research article clinical effects of gamma-radiation

Research ArticleClinical Effects of Gamma-Radiation-ResistantAspergillus sydowii on Germ-Free Mice ImmunologicallyProne to Inflammatory Bowel Disease

Alexander Rodriguez-Palacios12 Natalia Aladyshkina12 Mauricio Retuerto3

Christopher L Hager3 Sanja Ilic4 Mahmoud A Ghannoum35 and Fabio Cominelli125

1Division of Gastroenterology and Liver Diseases Department of Medicine Digestive Health Research InstituteCase Western Reserve University School of Medicine Cleveland OH 44106 USA2Cleveland Digestive Diseases Research Core Center Case Western Reserve University School of Medicine Cleveland OH 44106 USA3Center for Medical Mycology Department of Dermatology Case Western Reserve University School of Medicine ClevelandOH 44106 USA4Department of Human Sciences Human Nutrition College of Education and Human Ecology The Ohio State UniversityColumbus OH 43210 USA5Digestive Health Institute University Hospitals Case Medical Center Cleveland OH 44106 USA

Correspondence should be addressed to Alexander Rodriguez-Palacios axr503caseedu

Received 1 June 2016 Accepted 18 July 2016

Academic Editor Martin Hoenigl

Copyright copy 2016 Alexander Rodriguez-Palacios et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

We report and investigated a case of inadvertent contamination of 125mice (housed in two germ-free positive-pressurized isolators)with emerging human and coral pathogen Aspergillus sydowii The infected mice correspond to genetic line SAMP1YitFc whichhave 100 immune predisposition to develop Crohnrsquos disease-like spontaneous pathologies namely inflammatory bowel disease(IBD) Pathogen update based on a scoping review of the literature and our clinical observations and experimentation are discussedThe unwanted infection of germ-free mice (immunologically prone to suffer chronic inflammation) with human pathogen Asydowii resulted in no overt signs of clinical disease over 3-week exposure period or during DSS-induced colitis experimentsResults and observations suggest that A sydowii alone has limited clinical effect in immunocompromised germ-free mice or thatother commensal microbial flora is required for Aspergillus-associated disease to occur

1 Introduction

To date there are a few studies reporting the severityof primary and disseminated gastrointestinal aspergillosisin immunocompromised hosts including children [1] (seealso references in [2]) Although aspergilloses are primarilyconfined to aerobic surfaces systemic infections and rapidmortality are known to have occurred in humans and domes-tic animals [3] especially during disruption of the gastroin-testinal barrier and associated microbiota (dysbiosis) whichincreases susceptibility to fatal systemic fungal infections [2]Of all invasive fungal infections aspergilloses account for

24 of human patients With aspergillosis one in every fivepatients develops fatal invasive infection

Of epidemiological interest we report a well-knownfungal pathogen of marine corals and humans Aspergillussydowii as an unwanted contaminant of a germ-free (GF)colony of SAMPYitFc mice immunologically prone to sufferspontaneous inflammatory bowel disease (IBD Crohnrsquos-like intestinal disease) and other multiorgan inflammatorycomplications Such Crohnrsquos-like intestinal disease occurs in100 mice in both specific pathogen-free (SPF) and germ-free conditions Among the aspergilloses [3] A sydowiiis epidemiologically interesting because it is a pathogen

Hindawi Publishing CorporationJournal of PathogensVolume 2016 Article ID 5748745 7 pageshttpdxdoiorg10115520165748745

2 Journal of Pathogens

known for causing mortality in Sea Fan corals (Gorgoniaventalina animals that feed on plankton) threatening variousCaribbean reefs since the 1990s [4] where it seems to remaingeographically confined Infections cause circular purpleareas of necrosis and mortality that is not evidenced in othercorals Initially isolated fromhumans in theUSA in 1989 fromintegumentary (eg skin and nails) infections [5] A sydowiihas been isolated in recent years from patients in other moredistant regions (Czech Republic Russia) [6 7]

Mice under germ-free conditions have a very high risk ofexuberant infections by any commensal pathogenic or envi-ronmental microbes Here we report a case of 125 germ-freeSAMPYitFc mice inadvertently exposed to A sydowii viacommercial germ-free grade irradiated contaminated feedThe (SAMPYitFc) mice contaminated in this study wereparticularly deemed prone toA sydowii opportunistic fungalinfections because (i) they were germ-free and thereforelacked protective competitive surface microbial commensalflora (microbiota) in both gut and skin (ii) the mousegenetic line is immunologically prone to progressive chronicintestinal inflammation conjunctivitis and ulcerative der-matitis (iii) they have decrease clearance of pathogens likeSalmonella enteritidis [8] and (iv) because germ-free micehave no protective antibody-mediated adaptive (memory)immunity against microorganisms due to lack of exposure tocommensal or pathogenicmicrobes when raised in germ-freeconditions

