BASELINE CUTANEOUS BACTERIA OF FREE-LIVING NEW ZEALAND

NATIVE FROGS (LEIOPELMA ARCHEYI AND LEIOPELMA

HOCHSTETTERI) AND IMPLICATIONS FOR THEIR ROLE IN DEFENSE

AGAINST THE AMPHIBIAN CHYTRID

(BATRACHOCHYTRIUM DENDROBATIDIS)

Stephanie D. Shaw,1,2,6 Lee Berger,1 Sara Bell,1 Sarah Dodd,3 Tim Y. James,4 Lee F. Skerratt,1

Phillip J. Bishop,5 and Rick Speare1

1 One Health Research Group, School of Public Health, Tropical Medicine and Rehabilitation Sciences, James CookUniversity, DB-41 Angus Smith Drive, Townsville, Queensland 4811, Australia2 New Zealand Centre for Conservation Medicine, Auckland Zoo, 117 Motions Road, Western Springs, Auckland 1022,New Zealand3 Landcare Research, 231 Morrin Road, St. Johns, Auckland 1072, New Zealand4 Department of Ecology and Evolutionary Biology, University of Michigan, 830 N University, Ann Arbor,Michigan 48109, USA5 Department of Zoology, University of Otago, 340 Great King Street, Dunedin 9016, New Zealand6 Corresponding author (email: sshaw@rspcaqld.org.au)

ABSTRACT: Knowledge of baseline cutaneous bacterial microbiota may be useful in interpretingdiagnostic cultures from captive sick frogs and as part of quarantine or pretranslocation diseasescreening. Bacteria may also be an important part of innate immunity against chytridiomycosis, afungal skin disease caused by Batrachochytrium dendrobatidis (Bd). In February 2009, 92 distinctbacterial isolates from the ventral skin of 64 apparently healthy Leiopelma archeyi and Leiopelmahochstetteri native frogs from the Coromandel and Whareorino regions in New Zealand wereidentified using molecular techniques. The most-common isolates identified in L. archeyi werePseudomonas spp. and the most common in L. hochstetteri were Flavobacterium spp. Toinvestigate the possible role of bacteria in innate immunity, a New Zealand strain of Bd (KaikoraiValley-Lewingii-2008-SDS1) was isolated and used in an in vitro challenge assay to test forinhibition by bacteria. One bacterial isolate, a Flavobacterium sp., inhibited growth of Bd. Theseresults imply that diverse cutaneous bacteria are present and may play a role in the innate defensein Leiopelma against pathogens, including Bd, and are a starting point for further investigation.

Key words: Amphibian chytrid, bacteria, Batrachochytrium dendrobatidis, innate immunity,Leiopelma archeyi, Leiopelma hochstetteri, quarantine, translocation.

INTRODUCTION

New Zealand native frog fauna iscomprised of four species of extant Leio-pelmatids with the following classifications:Leiopelma archeyi (Archey’s frog; criticallyendangered), Leiopelma hamiltoni (Hamil-ton’s frog; endangered), Leiopelma hoch-stetteri (Hochstetter’s frog; vulnerable) andLeiopelma pakeka (Maud Island frog;vulnerable) (IUCN 2011). They are all alsolisted in the top 100 amphibian species ofthe most evolutionarily distinct and globallyendangered list, with L. archeyi holdingthe top position (Zoological Society ofLondon 2011). All are nocturnal, terrestrialfrogs except L. hochstetteri, which issemiaquatic.

In 1996, one of the two known popula-tions of L. archeyi underwent a severepopulation crash in the Coromandel re-gion (Bell et al. 2004). The cause of thedecline was thought to be chytridiomyco-sis, as has occurred in many amphibianpopulations worldwide (Berger et al. 1998;Lips 1999; Daszak et al. 2000; Skerrattet al. 2007; Vredenburg et al. 2010). Thisfinding sparked the testing of populationsof L. archeyi in the Whareorino regionand the 22 known populations of L.hochstetteri (Baber et al. 2006) for Ba-trachochytrium dendrobatidis (Bd) (Shawet al. 2013). The Whareorino population ofL. archeyi was positive for Bd, butpopulation monitoring that occurred every6 mo since 2005 has shown that the

DOI: 10.7589/2013-07-186 Journal of Wildlife Diseases, 50(4), 2014, pp. 723–732# Wildlife Disease Association 2014

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population size is stable (Shaw et al. 2013).Monitoring of the L. hochstetteri popula-tions has been sporadic, but Bd has notbeen detected and their populations alsoappear to be stable (Whitaker and Alspach1999; Baber et al. 2006; Shaw et al. 2013).

