6,7/

5/06

7/6/

06N

ecro

psy

Neg

ativ

e27

8,27

1C

omm

onm

urr

e2

7/13

/06,

7/14

/06

7/15

/06

Nec

rop

syN

egat

ive

102,

43D

oubl

e-cr

este

dco

rmor

ant

(Pha

lacr

ocor

axau

ritu

s)2

7/6/

06,

7/18

/06

7/30

/06

Nec

rop

syN

egat

ive

69,

0

Com

mon

mu

rre

27/

1/07

,7/

5/07

7/5/

07N

ecro

psy

Neg

ativ

e60

,59

Com

mon

mu

rre

310

/2/0

6,10

/11/

06,

10/1

6/06

10/1

9/06

Nec

rop

syN

egat

ive

59,

186,

142

Com

mon

mu

rre

27/

27/0

6,8/

10/0

68/

10/0

6N

ecro

psy

Neg

ativ

e30

,81

Com

mon

mu

rre

25/

5/06

,5/

7/06

5/9/

06N

ecro

psy

Neg

ativ

e14

9,64

Wes

tern

greb

e(A

echm

opho

ruso

ccid

enta

lis)

12/

28/0

63/

6/06

Nec

rop

syN

egat

ive

30C

omm

onm

urr

e1

7/27

/06

7/27

/06

Nec

rop

syN

egat

ive

134

Com

mon

mu

rre

18/

7/06

8/8/

06N

ecro

psy

Neg

ativ

e30

5C

omm

onm

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e1

7/27

/06

7/27

/06

Nec

rop

syN

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ive

745

Com

mon

mu

rre

110

/3/0

610

/5/0

6N

ecro

psy

Neg

ativ

e53

Com

mon

mu

rre

13/

13/0

63/

14/0

6N

ecro

psy

Neg

ativ

e26

2W

este

rngr

ebe

15/

21/0

65/

23/0

6N

ecro

psy

Neg

ativ

e80

Com

mon

mu

rre

15/

16/0

65/

19/0

6N

ecro

psy

Neg

ativ

e52

Cla

rk’s

greb

e(A

echm

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186 J. D. Burco et al.

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.

b-D-glucan and avian aspergillosis 187

into overall samples and late disease samples to give an indication ofhow the severity of disease affected BG measurements. Based on theROC analysis, which aims to optimize the sensitivity and specificityof a test, the recommended cut-off for detection of aspergillosiswas $669 pg/dl for Japanese quail, $461 pg/dl for seabirds (overalland late testing), and $461 pg/dl for all avian species combined(Table 3). Sensitivity increased in samples from birds late in thecourse of infection; this increased the negative predictive value andthe percentage of samples that were correctly classified.

DISCUSSION

Aspergillosis remains a challenging disease to diagnose and treat.Our data indicate that the Fungitell assay, measuring circulating BG,may be a useful serologic test to aid in the diagnosis of disease in theavian patient. Interestingly, even birds without confirmed aspergil-losis had relatively high levels of circulating BG (M 5 208.7 6

36.5 pg/dl), considering that the manufacturer’s recommended cut-off in humans is $80 pg/dl. One reason for this elevated baselinelevel may include that of increased exposure to Aspergillus spp. fromthe type of housing or local environmental conditions the birds areexposed to. For instance, none of the SPF chickens had BGconcentrations higher than 80 pg/dl, whereas the Hopkins chickensthat were housed in a more traditional environment with someorganic matter (i.e., pine shavings) had much higher and widelyvariable plasma BG concentrations. The low BG concentrations inthe SPF chickens is likely due to the strict housing requirements and

limited pathogen exposure; these conditions made the SPF chickensan excellent negative control group for comparison to all otheravian groups. Other postulated possibilities concerning the highBG measurements in birds include diet preparation (44) as wellas environmental contamination, asymptomatic yeast or fungal

Fig. 2. Repeated measurements of b-D-glucan concentrations, overtime, in two cases of aspergillosis in common murres (Uria aalge) thatdied of systemic aspergillosis.