2 Case

To historically contextualize the relevance of the presentreport we first conducted a systematic scoping review ofpublications onA sydowii since its discovery in plants in 1980[9] until February 15 2016 using PubMed and ldquoAspergillussydowiirdquo as search keyword term We then examined the100 top hits in Google Scholar (ie articles with the highestcitations) and secondarily reviewed the title relevance oftheir Google citations referencing the papers for the years2013ndash2015 to capture recently published studies with lowcitation indexes Analysis of titles abstracts and content of97 selected research studies indicates that the frequency ofstudies on emerging bioengineering and basic research onA sydowii is growing exponentially since its isolation fromaffected Sea Fan corals was linked to the near extinctionof some reefs in the West Indies in the 1990s (Figure 1)With the scoping review it also became evident that little isknown about the intestinal colonization and pathogenicityof A sydowii in mammals Because the putative role of themycobiome as disease contributor in human inflammatorybowel disease (IBD) is increasing we deemed pertinent toreport measurable mortality and clinical morbidity outcomesduring this contamination event to further understand therole of the mycobiome in animals Of relevance to IBDthe affected genetic mouse line is one of only two availablespontaneous mouse models for Crohnrsquos disease-like enteritisprimarily affected by ileitis (inflammation of ileum ie thelast segment of the small intestine)

Fungal infections are opportunistic and commonly occurin immunocompromised hosts [4 6] in both humans and

0

2

4

6

8

10

12

1980

1985

1990

1995

2000

2005

2010

2015

Humans

Infected Sea Fanrepresentation

Plants

Rese

arch

artic

les P

ubM

ed n

See Fans

Year of publication

Mice this studyJuly 17th

2016

R2 = 08879

y = 00008x3 minus 46467x2 + 92462x minus 6E + 06

Figure 1 The frequency of peer-review studies on Aspergillussydowii has increased exponentially over the last decade with thecurve shoulder starting after the fungus was linked to emergingepidemic infections in Caribbean corals The first report was inplants in Egypt 1980 [9] Systematic scoping review of articles inPubMed until February 2016 Two-step moving average (solid line)and polynomial estimation curve (dashed line 119910 = 119899 of publicationsestimate 119909 year)

animals Of immunological relevance the SAMP1YitFcmicecorrespond to a genetic line known for suffering spontaneousprogressive intestinal inflammation with unique three-dimensional structural similarity to human Crohnrsquos diseaselesions [10] SAMP1YitFc mice also have complex immuno-logical abnormalities with 100 penetrance Although par-tially understood the immunological abnormality in thesemice appears to transition from T-helper-1 (Th1) driveninflammation at young age to an inflammatory process drivenby a combinedTh1Th2 alteration in animal adulthood whichaccentuates as disease worsens Mechanistically mice havegut epithelial barrier and pathogen clearance dysfunctions[8 11] which could enhance their predisposition in GFconditions to overt fungal infections

Herein we describe the unexpected rapid and simultane-ous contamination of 125 mice housed in two GF positive-pressurized isolators with A sydowii in an established GFbreeding facility (Cleveland OH USA) in June of 2015 Anoverview of the timeline during the contamination event andsubsequent experimentation is presented in Figure 2 (iefungus detection colony dismantling and monitoring micefor signs of disease using an in-house caging system)

In brief two GF isolators having weekly husbandryand high mouse density (2-3 breederscage lt10 unweanedmicecage 12ndash14 cagesisolator) were observed at main-tenance to have unexpected condensation and increasedhumidity as well as containing feed pellets covered withmold on a day of routine inspection in June 2015 (day 0)Following removal surface disinfection with Spor-Klenz(1 hydrogen peroxide 10 acetic and 008 paracetic acidSteris Corp Catalog 6525) and quarantine of all cages (no

Journal of Pathogens 3

Gnotobiotic housing in cages

Rout

ine f

eed

repl

aced

Mol

d in

one

pell

et-c

age

All

cage

s affe

cted

Col

ony

dism

antle

d 12

5 m

ice t

rans

ferr

ed to

in

-hou

se D

CTB

cagi

ngF a

cilit

y di

sinfe

ctio

nFungal traps

testing Col

ony-

anim

als d

estro

yed

DSS

expe

rimen

t

Isol

ator

s tes

ted

nega

tive

Qua

rant

ine a

nd te

sting

ex

tend

ed fo

r 8 w

eeks

for 1

5 y

ears

(Week) 0 1 2 3Increasing humidity

Germ-free pressurized isolators

Controlled humidity

Col

ony

teste

d ne

gativ

e

Figure 2 Timeline of A sydowii contamination of germ-freemouse facility and implementation of germ-free in-house cage-based housing system to monitor 125 animals and conduct DSScolitis experimentation

handlingopening of lids) was initiated Visual inspection offeed and bedding material revealed that most cages in bothisolators exhibited various degrees of contamination withina week of mold detection Due to the severity extent andthe rate of feed contamination the colony was dismantledand tests for fungal identification were performed All micewere transferred to an in-house cage system and each cageand animal were monitored for overt signs of depressiondiarrhea and ocular skin or respiratory disease at thepopulation level (using available scoring systems if needed)to determine the role of the infection in the overall healthstatus of the mice All mice were inspected daily maintaininggerm-free grade sterility to prevent contamination by othermicrobes