Amphibians are frequently brought intocaptivity and transferred between institu-tions for captive reproduction and treat-ment for conservation purposes. Current-ly, routine bacterial skin cultures are notcollected as part of quarantine procedures(Pessier and Mendelson 2010), and thereare little data on the baseline cutaneousbacterial flora in free-living amphibians.Therefore, when skin cultures from sickanimals are analyzed (Pessier 2002), it isdifficult to tell what organisms are likely tobe pathogens and which are part of thenormal bacterial microbiota. Bacterialcultures have been performed before fromthe dorsal skin surface on both captiveand free-living L. archeyi from both theCoromandel and Whareorino populations(Potter and Norman 2006). Those authorsidentified 41 bacterial isolates using stan-dard morphologic and biochemical testsand found that the bacterial skin floradiffered between captive and free-livingfrogs and between locations of free-livingfrogs. However, as the bacterial swabswere taken only from the dorsal skinsurface, the results may not be a trueindication of the full spectrum of bacterialspecies present (Culp et al. 2007).

Amphibian species vary in their abilityto resist Bd infection and their suscepti-bility to population declines. For example,in the case of New Zealand frogs, when L.archeyi and L. pakeka were experimental-ly infected with Bd they rapidly self-curedand did not show clinical signs (Shaw et al.2010; Ohmer et al. 2013). Similar exper-iments in L. hochstetteri have shownequivocal results and indicate they arelikely resistant to infection (Ohmer et al.2013). Adaptive (acquired) immunity hasnot been found to play a role in Bddefense (Rosenblum et al. 2009; Stice andBriggs 2010).

Many factors can contribute to hostvulnerability, such as Bd strain, climate,and habitat, as well as innate immunity(Berger et al. 2004, 2005; Rollins-Smith etal. 2006; Ramsey et al. 2010; Puschendorfet al. 2011). Antimicrobial skin peptidesfrom L. archeyi have higher in vitroactivity against Bd than do those from L.hochstetteri and L. pakeka, and these maybe vital in their initial defense (Melzer andBishop 2010). Another aspect of innatedefense is the cutaneous bacterial flora,and many bacterial species produce me-tabolites that inhibit growth of Bd onnutrient agar (Harris et al. 2009). In somefrog species, individuals with inhibitorybacteria resist Bd while those individualswithout these beneficial bacteria maybecome infected (Harris et al. 2009).Using probiotic symbiotic bacteria as atreatment to protect amphibians againstchytridiomycosis has had mixed success(Harris et al. 2009; Becker et al. 2011;Woodhams et al. 2011).

Our objectives were to obtain baselinecutaneous bacterial flora data from theventral skin of L. archeyi and L. hoch-stetteri and to test the bacteria against aNew Zealand isolate of Bd in vitro to see ifbacterial metabolites were produced thatprevented Bd growth. We hoped to gaininsight into the apparent immunity to Bdin leiopelmatid frogs and aid furtherdevelopment of bacteria as a bioaugmen-tation tool in amphibian species suscepti-ble to chytridiomycosis.

MATERIALS AND METHODS

Sample collection for cutaneous bacteria

In February 2009, New Zealand Departmentof Conservation staff collected swab samplesfrom 33 L. archeyi and 20 L. hochstetteri in theWhareorino forest (38u23959.90S, 174u48900E)and 11 L. archeyi from the CoromandelPeninsula (38u23959.90S, 174u48900E) of NewZealand. To remove surface dirt and transient,environmental bacteria from the skin, theentire ventral surface of all frogs was washedtwice with 10 mL of sterile water (10-mL plasticvials; Astra Zeneca Ltd., North Ryde, Australia)in the Coromandel, or with rainwater in the

724 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 4, OCTOBER 2014

Whareorino. Frogs were swabbed to collectskin bacteria using a sterile transport swab(Copan 157C, Copan, Via F., Perotti, Brecia,Italy) which was placed into sterile collectionmedia, transported to the lab in a chilledcontainer, and stored at 4 C until plated onnutrient agar within 48 hr of collection.