Table 2. (1R3)-b-D-glucan (BG) concentrations from Japanesequail (Coturnix japonica) experimentally inoculated with Aspergillusfumigatus and relationship to fungal load status.A

Voriconazoledose (mg/kg)

Days toeuthanasia

BG(pg/dl)

Fungal load status

GMSlobe A

GMSlobe B

log10

CFU/gram lung

0 5 4839 3+ 3+ 5.180 5 1666 4+ 4+ 2.70 10 106 2+ 1+ 0

20 5 51 Negative 1+ NP20 5 1254 2+ 1+ 020 5 358 Negative Negative 040 5 115 4+ Negative 1.6340 5 3968 3+ Negative 4.0840 5 669 1+ 1+ 5.1320 10 846 Negative Negative 1.3220 10 1060 2+ Negative 020 10 143 Negative Negative 1.720 10 338 Negative NP 1.3440 10 87 Negative NP 1.640 10 765 Negative 1+ 1.940 10 924 3+ 2+ 040 10 942 None NP 2.35

AIndividuals were treated with varying dosages of oral voriconazole(0 mg/kg [n 5 3], 20 mg/kg [n 5 7], and 40 mg/kg [n 57]) andeuthanasia was performed on day 5 or 10; plasma BG concentrationswere measured on plasma samples obtained immediately prior toeuthanasia. Fungal load status was evaluated by histopathology usingGMS stain; severity of fungal infection was classified as rare fragments(1+), light hyphae (2+), moderate hyphae (3+), and heavy hyphae (4+)and counts of CFU (log10) per gram of lung tissue. NP 5 notperformed.

Fig. 3. ROC curves for evaluation of the Fungitell assay in diagnosingaspergillosis in multiple avian groups. Separate ROC curves represent allavian groups (‘‘Overall avian’’) and are then broken down into seabirds incare at a rehabilitation center and Japanese quail (Coturnix japonica). ‘‘Allseabird’’ refers to all samples taken from the birds during their stay in therehabilitation center and ‘‘late disease seabird’’ indicates the last samplestaken before euthanasia or death due to aspergillosis.

188 J. D. Burco et al.

infection, or both (4). On average, seabirds with aspergillosis hadthe highest concentration of circulating BG. This is likely due to theincreased incidence of acute systemic disease, which often invadessurrounding tissues and vasculature and creates a miliary, consoli-dated pneumonia with large numbers of branching hyphae. Thesehyphae or hyphal fragments are likely shed into the blood andcontribute to the higher concentration of BG. Other birds may havemore localized and chronic infections that can result in walled-offgranulomas within air sac membranes or the syrinx, therebyminimizing hematogenous spread of the fungus and a comparativedecrease in the release of fungal cell wall components to thecirculation. This difference in BG concentration was demonstratedby a subset of seabirds with solitary walled-off lesions vs. those thathad systemic, multifocal lesions. The high BG concentrations inseabirds with natural infections, when compared to the experimen-tally infected Japanese quail and birds entering the CAPE service,may also be related to sampling, as all of the seabirds had diseasesevere enough that they died or were euthanatized. In contrast, theInfected Quail were euthanatized at set time-points, and some of theCAPE birds may have had milder forms of infection or the BG wasdetected earlier in the disease process.

As depicted from the serial sampling of two common murres thatdeveloped acute systemic disease, BG concentrations rose exponen-tially as the disease process progressed (Fig. 2a, b). Increased presenceof tissue invasion and fungal loads in inoculated quail with elevatedBG concentrations also confirmed this pattern. Importantly, thesedata illustrate that BG levels may not rise to detectable or significantlevels until late in the disease process. Consequently, the BG assay maynot be ideal for detecting early, localized, or subclinical infections.