Over the following 2-week period the mold was con-firmed as A sydowii and the animals were determined to beclinically sound indicating that heavy exposure toA sydowiispores do not necessarily result in mortality or overtlyrespiratory ocular dermatological infections or diarrheain GF mice prone to immune deficiencies and intestinaldisease Single colony PCR and Sanger sequencing of 18Sinternal transcribed spacers (ITS1ndash4) indicated that threefungal isolates from both isolators were highly similar toA sydowii sequences deposited in GenBank (Figures 3(a)ndash3(e)) Primers ITS1 (51015840TCC GTA GGT GAA CCT GCG G)and ITS4 (51015840TCC TCC GCT TAT TGA TAT GC) were usedin a PCR amplification protocol described in detail in [12]using DreamTaq Green PCRMasterMix (Thermoscientific)01 gL bovine serum albumin 1 of dimethyl sulfoxide6mMMgCl

2 and a final primer concentration of 400 nM

Initial denaturation at 94∘C for 3min was followed by 35cycles of denaturation for 30 s each at 94∘C annealing at50∘C for 30 s and extension at 72∘C for 1min Upon cyclingcompletion there was a final extension time of 5min at 72∘CPCR amplification of the ribosomal internal transcribedspacer 1 (ITS-1) and ITS-2 regions has been recommendedas targets for molecular identification of medically importantAspergillus and as deposition of sequences at GenBank [13]

The ITS region 4 was also sequenced in this study sequencesfrom this report are available in GenBank under accessionnumbers KU757469 KU757470 and KU757471 ldquoFungaltrapsrdquo (soiled moistened mouse cages incubated for 3 weeksat 23∘C) with cages used during the clinical observationperiod confirmed the presence of A sydowii (Figure 3(f))supporting the lack of disease induction in these immune-defective inflammation-prone mice despite the presence ofmold in the fecesbedding material

Given the reported risk for integumentary infections inhumans the rapid growth of the mold and the critical needto eradicate it from the GF equipment and facility all animalswere euthanized after the clinical observation period leavinga small cohort of mice for BSL-2 grade experimentation todetermine the role of the fungal infection on the severity ofacute colitis induction with dextran sulfate sodium (DSS)DSS colitis is a valid established method to assess the roleof the gut microbiome in infection pathogenicity includingCandida spp and the immunology of inflammatory boweldiseases [10 14] In brief 7 days of dextran sulfate sodiumsupplementation (DSS) in the drinking water (3 wv) leadsto the colitis in the presence of gut microbiota Compared toSAMPYitFc mice colonized with regular specific pathogen-free gut microbiota the GF mice contaminated in this studydid not exhibit clinical signs of acute colitis based on clinicalsigns that are typical for DSS-induced diarrhea body weightloss or reduction of the cecum size (Figures 4(a)ndash4(c))

After the dismantling of the isolators and the euthanasiaof the entire GF mouse colony stringent disinfection proto-cols have been implementedThese were based on quaternaryammonium-based cleaner to remove organic matter 70ethanol to remove grease and dehydrate and Spor-Klenz toeliminate microbial spores (bacterial and fungal) on delicateGF equipment Floors and other surfaces were disinfectedwith Spor-Klenz and Clidox (chlorine dioxide PharmacalCatalog 96120F) Contaminated cages were handled andanimals manipulated (eg for fecal sample collection andbody weight measurements) exclusively in biosafety hoodsequipped with new high-efficiency particulate air (HEPA)filters routinely sterilized with chlorine gas By July 2016a year after the contamination event the GF colony hasbeen reestablished with amended protocols for autoclavingnutritionally balanced irradiated diet to increase feed safetyNo further contamination events with this mold have beenobserved

3 Discussion

This is the first report of a contamination event in germ-freeanimals with increased susceptibility to inflammatory boweldisease by any fungal pathogen We report A sydowii feed-mediated exposure for the first time in GF animals devoidof natural protective commensal (intestinal and cutaneous)microbiota in a genetic background with increased suscepti-bility to microbial infections In humans this pathogen cancause peritonitis [15] but no intestinal diseases have beenreported for this microorganism Although the intestinalpassage of A sydowii in snails (vectors for Sea Fan disease)results in excretion of viable spores [16] we did not detect


Mouse cage floor - ldquofungal traprdquo

(f)

(a)

(c)

(d)

(e)

(b)

AY373869Peritonitis18(KU757471)15(KU757469)17(KU757470)Consensus

Conservation

0180

Bootstrap 96

100

GGGGGGGGGGGGGGGGG

G GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGG

TTTTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTT

TTTTTTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TT

T

T T

T

TTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTTTTTTTTTTTTTTT

TTTTTT

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAA

AAAAAA

AAA

AAA

AAAAAA

AAAA

AAAAAA

AAAA

AAA A AA

A

CCCCCCCCCCC

CCCCCCCCCC

CCCCCCCCCC

CCCCCC

CCCCCC

CCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCCC

C

CCCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCC

CCCC

CCCCCCCCCCC

C

CCCC

CCC CCCC

C

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CC

CCCCCCCCCCCCCCC

CCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCC

CCTAACACTG TTGCTTCGGC GGG------- ---------- -----

------- ---------- ------------ - --------- ------------ - --------- ------------ - --------- -