Bacterial culture and identification

Bacteria were transferred from the swabsonto DifcoTM R2A agar plates (Bd NewZealand, Mt. Wellington, Auckland, NewZealand; Harris et al. 2006) within a laminarflow cabinet at Landcare Research (Auckland,New Zealand). Swabs were wiped over thesurface of the agar in the plate while rotatingthe tip of the swab to ensure completetransfer. Agar plates were incubated in thedark at 18 C to simulate normal growthconditions of the ventral surface of L. archeyi.Plates were checked daily and obvious singlecolonies of bacteria were transferred to afresh agar plate and isolated to pure culture.Each pure culture was numbered and storedon Difco R2A agar slants at 4 C. Pure cultureswere compared and, for each frog species andsite, those bacteria that had similar morphol-ogy were grouped. Given financial con-straints, only one representative from eachof the morphologically distinct groups wasidentified by 16S rRNA sequencing (Land-care Research). DNA was extracted using aSigma REDExtract-N-AmpTM tissue kit fol-lowing the manufacturer’s instructions (Sig-ma-Aldrich, Castle Hill, New South Wales,Australia). The extracted DNA samples wereamplified using the bacterial 16S rRNAprimers 1F and 1509R and the followingPCR conditions: 95 C for 4 min; 95 C for30 sec, 53 C for 30 sec, and 72 C for 1 min for25 cycles; and 72 C for 10 min. Successfulamplifications were confirmed by running thePCR products on a 1.5% (wt/vol) agarose gelat 150V for 30 min, staining with ethidiumbromide, and visualizing under ultravioletlight. The PCR products were sequencedusing an ABI Genetic Analyzer 3130xLsequencing machine (Applied Biosystems,Mulgrave, Victoria, Australia). Sequence datawere analyzed using the Sequencher softwarev. 5.0 (Gene Codes Corp., Ann Arbor,Michigan, USA) and identities confirmedusing the Basic Local Alignment Search Tool(NCBI 2014) using the program Geneious(v.5.65) (Biomatters, Ltd., Auckland, NewZealand).

To assess if location or species affected thepresence or frequency of bacterial genera

identified, the data were analyzed usingFisher’s exact tests with the WINPEPI statis-tical program, v. 11.20 (Brixton Health 2013).

Nucleotide sequence accession number

All 16S rRNA gene sequences of thebacterial species isolated in this study weredeposited in the NCBI GenBank databaseunder accessions KC306404–KC306502. Thebacterial isolates have been cryopreserved at270 C at the Auckland Zoo and the Bd isolatehas been cryopreserved at 270 C at LandcareResearch (Boyle et al. 2003).

In vitro Bd-bacterial challenge assay

Thirty-one bacterial isolates from the Cor-omandel population of L. archeyi werechallenged against Bd using the techniquedescribed by Harris et al. (2006). Allprocedures were performed in a class-twobiosafety cabinet. A New Zealand isolate ofBd was cultured by standard methods(Berger et al. 2005) and identified as aunique genotype (Kaikorai Valley-Lewingii-2008-SDS1) using methods described byJames et al. (2009; Fig. 1). Actively growingBd cultures in TGhL broth were passaged toTGhL agar plates (Berger et al. 2009) andincubated at 15 C. After 3 days, zoosporeswere collected by flushing plates with 6 mLsterile distilled water. Zoospores were count-ed using a Neubauer hemocytometer andresuspended using sterile distilled water to aconcentration of 43106 zoospores/mL. Onemilliliter of the zoospore suspension wasspread evenly on a new TGhL plate and air-dried in a sterile biohazard cabinet until theplate appeared dry, but still glistening. Onestreak of freshly cultured and identifiedchallenge bacterium was made on the leftside of the plate while a sterile loop with nobacteria was streaked on the right side of theplate as a negative control. This process wasrepeated until a bacterium that did notinhibit Bd was found (Chryseobacterium sp.3A blue). This was used as a negativebacterial control.

The plates were incubated at 15 C andinspected 24, 48, and 72 hr after inoculation.They were scored as 1) positive inhibition ifthere was Bd growth and a zone of inhibitionaround the bacterial streak, 2) negativeinhibition if there was Bd growth up to thebacterial streak, or 3) indeterminate if Bd didnot grow at all or if the bacterial streakovertook the whole plate. If an indeterminateresult was obtained, the experiment wasrepeated up to two more times before beingrecorded as indeterminate.

SHAW ET AL.—CUTANEOUS BACTERIA OF NEW ZEALAND NATIVE FROGS 725

RESULTS

Bacterial culture and identification

Thirty-one of the bacterial isolatesobtained from the 11 L. archeyi at theCoromandel site were distinct isolates and21 of the 31 isolates (68%) were Pseudo-

monas spp. (Table 1) Thirty-four of the 62isolates from the 33 L. archeyi at theWhareorino site were distinct and 24 ofthe 34 (71%) were Pseudomonas spp.(Table 1). Thirty-one of the 50 bacterialisolates from the 20 L. hochstetteri at theWhareorino site were distinct. Flavobac-

FIGURE 1. Dendrogram of global isolates of Batrachochytrium dendrobatidis as of 2009. The arrowindicates the New Zealand isolate used in this study.