ROC curve analysis is often used for its ability to determineoptimum diagnostic cut-off values by maximizing both sensitivityand specificity of a diagnostic test and provides a comparison of howwell a test predicts disease comparing the area under the curve(AUC) value. Based on our analysis of the present data set, werecommend a cut-off concentration of $461 pg/dl in seabirds,$669 pg/dl for Japanese quail, and $461 pg/dl for overall avianspecies. These values correctly classified disease 81.4%, 80.0%, and83.2% of the time, respectively. As demonstrated by the higher AUCvalue of late-disease seabirds vs. early disease seabirds, BG testing ismore accurately predictive later in the disease process (Table 3).Sensitivity of the BG test was similar to that estimated for evaluationof another fungal cell wall antigen test, galactomannan, in live birdswith suspect infections in one study (sensitivity [Se] 5 67%), but ismuch improved when compared to the utility of galactomannan inraptors (Se 5 12%) (3,8). In our study, we found a strong, positivepredictive value with the recommended cut-off value indicating thatthere is confidence in a positive result. However, in our study

animals, we did not have many individuals that had other fungal orbacterial diseases or were on long-term antibiotics that may haveemphasized the non-specificity of the Fungitell assay. Human studieshave demonstrated higher negative predictive values, indicating thatBG is a good test to rule out invasive fungal disease (30). TheFungitell BG test could serve a similar purpose in avian medicinewith a lower cut-off value. Also, it might be advisable to have a lessconservative, somewhat lower BG cut-off value to detect moresubclinical disease or to use BG as an adjunctive confirmatory test inconjunction with screening with another diagnostic such as plasmaprotein electrophoresis. As evidenced by ROC analysis, late-diseasesamples more accurately reflected the true disease status, likelyindicating that BG may not be observed at high concentrations in theblood until the disease has progressed significantly. The comparisonof type of infection (localized vs. systemic) also indicates that BGdoes not appear to be an ideal detection method for diagnosing earlyor localized infections.

The recommended BG cut-off values determined from our studyare general guidelines and should be interpreted with caution inother avian patients, as these patients received varying doses ofantifungals for prophylaxis and treatment of disease, and mostaspergillosis-positive birds had very severe disease resulting in deathor euthanasia. However, susceptible bird species are frequentlystarted on prophylactic antifungals, and many of the terrestrial birdshad initial treatment based on a presumptive diagnosis which couldaffect later BG assay results. Studies done in murine models havedemonstrated a decrease in BG levels with antifungal treatment (28).

Another issue to consider with the use of BG as a diagnostic foraspergillosis is its lack of specificity for detecting Aspergillus spp., as italso detects other fungal infections such as Candida spp. Candidiasisoccurs relatively frequently in stressed, captive birds but is usuallydistinguishable from aspergillosis based on clinical presentationand ease of diagnosis with crop or gastrointestinal cultures (18). Inaddition, BG values have been shown to be affected by somebacterial infections (2) and certain antibiotics such as amoxicillinand clavulinic acid in humans (27). Consequently, future studiesshould examine this potential of cross-reactivity with other clinicallyimportant fungal infections as well as false-positive reactivity tovarious antimicrobials commonly used in avian medicine.

Finally, sample handling and processing of specimens need tobe taken into consideration when using and evaluating BG con-centrations as some medical supplies, such as cotton balls, gauze,etc., have been shown to affect BG concentrations (17). Similarly,exposing a plasma sample to a contaminated fungal environmentmay also impact results.

In summary, the Fungitell assay, measuring BG, appears to beuseful as an adjunctive diagnostic for aspergillosis in a variety of

Table 3. ROC curve values and recommended cut-off levels for detection of aspergillosis in seabirds, Japanese quail (Coturnix japonica), andoverall avian groups.AB

Avian groups Obs Cut-off (pg/dl) AUCAsymptotic normal

(95% CI)% Correctly

classified Se Sp PPV NPV

Seabirds

All samples 60 $461 0.854 0.756–0.952 81.4 54.6 97.3 91.7 76.6Late disease 52 $461 0.923 0.848–0.999 88.5 66.7 97.3 90.7 87.8

Quail 40 $669 0.762 0.587–0.938 80 58.8 100 100 76.7Overall avianA 182 $461 0.827 0.761–0.877 83.0 60.0 92.7 81.4 83.4

ARepresented by naturally infected (seabirds undergoing rehabilitation and companion and raptor species entering a veterinary teaching hospital)and experimentally infected Japanese quail. Negative control samples included negative seabirds, healthy quail, SPF chickens and Hopkins chickens(normal chickens).