-

-

-

- -

-

-

-

----------- - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ ---------- -------------- --- ---------- ---

GAGCT ----

----------------------------------------

CCCTC

280

100

0

G

G

G

G

G T G A T C A T C G A G T C T T T G A

T

T

T

T

A

A

A

A

A A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

C

C

C

C

C

C

C

C

C

C

C

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T T

T

T

T

G

G

G

G

G

G

G

G

G

G G

G

G

G

G

17_FP_1(Asydowii)21218_FP_1(Asydowii)220

15_FP_1(Asydowii)100A sydowii_GU941332_Cruise_CHINAA sydowii_KP329836_houseDust_NETHERLANDS

Aspergillus sydowii_KP973600_Rock_ATACAMAdesertA sydowii_FR733852_CultureCollection_Patient_CZECH_A sydowii_KP329840_houseDust_NETHERLANDSA sydowii_gb|KP3298401A sydowii_AY373869 (Peritonitis)

16_FP_1(Penicillium)212

Penicillium granulatum_GU134897_Plants_CHINAPenicillium oxalicum_DQ681335_Plants_SPAIN

Penicillium chrysogenum_JF807949_Plants_CHINAPenicillium chrysogenum_KM232481_SeaFloor_FRANCE

Penicillium rubens_KP329843_houseDust_NETHERLANDSCandida tropicalis_EF216862

Figure 3 Culture and molecular identification of Aspergillus sydowii (a) A sydowii in potato dextrose agar supplemented with gentamicinand chloramphenicol (30 and 125mgL)with typical blue-green colonies (solid triangles three distinct isolates fromGF-irradiated rodent feedin two GF isolators) (b) Coculture with a Penicillium feed isolate (open triangle) to emphasize colony morphology and pigment productionby A sydowii (transagar pigmentation) (c) Adhesive tape preparation of A sydowii colony to illustrate conidiophores conidia and typicalfruiting head vesicle arrangement with nonseptate conidiophore (400x phase-contrast microscopy on Calcofluor White from Remel AlexaFluor 430 emission 541 absorption 433 nm scale bar 30120583m) (d) See distinct types of conidiophore formation by A sydowii (e) Neighbor-Joining phylogram of A sydowii isolates in the context of other published 18S internal transcribed spacers ITS1ndash4 sequences and Penicilliumspp Notice the close-up of region where strains have sequence dissimilarity (f) Panoramic photo of mouse cage bedding (set as ldquofungal traprdquoie mouse cage incubated for 3 weeks at 23∘C) at the end of clinical observation period confirming mice were still exposed or shedding Asydowii


0 2 4 6 8minus15

minus10

minus5

0

5

10

SAMP-GFree_food floraSAMP regular SPF flora

Days with DSS in water

Body

wei

ght c

hang

e (

ver

sus d

ay 0

)

Wat

er le

ft in

bottl

e (m

L)330310290270250230210190

0 2 4 6 8

(a) (b)

Expected size

SPF flora A sydowiimice

for GF

0

2

4

6

8

Cec

um si

ze (

of B

W aft

er D

SS)

(c)

Figure 4 DSS treatment did not induce typical signs of DSS colitis in contaminatedmice (a) Bodyweight ofmice (8 weeks old) contaminatedwithA sydowii did not lose weight compared tomice harboring regularmouse SPFmicrobiota (119899 = 4group 2cage) Inset plot water contentin each cagewater bottle indicates that allmice consumed the same amount ofDSS supplementedwater (b) PostmortemafterDSS experimentNote the small liver and large spleen and cecum (left) in A sydowii mice as it is expected in mice in GF conditions (c) Note the expectedreduction in cecum size in SPF mice after DSS treatment due to diarrhea and appetite loss

their presence in mouse feces using culture agar platesindicating that intestinal colonization was negligible or thatalternatively the intestinal transit andor metabolites couldmake spores superdormant a phenomenon known to occurby other means in other spore-forming microorganismsnamely Bacillus spp and Clostridium difficile [17] Moleculardetection methods [14] were not employed to detect fungalspores in feces but (i) the presence of A sydowii at theend of the observation period in the mouse cages and fecalexcrements using fungal traps (ii) the lack of induction ofclinical signs in the DSS colitis experiment and (iii) the lackof mortality or evident clinical morbidity when the cages hadthe highest load of spores inside the isolators and duringthe monitoring period (housed inside gnotobiotic cages)in none of the 125 animals suggest that this pathogen haslimited clinical effect on immunocompromised mice devoidof commensal flora