726 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 4, OCTOBER 2014

terium spp. was the most common generaidentified and comprised 12 of the 31bacterial isolates (39%) (Table 1).

Three isolates of Pseudomonas werefound in more than one location (Pseudomo-nas putida isolate PSB31, Pseudomonas sp.BR6-10, and Pseudomonas sp. 29H), whichmade the total distinct isolates identified 92(Table 1). Flavobacterium species were sig-nificantly more prevalent in the WhareorinoL. hochstetteri compared with the Whareor-ino L. archeyi (Fisher’s exact test, P50.02[odds ratio 6.3 with 95% confidence interval[CI] 1.3–33.1]) and when compared with allL. archeyi at both the Whareorino andCoromandel locations together (P50.01[odds ratio 6.4 with 95% CI 1.5–29.2]).

In vitro Bd-bacterial challenge assay

The Bd-bacterial challenge assay wasonly performed for bacterial species fromL. archeyi from the Coromandel becauseit was difficult to obtain consistent resultsusing the technique developed by Harriset al. (2006). From 31 bacterial challenges,one was positive, (Flavobacterium sp.XAS590; Fig. 2), 20 were negative, and10 were indeterminate despite repeatedattempts to get a definitive result. Thereasons for a test being indeterminatewere 1) the Bd agar plate was too dry, thuskilling the zoospores, or 2) the plate wasnot dry enough, so some mucoid bacteria(e.g., Pseudomonas) took over the entireplate within 24 hr so that a 24-hr readingcould not be obtained.

DISCUSSION

We isolated and identified 92 uniquebacterial isolates from 44 L. archeyi and20 L. hochstetteri in the Coromandel andWhareorino regions. One of 31 isolateschallenged, Flavobacterium sp. XAS590inhibited the growth of Bd in vitro.Flavobacterium spp. occurred more fre-quently in L. hochstetteri than in L.archeyi.

Baseline data on the cutaneous bacteriain healthy, free-ranging L. archeyi and L.

hochstetteri could be used to interpretbacterial culture results as part of adiagnostic work-up in sick frogs. The dataalso may be useful when interpretingbacterial skin cultures from pretransloca-tion or quarantine disease screening,where abnormal results can jeopardizean entire movement of frogs. Whencomparing our results to those of Potterand Norman (2006), only Serratia spp.were found in both studies. This differ-ence in bacterial isolates found couldreflect the more-precise molecular DNAidentification techniques used in our study(Ludwig 2008) or differences between thebacterial flora on the dorsal and ventralskin surfaces (Culp et al. 2007). Forbacterial culture, we used Difco R2A agarplates and lower incubation temperaturesto simulate conditions in wild frogs andalso those favorable to Bd growth (Bergeret al. 2004); thus, our methods could haveselected for different bacteria. Anotherdifference between this study and Potterand Norman (2006) is that we did notidentify all the bacterial isolates. Bygrouping morphologically similar isolateswe expected to identify most of the flora.However, as bacteria are difficult todistinguish solely by gross morphology,or were unculturable, we likely missedspecies that would only be detected bymolecular screening (e.g., with next-gen-eration sequencing; MacLean et al. 2009).

Flavobacterium spp. were isolated sig-nificantly more frequently in L. hochstet-teri than in L. archeyi in both theWhareorino location and when combiningboth the Coromandel and Whareorinolocations. Flavobacterium XAS590 fromL. archeyi was the only bacterial isolatethat showed anti-Bd properties in ourexperiments. Although the Bd-bacterialchallenge was not complete for theFlavobacterium spp. isolated in L. hoch-stetteri, this is the first time that bacteriafrom Leiopelma spp. have been shown toexhibit in vitro anti-Bd properties.

If Flavobacterium spp. are important ininnate immunity against chytridiomycosis

SHAW ET AL.—CUTANEOUS BACTERIA OF NEW ZEALAND NATIVE FROGS 727

TABLE 1. Closest taxonomic affiliation from GenBank for all unique 16s rDNA sequences for baselinecutaneous bacteria from New Zealand native frogs used in a challenge study to measure inhibition of growthof Batrachochytrium dendrobatidis (Bd) by cutaneous bacteria of frogs in 2009. Numbers of frogs possessingeach unique sequence are shown by species: Leiopelma archeyi (La) and Leiopelma hochstetteri (Lh) and site:Coromandel Pahi Moehau (Coro) and Whareorino (Whare). Dashes indicate zero.