BObs 5 observed total positive and negative control samples; AUC 5 area under the curve; Se 5 sensitivity; Sp 5 specificity; PPV 5 positivepredictive value; NPV 5 negative predictive value.

b-D-glucan and avian aspergillosis 189

avian species. However, the cost of the testing, wide variation ofresults among avian groups and stages of disease, high baseline BGvalues in healthy birds, and potential for cross-reactivity make thistest less practical for the average avian practitioner with individualpatients. Where the BG assay may be more useful is in the setting ofoutbreaks in flocks, or in the rehabilitation settings described in thispaper, where large numbers of birds are at risk or potentially havedisease. Avian BG concentrations were found to be much higher thanthose found in human samples, indicating a variation in the pathogenesisof disease and the sources of exposure to BG in birds. The utility of theFungitell BG assay to detect early disease may be limited, but showspromise as a noninvasive confirmatory test, particularly as a negativepredictor, when combined with other diagnostic tests and clinicaljudgment. Additional studies with larger numbers of birds of differentspecies are needed to substantiate the utility of the BG assay as anadjunctive diagnostic test for avian aspergillosis.

REFERENCES

1. Akan, M., R. Haziroglu, Z. Ilhan, B. Sareyyupoglu, and R. Tunca. Acase of aspergillosis in a broiler breeder flock. Avian Dis. 46:497–501. 2002.

2. Albert, O., D. Tobias, C. Strady, J. Cousson, C. Delmas, V. Vernet,and I. Villena. Reactivity of (1R3)-b-D-glucan assay in bacterial blood-stream infections. Eur. J. Clin. Microbiol. Infect. Dis. 11:1453–1460. 2011.

3. Arca-Ruibal, B., U. Wernery, R. Zachariah, T. A. Bailey, A. DiSomma, C. Silvanose, and P. McKinney. Assessment of a commercialsandwich ELISA in the diagnosis of aspergillosis in falcons. Vet. Rec.158:442–444. 2006.

4. Cafarchia, C., A. Camarda, D. Romito, M. Campolo, N. C. Quaglia,D. Tullio, and D. Otranto. Occurrence of yeasts in cloacae of migratorybirds. Mycopathologia 161:229–234. 2006.

5. Clancy, C. J., R. A. Jaber, H. L. Leather, J. R. Wingard, B. Staley,L. J. Wheat, C. L. Cline, K. H. Rand, D. Schain, M. Baz, and M. H.Nguyen. Bronchoalveolar lavage galactomannan in diagnosis of invasivepulmonary aspergillosis among solid-organ transplant recipients. J. Clin.Microbiol. 45:1759–1765. 2007.

6. Cray, C., and L. Tatum. Application of protein electrophoresis inavian diagnostics. J. Avian Med. Surg. 12:4–10. 1998.

7. Cray, C., D. Reavill, A. Romagnano, F. V. Sant, D. Champagne, R.Stevenson, V. Rolfe, C. Griffin, and S. Clubb. Galactomannan assay andplasma protein electrophoresis findings in psittacine birds with aspergillosis.J. Avian Med. Surg. 23:125–135. 2009.

8. Cray, C., T. Watson, M. Rodriguez, and K. L. Arheart. Applicationof galactomannan analysis and protein electrophoresis in the diagnosis ofaspergillosis in avian species. J. Zoo Wildl. Med. 40:64–70. 2009.

9. Dalhousen, R. D. Implications of mycoses in clinical disorders.In: Clinical avian medicine, vol 2. G. J. Harrison, and T. L. Lightfoot, eds.Spix Publishing, Inc., Palm Beach, FL. pp. 691–703. 2004.

10. Daoust, P.-Y., G. Conboy, S. McBurney, and N. Burgess. Interactivemortality factors in common loons from Maritime Canada. J.Wildl. Dis.34:524–531. 1998.

11. Deem, S. Fungal diseases of birds of prey. Vet. Clin. N. Am. ExoticAnim. Pract. 6:363–376. 2003.

12. Fawcett, T. An introduction to ROC analysis. Pattern Recog. Lett.27:861–874. 2006.

13. Femenia, F. O., J.-J. Fontaine, S. Lair-Fulleringer, N. Berkova, D.Huet, N. Towanou, F. Rakotovao, O.-I. Granet, G. L. Loc’h, P. Arne, andJ. Guillot. Clinical, mycological and pathological findings in turkeysexperimentally infected by Aspergillus fumigatus. Avian Pathol. 36:213–219. 2007.