Maintaining GF animals is an expensive and labor-intensive practice highly prone to undesirable contaminantsAlthough Aspergillus could be classified either as sapro-trophic (decaying plant matter) or as clinical strains [7]the major risk for GF contamination is saprotrophic strainsThe source of A sydowii presence in our irradiated diet isuncertain but it is possible that a batch of contaminated rawplant material (because A sydowii is a saprophytic fungi ofdecaying plant matter that could be used for animal feedpreparation) [9] served as source of spores that survivedirradiation [18] The amount of energy required to kill90 (1 Log

10) of the microbial population (LD10) for most

Aspergillus spp spores has been reported to be between028 and 060 kGy [19] Irradiation doses of 20ndash30 kGy arecommonly used to treat diets intended for SPF animals anddoses of 40ndash50 kGy are recommended for diets intended forgnotobiotic or germ-free animals [20] Although it was not


possible to document the irradiation protocol used commer-cially by the supplier of feed to our GF colony the resistanceto irradiation is of bioengineering interest because it isincreasingly known that dark pigmented fungi via melaninmolecules seem to protect them in highly gamma-radiatedenvironments including outer space Aspergillus sydowii isthe third most common fungus in the NASA internationalspace station [18] With respect to pathogenicity in GF miceour findings cannot be extended to other A sydowii strainsbecause virulence seems to be strain-dependent based onstudies characterizing clinical and environmental isolates [7]Fungi (initially thought to be exclusively aerobic microor-ganisms) could however modify host-microbial responses inintestinal diseases due to their production of mycotoxinsespecially if certain strains were capable of colonizing theanaerobic intestinal tract (eg as it commonly occurs in theforestomach of ruminants) In addition A sydowii couldmodify host-microbial responses in the gut in the presenceof other commensal flora via epigenetic modifiers that alterthe production of novel anti-inflammatory (sesquiterpenoid)molecules [21 22] IfAspergilluswas capable of colonizing theanaerobic gut it couldmodulate themicrobiota and intestinalinflammation by the production of secretory molecules Thiscase report only highlights that the relevance of this andother fungi in IBD requires further studies to control forinteractions with other elements important in the function ofthe digestive system including the presence of various typesof commensal and dysbiotic gut microbiota

Competing Interests

The authors declare no conflict of interests

Acknowledgments

Special thanks are due to the present and past members ofthe personnel at the Animal Resource Center at CaseWesternReserve University (CWRU) for their constant support withanimal husbandry and protocols High School student JessicaEzeji for technical assistance Mrs Simone Edelheit MilenaRajak and Tina Nunn at the Genomics Core at CWRUfor Sanger Sequencing Mrs Alicia DePlatchett for editorialsuggestions and Dr Mikhail Khoretonenko at Departmentof Biology Lakeland Community College for scientific dis-cussions and editorial suggestions This work was supportedby National Institutes of Health Grants DK091222 andDK055812 to Fabio Cominelli and by the Crohnrsquos and ColitisFoundation of America via a Career Development Awardto Alexander Rodriguez-Palacios The authors acknowledgethe Mouse Models Core of the Cleveland Digestive DiseaseResearch Core Center (DK097948)

References

[1] S A Cha M H Kim T S Lim et al ldquoInvasive primary colonicaspergillosis in the immunocompetent host without classicalrisk factorsrdquoYonseiMedical Journal vol 56 no 5 pp 1453ndash14562015

[2] C Ekwueme A A Otu S Chinenye et al ldquoHaemoptysisin a female with diabetes mellitus a unique presentation ofchronic pulmonary aspergillosis pulmonary tuberculosis andKlebsiella peumoniae co-infectionrdquo Clinical Case Reports vol 4no 4 pp 432ndash436 2016

[3] S Seyedmousavi J Guillot P Arne et al ldquoAspergillus andaspergilloses in wild and domestic animals a global healthconcernwith parallels to humandiseaserdquoMedicalMycology vol53 no 8 pp 765ndash797 2015

[4] DM Geiser JW Taylor K B Ritchie andGW Smith ldquoCauseof sea fan death in the West Indiesrdquo Nature vol 394 no 6689pp 137ndash138 1998

[5] R C Summerbell J Kane and S Krajden ldquoOnychomycosistinea pedis and tinea manuum casused by non-dermatophyticfilamentous fungirdquoMycoses vol 32 no 12 pp 609ndash619 1989

[6] V Hubka A Kubatova N Mallatova et al ldquoRare and newetiological agents revealed among 178 clinicalAspergillus strainsobtained from Czech patients and characterized by molecularsequencingrdquoMedical Mycology vol 50 no 6 pp 601ndash610 2012

[7] O E Marfenina G M Fomicheva M V Gorlenko and NM Svirida ldquoEcophysiological differences between saprotrophicand clinical strains of the microscopic fungus Aspergillussydowii (Bainier amp Sartory)ThomampChurchrdquoMicrobiology vol82 no 1 pp 85ndash90 2013

[8] D Corridoni T Kodani A Rodriguez-Palacios et al ldquoDys-regulated NOD2 predisposes SAMP1YitFc mice to chronicintestinal inflammationrdquo Proceedings of the National Academyof Sciences of the United States of America vol 110 no 42 pp16999ndash17004 2013

[9] F T El-Hissy S I Abdel-Hafez and M I Abdel-KaderldquoRhizosphere fungi of five plants in Egyptrdquo Zeitschrift furAllgemeine Mikrobiologie vol 20 no 3 pp 177ndash184 1980

[10] A Rodriguez-Palacios T Kodani L Kaydo et al ldquoStereomi-croscopic 3D-pattern profiling of murine and human intestinalinflammation reveals unique structural phenotypesrdquo NatureCommunications vol 6 article 7577 2015

[11] C De Salvo X M Wang L Pastorelli et al ldquoIL-33 driveseosinophil infiltration and pathogenic type 2 helper T-cellimmune responses leading to chronic experimental ileitisrdquoTheAmerican Journal of Pathology vol 186 no 4 pp 885ndash898 2016

[12] T J White T Bruns S Lee and J W Taylor ldquoAmplificationand direct sequencing of fungal ribosomal RNA genes forphylogeneticsrdquo in PCR Protocols A Guide to Methods andApplications M A Innis D H Gelfand J J Sninsky and T JWhite Eds pp 315ndash322 Academic Press New York NY USA1990

[13] H P Hinrikson S F Hurst T J Lott D W Warnock andC J Morrison ldquoAssessment of ribosomal large-subunit D1-D2internal transcribed spacer 1 and internal transcribed spacer2 regions as targets for molecular identification of medicallyimportant Aspergillus speciesrdquo Journal of Clinical Microbiologyvol 43 no 5 pp 2092ndash2103 2005

[14] I D Iliev V A Funari K D Taylor et al ldquoInteractions betweencommensal fungi and the C-type lectin receptor Dectin-1influence colitisrdquo Science vol 336 no 6086 pp 1314ndash1317 2012

[15] Y-L Chiu S-J Liaw V-C Wu and P-R Hsueh ldquoPeritonitiscaused by Aspergillus sydowii in a patient undergoing continu-ous ambulatory peritoneal dialysisrdquo Journal of Infection vol 51no 3 pp e159ndashe161 2005

[16] K L Rypien and D M Baker ldquoIsotopic labeling and antifungalresistance as tracers of gut passage of the sea fan pathogen


Aspergillus sydowiirdquo Diseases of Aquatic Organisms vol 86 no1 pp 1ndash7 2009

[17] A Rodriguez-Palacios and J T LeJeune ldquoMoist-heat resis-tance spore aging and superdormancy in Clostridium difficilerdquoApplied and Environmental Microbiology vol 77 no 9 pp3085ndash3091 2011

[18] E Dadachova andA Casadevall ldquoIonizing radiation how fungicope adapt and exploit with the help of melaninrdquo CurrentOpinion in Microbiology vol 11 no 6 pp 525ndash531 2008

[19] G Blank and D Corrigan ldquoComparison of resistance of fungalspores to gamma and electron beam radiationrdquo InternationalJournal of Food Microbiology vol 26 no 3 pp 269ndash277 1995

[20] C D Caulfield J P Cassidy and J P Kelly ldquoEffects of 120574irradiation and pasteurization on the nutritive composition ofcommercially available animal dietsrdquo Journal of the AmericanAssociation for Laboratory Animal Science vol 47 no 6 pp 61ndash66 2008

[21] Y-M Chung C-K Wei D-W Chuang et al ldquoAn epige-netic modifier enhances the production of anti-diabetic andanti-inflammatory sesquiterpenoids from Aspergillus sydowiirdquoBioorganic and Medicinal Chemistry vol 21 no 13 pp 3866ndash3872 2013

[22] B Singh and A Kaur ldquoAntidiabetic potential of a peptideisolated from an endophytic Aspergillus awamorirdquo Journal ofApplied Microbiology vol 120 no 2 pp 301ndash311 2016

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Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


known for causing mortality in Sea Fan corals (Gorgoniaventalina animals that feed on plankton) threatening variousCaribbean reefs since the 1990s [4] where it seems to remaingeographically confined Infections cause circular purpleareas of necrosis and mortality that is not evidenced in othercorals Initially isolated fromhumans in theUSA in 1989 fromintegumentary (eg skin and nails) infections [5] A sydowiihas been isolated in recent years from patients in other moredistant regions (Czech Republic Russia) [6 7]

Mice under germ-free conditions have a very high risk ofexuberant infections by any commensal pathogenic or envi-ronmental microbes Here we report a case of 125 germ-freeSAMPYitFc mice inadvertently exposed to A sydowii viacommercial germ-free grade irradiated contaminated feedThe (SAMPYitFc) mice contaminated in this study wereparticularly deemed prone toA sydowii opportunistic fungalinfections because (i) they were germ-free and thereforelacked protective competitive surface microbial commensalflora (microbiota) in both gut and skin (ii) the mousegenetic line is immunologically prone to progressive chronicintestinal inflammation conjunctivitis and ulcerative der-matitis (iii) they have decrease clearance of pathogens likeSalmonella enteritidis [8] and (iv) because germ-free micehave no protective antibody-mediated adaptive (memory)immunity against microorganisms due to lack of exposure tocommensal or pathogenicmicrobes when raised in germ-freeconditions