14. Flach, E., M. Stevenson, and G. Henderson. Aspergillosis in gentoopenguins (Pygoscelis papua) at Edinburgh Zoo, 1964 to 1988. Vet. Rec.126:81–85. 1990.

15. Friend, M. Aspergillosis. In: Field manual of wildlife diseases: generalfield procedures and diseases of birds. National Wildlife Health Center,USGS, Madison, WI. pp. 129–134. 2001.

16. Garcia-Borboroglu, P., P. D. Boersma, V. Ruoppolo, L. Reyes, G. A.Rebstock, K. Griot, S. R. Heredia, A. C. Adornes, and R. P. da Silva.Chronic oil pollution harms Magellanic penguins in the Southwest Atlantic.Marine Pollution Bulletin 52:193–198. 2006.

17. Kanamori, H., K. Kanemitsu, T. Miyasaka, K. Ameku, S. Endo, T.Aoyagi, K. Inden, M. Hatta, N. Yamamoto, H. Kunishima, H. Yano, K.Kaku, Y. Hirakata, and M. Kaku. Measurement of (1R3)-b-D-glucanderived from different gauze types. Tohoku J. Exp. Med. 217:117–121.2009.

18. Hubbard, G. B., R. E. Schmidt, D. L. Eisenbrandt, W. M. Witt, andK. C. Fletcher. Fungal infections of ventriculi in captive birds. J. Wildl. Dis.21:25–28. 1985.

19. Ivey, E. Serologic and plasma protein electrophoretic findings in 7psittacine birds with aspergillosis. J. Avian Med. Surg. 14:103–106. 2000.

20. Jones, P., and S. Orosz. The diagnosis of aspergillosis in birds. Semin.Avian Exot. Pet 9:52–58. 2000.

21. Khan, Z., M. Pal, D. Paliwal, and V. Damodaran. Aspergillosis inimported penguins. Sabouraudia 15:43–45. 1977.

22. Klont, R. R., M. A. S. H. Mennink-Kersten, and P. E. Verweij.Utility of Aspergillus antigen detection in specimens other than serumspecimens. Clin. Infect. Dis. 39:1467–1474. 2004.

23. Lightfoot, T. Major anatomical and physiological differences betweenbirds and mammals. In: Atlantic Coast Veterinary Conference, Atlantic City,NJ. 2001.

24. Marty, F. M., and S. Koo. Role of (1R3)-b-D-glucan in thediagnosis of invasive aspergillosis. Med. Mycol. 47:S233–S240. 2009.

25. Massey, J. G. Summary of an oiled bird response. J. Exot. Pet Med.15:33–39. 2006.

26. Mayahi, M., S. Esmaeilzadeh, R. Kiani, and M. Fatahinia. Aspergillusfumigatus infection in a green parrot. Comp. Clin. Pathol. 17:279–281.2008.

27. Mennink-Kersten, M. A. S. H., A. Warris, and P. E. Verweij. (1R3)-b-D-glucan in patients receiving intravenous amoxicillin clavulanic acid.New Engl. J. Med. 354:2834–2835. 2006.

28. Mitsutake, K., S. Kohno, T. Miyazaki, Y. Yamamoto, K. Yanagihara,H. Kakeya, A. Hashimoto, H. Koga, and K. Hara. Detection of (1R3)-beta-D-glucan in a rat model of aspergillosis. J. Clin. Lab. Anal. 9:119–122.1995.

29. Morris, G., M. H. Kokki, K. Anderson, and M. D. Richardson.Sampling of Aspergillus spores in air. J. Hosp. Infect. 44:81–92. 2000.

30. Obayashi, T., K. Negishi, T. Suzuki, and N. Funata. Reappraisal ofthe serum (1R3)-b-D-glucan assay for the diagnosis of invasive fungalinfections—a study based on autopsy cases from 6 years. Clin. Infect. Dis.46:1864–1870. DOI: 10.1086/588295. 2008.