2 Case

To historically contextualize the relevance of the presentreport we first conducted a systematic scoping review ofpublications onA sydowii since its discovery in plants in 1980[9] until February 15 2016 using PubMed and ldquoAspergillussydowiirdquo as search keyword term We then examined the100 top hits in Google Scholar (ie articles with the highestcitations) and secondarily reviewed the title relevance oftheir Google citations referencing the papers for the years2013ndash2015 to capture recently published studies with lowcitation indexes Analysis of titles abstracts and content of97 selected research studies indicates that the frequency ofstudies on emerging bioengineering and basic research onA sydowii is growing exponentially since its isolation fromaffected Sea Fan corals was linked to the near extinctionof some reefs in the West Indies in the 1990s (Figure 1)With the scoping review it also became evident that little isknown about the intestinal colonization and pathogenicityof A sydowii in mammals Because the putative role of themycobiome as disease contributor in human inflammatorybowel disease (IBD) is increasing we deemed pertinent toreport measurable mortality and clinical morbidity outcomesduring this contamination event to further understand therole of the mycobiome in animals Of relevance to IBDthe affected genetic mouse line is one of only two availablespontaneous mouse models for Crohnrsquos disease-like enteritisprimarily affected by ileitis (inflammation of ileum ie thelast segment of the small intestine)

Fungal infections are opportunistic and commonly occurin immunocompromised hosts [4 6] in both humans and

0

2

4

6

8

10

12

1980

1985

1990

1995

2000

2005

2010

2015

Humans

Infected Sea Fanrepresentation

Plants

Rese

arch

artic

les P

ubM

ed n

See Fans

Year of publication

Mice this studyJuly 17th

2016

R2 = 08879

y = 00008x3 minus 46467x2 + 92462x minus 6E + 06

Figure 1 The frequency of peer-review studies on Aspergillussydowii has increased exponentially over the last decade with thecurve shoulder starting after the fungus was linked to emergingepidemic infections in Caribbean corals The first report was inplants in Egypt 1980 [9] Systematic scoping review of articles inPubMed until February 2016 Two-step moving average (solid line)and polynomial estimation curve (dashed line 119910 = 119899 of publicationsestimate 119909 year)

animals Of immunological relevance the SAMP1YitFcmicecorrespond to a genetic line known for suffering spontaneousprogressive intestinal inflammation with unique three-dimensional structural similarity to human Crohnrsquos diseaselesions [10] SAMP1YitFc mice also have complex immuno-logical abnormalities with 100 penetrance Although par-tially understood the immunological abnormality in thesemice appears to transition from T-helper-1 (Th1) driveninflammation at young age to an inflammatory process drivenby a combinedTh1Th2 alteration in animal adulthood whichaccentuates as disease worsens Mechanistically mice havegut epithelial barrier and pathogen clearance dysfunctions[8 11] which could enhance their predisposition in GFconditions to overt fungal infections

Herein we describe the unexpected rapid and simultane-ous contamination of 125 mice housed in two GF positive-pressurized isolators with A sydowii in an established GFbreeding facility (Cleveland OH USA) in June of 2015 Anoverview of the timeline during the contamination event andsubsequent experimentation is presented in Figure 2 (iefungus detection colony dismantling and monitoring micefor signs of disease using an in-house caging system)

In brief two GF isolators having weekly husbandryand high mouse density (2-3 breederscage lt10 unweanedmicecage 12ndash14 cagesisolator) were observed at main-tenance to have unexpected condensation and increasedhumidity as well as containing feed pellets covered withmold on a day of routine inspection in June 2015 (day 0)Following removal surface disinfection with Spor-Klenz(1 hydrogen peroxide 10 acetic and 008 paracetic acidSteris Corp Catalog 6525) and quarantine of all cages (no



Rout

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Controlled humidity

Col

ony

teste

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3 Discussion




(f)

(a)

(c)

(d)

(e)

(b)


Conservation

0180

Bootstrap 96

100

GGGGGGGGGGGGGGGGG

G GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGG

TTTTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTT

TTTTTTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TT

T

T T

T

TTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTTTTTTTTTTTTTTT

TTTTTT

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAA

AAAAAA

AAA

AAA

AAAAAA

AAAA

AAAAAA

AAAA

AAA A AA

A

CCCCCCCCCCC

CCCCCCCCCC

CCCCCCCCCC

CCCCCC

CCCCCC

CCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCCC

C

CCCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCC

CCCC

CCCCCCCCCCC

C

CCCC

CCC CCCC

C

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CC

CCCCCCCCCCCCCCC

CCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCC


------- ---------- ------------ - --------- ------------ - --------- ------------ - --------- -

-

-

-

- -

-

-

-

----------- - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ ---------- -------------- --- ---------- ---

GAGCT ----

----------------------------------------

CCCTC

280

100

0

G

G

G

G


T

T

T

T

A

A

A

A

A A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

C

C

C

C

C

C

C

C

C

C

C

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T T

T

T

T

G

G

G

G

G

G

G

G

G

G G

G

G

G

G










0 2 4 6 8minus15

minus10

minus5

0

5

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Body

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ver

sus d

ay 0

)

Wat

er le

ft in

bottl

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0 2 4 6 8

(a) (b)

Expected size


for GF

0

2

4

6

8

Cec

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ze (

of B

W aft

er D

SS)

(c)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Rout

ine f

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Mol

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All

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5 m

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testing Col

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expe

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Qua

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nd te

sting

ex

tend

ed fo

r 8 w

eeks

for 1

5 y

ears



Controlled humidity

Col

ony

teste

d ne

gativ

e









3 Discussion




(f)

(a)

(c)

(d)

(e)

(b)


Conservation

0180

Bootstrap 96

100

GGGGGGGGGGGGGGGGG

G GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGG

TTTTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTT

TTTTTTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TT

T

T T

T

TTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTTTTTTTTTTTTTTT

TTTTTT

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAA

AAAAAA

AAA

AAA

AAAAAA

AAAA

AAAAAA

AAAA

AAA A AA

A

CCCCCCCCCCC

CCCCCCCCCC

CCCCCCCCCC

CCCCCC

CCCCCC

CCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCCC

C

CCCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCC

CCCC

CCCCCCCCCCC

C

CCCC

CCC CCCC

C

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CC

CCCCCCCCCCCCCCC

CCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCC


------- ---------- ------------ - --------- ------------ - --------- ------------ - --------- -

-

-

-

- -

-

-

-

----------- - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ ---------- -------------- --- ---------- ---

GAGCT ----

----------------------------------------

CCCTC

280

100

0

G

G

G

G


T

T

T

T

A

A

A

A

A A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

C

C

C

C

C

C

C

C

C

C

C

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T T

T

T

T

G

G

G

G

G

G

G

G

G

G G

G

G

G

G










0 2 4 6 8minus15

minus10

minus5

0

5

10



Body

wei

ght c

hang

e (

ver

sus d

ay 0

)

Wat

er le

ft in

bottl

e (m

L)330310290270250230210190

0 2 4 6 8

(a) (b)

Expected size


for GF

0

2

4

6

8

Cec

um si

ze (

of B

W aft

er D

SS)

(c)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















(f)

(a)

(c)

(d)

(e)

(b)


Conservation

0180

Bootstrap 96

100

GGGGGGGGGGGGGGGGG

G GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGGG

GGGGGGGGGGGGGGGG

TTTTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTTTTTTTT

TTTTTTTTTT

TTTTTTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTT

TT

T

T T

T

TTTT

TTTTTT

TTTTTT

TTTTTT

TTTTTTTTTTTTTTTTTTT

TTTTTT

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAAAA

AAAA

AAAAAA

AAA

AAA

AAAAAA

AAAA

AAAAAA

AAAA

AAA A AA

A

CCCCCCCCCCC

CCCCCCCCCC

CCCCCCCCCC

CCCCCC

CCCCCC

CCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCCC

C

CCCCCCCCCCCCCC

C

CCCCCCCCCCCCCC

C

CCC

CCCC

CCCCCCCCCCC

C

CCCC

CCC CCCC

C

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CCCCCC

CC

CCCCCCCCCCCCCCC

CCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCCCCCCCC

CCCCCCCCCC


------- ---------- ------------ - --------- ------------ - --------- ------------ - --------- -

-

-

-

- -

-

-

-

----------- - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ - --------- ------------ ---------- -------------- --- ---------- ---

GAGCT ----

----------------------------------------

CCCTC

280

100

0

G

G

G

G


T

T

T

T

A

A

A

A

A A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

C

C

C

C

C

C

C

C

C

C

C

T

T

T

T

T

T

T

T

T

T

T

T

T

T

T T

T

T

T

G

G

G

G

G

G

G

G

G

G G

G

G

G

G










0 2 4 6 8minus15

minus10

minus5

0

5

10



Body

wei

ght c

hang

e (

ver

sus d

ay 0

)

Wat

er le

ft in

bottl

e (m

L)330310290270250230210190

0 2 4 6 8

(a) (b)

Expected size


for GF

0

2

4

6

8

Cec

um si

ze (

of B

W aft

er D

SS)

(c)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 2 4 6 8minus15

minus10

minus5

0

5

10



Body

wei

ght c

hang

e (

ver

sus d

ay 0

)

Wat

er le

ft in

bottl

e (m

L)330310290270250230210190

0 2 4 6 8

(a) (b)

Expected size


for GF

0

2

4

6

8

Cec

um si

ze (

of B

W aft

er D

SS)

(c)








Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Competing Interests


Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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