31. Oiled Wildlife Care Network. Protocols for the care of oil-affectedbirds. Wildlife Health Center, University of California, Davis, Davis, CA.pp. 23–25. 2000.

32. Penack, O., P. Rempf, B. Graf, I. W. Blau, and E. Thiel. Aspergillusgalactomannan testing in patients with long-term neutropenia: implicationsfor clinical management. Ann. Oncol. 19:984–989. 2008.

33. Persat, F., S. Ranque, F. Derouin, A. Michel-Nguyen, S. Picot, andA. Sulahian. Contribution of the (1R3)-b-D-glucan assay for diagnosis ofinvasive fungal infections. J. Clin. Microbiol. 46:1009–1013. 2008.

34. Pickering, J. W., H. W. Sant, C. A. P. Bowles, W. L. Roberts, andG. L. Woods. Evaluation of a (1R3)-b-D-glucan assay for diagnosis ofinvasive fungal infections. J. Clin. Microbiol. 43:5957–5962. 2005.

35. Redig, P. T. Avian emergencies. In: Manual of raptors, pigeons andwaterfowl. Iowa State University Press, Ames, IA. pp. 30–41. 1996.

36. Shivaprasad, H. L., M. Franca, and C. Cray. Study of aspergillosisserodiagnostics in naturally infected chickens and turkeys. Proc. of theAnnual Conference of the Association of Avian Veterinarians.. 33–34. 2011.

37. Skerratt, L. F., J. C. Franson, C. U. Meteyer, and T. E. Hollmen.Causes of mortality in sea ducks (Mergini) necropsied at the USGS–National Wildlife Health Center. Waterbirds 28:193–207. 2005.

38. Stocker, L. Avian wildlife disease. In: Practical wildlife care. L.Stocker, ed. Blackwell Publishers, Malden, MA. p. 89. 2005.

39. Stoute, S. T., A. A. Bickford, R. L. Walker, and B. R. Charl-ton. Mycotic pododermatitis and mycotic pneumonia in commercial

190 J. D. Burco et al.

turkey poults in northern California. J. Vet. Diagn. Invest. 21:554–557.2009.

40. Tell, L. A. Aspergillosis in mammals and birds: impact on veterinarymedicine. Med. Mycol. 43:71–73. 2005.

41. Tell, L. A., K. V. Clemons, Y. Kline, L. Woods, P. H. Kass, M.Martinez, and D. A. Stevens. Efficacy of voriconazole in Japanese quail(Coturnix japonica) experimentally infected with Aspergillus fumigatus. Med.Mycol. 48:234–44. 2009.

42. Xavier, M. O., M. P. Soares, A. R. M. Meinerz, M. O. Nobre, L. G.Osorio, R. P. d. Silva Filho, and M. C. A. Meireles. Aspergillosis: a limitingfactor during recovery of captive magellanic penguins. Braz. J. Microbiol.38:480–484. 2007.

43. Zafra, R., J. Perez, R. Perez-Ecija, C. Borge, R. Bustamante, A.Carbonero, and C. Tarradas. Concurrent aspergillosis and ascites with highmortality in a farm of growing broiler chickens. Avian Dis. 52:711–713.2008.

44. Zekovic, D. B., S. Kwiatkowski, M. M. Vrvic, D. Jakovljevic, andC. A. Moran. Natural and modified (1R3)-b-D-glucans in healthpromotion and disease alleviation. Crit. Rev. Biotechnol. 25:205–230. 2005.

ACKNOWLEDGMENTS

Partial funding for this project was provided by the Oiled WildlifeCare Network. We thank Dr. Barbara Byrne at the University ofCalifornia Veterinary Medical Teaching Hospital (VMTH) microbiol-ogy laboratory for technical advice and expertise. We thank theclinicians and staff from the University of California, Davis VMTH andthe San Francisco Bay Oiled Wildlife Care and Education center inFairfield, CA. We also gratefully acknowledge Dr. Malcolm Finkelmanand Associates of Cape Cod Inc. for analysis of samples from quail,chicken, and VMTH cases at no cost.

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