Taxonomy and GenBank closest matchaGenBankaccession

Similarity%

Coro Whare

La(n510)

La(n524)

Lh(n528)

Bacteroidetes

Flavobacteria

Chryseobacterium sp. 3Ablueb EU057843 98.9 1 — —Chryseobacterium jejunensec AB682422 99.5 1 — —Flavobacterium columnare AY747592 99.4 — 1 —Flavobacterium sp. DB 2.3-10 AM493386 99.2 — — 1Flavobacterium sp. EP 372 AF493653 99.8 — — 1Flavobacterium sp. KOPRI 25403 GU062496 92.2 — 1 —Flavobacterium sp. KOPRI 25403 GU062496 99.1 — 1 —Flavobacterium sp. LM-20-Fp HE573273 97.9 — — 1Flavobacterium sp. Sa CS2 JQ806423 99.7 — — 1Flavobacterium sp. WB 3.1-53 AM934654 99.6 — — 1Flavobacterium sp. WB 3.1-78 AM177614 98.9 — — 1Flavobacterium sp. WB 3.1-79 AM934656 99.0 — — 1Flavobacterium sp. WB 3.2-28 AM934659 99.8 — — 1Flavobacterium sp. WB 3.3.45 AM177620 88.8 — 1 —Flavobacterium sp. WB 3.4.10 AM177622 98.5 — — 1Flavobacterium sp. WB 4.4-22 AM177636 99.7 — — 1Flavobacterium sp. WB 4.3-36 AM934669 99.3 — — 1Flavobacterium sp. XAS590d GQ395239 98.5 1 — —Flavobacterium sp. YO51c DQ778315 99.9 1 — —Flavobacterium sp. III-082-7 FJ786051 99.8 — 1 —Flavobacterium sp. III-082-7 FJ786051 99.3 — — 1

Proteobacteria

Betaproteobacteria

Duganella zoogloeoides strain IAM 12670 NR025833 99.5 — 1 —

Gammaproteobacteria

Acinetobacter sp. AW 1-18 JQ316540 99.1 — — 1Acinetobacter sp. LB BR 12338 JQ247320 99.7 — — 1Acinetobacter sp. LD BR 12340 JQ247322 99.6 — — 1Aeromonas tecta strain CECT7082 HQ832416 99.9 — — 1Aeromonas veronii strain MTTSA 14 JQ795738 100.0 — — 1Aeromonas sp. F518 AJ458402 100.0 — — 1Enterobacteriaceae bacterium JL6J JX162035 99.7 — — 1Hafnia sp. NP33c EU196322 98.9 1 — —Pseudomonas brenneric AM933513 100.0 1 — —Pseudomonas fluorescens AB680968 100.0 — 1 —Pseudomonas fluorescens strain DLJ1c FJ407181 100.0 1 — —Pseudomonas fluorescens strain F32 HQ647251 100.0 — 1 —Pseudomonas fluorescens Pf0-1 CP000094 99.9 — 1 —Pseudomonas fragic AB685586 99.9 1 — —Pseudomonas koreensis AB495131 99.8 — 1 —Pseudomonas palleroniana strain POT2 JQ281539 99.9 1 — —Pseudomonas poae strain BCHCNZ253 GU188947 100.0 — 1 —Pseudomonas poae strain YUST-DW11 HM640290 99.9 — 1 —Pseudomonas putidac AB681704 99.7 1 — —Pseudomonas putida AB681704 99.9 1 — —Pseudomonas putida isolate PSB31 HQ242744 99.4 — 1 1Pseudomonas putida isolate PSB31 HQ242744 99.6 — — 1

728 JOURNAL OF WILDLIFE DISEASES, VOL. 50, NO. 4, OCTOBER 2014

Taxonomy and GenBank closest matchaGenBankaccession

Similarity%

Coro Whare

La(n510)

La(n524)

Lh(n528)

Pseudomonas putida strain CICR-GV2 JX276777 89.5 — 1 —Pseudomonas putida strain LCB43 JN650580 99.3 — 1 —Pseudomonas tolaasii strain 93 JX417438 99.8 — 1 —Pseudomonas tolaasii strain 93 JX417438 99.9 — 1 —Pseudomonas vancouverensis strain A-18c HQ202824 100.0 1 — —Pseudomonas sp. A8(2011)c JN228275 99.7 1 — —Pseudomonas sp. A8(2011) JN228275 99.7 — 1 —Pseudomonas sp. A8(2011) JN228275 99.6 — 1 —Pseudomonas sp. A8(2011) JN228275 99.5 — — 1Pseudomonas sp. A17(2011) JN228277 100.0 1 — —Pseudomonas sp. A17(2011) JN228277 99.9 1 — —Pseudomonas sp. BCRC 80328c JQ361087 100.0 1 — —Pseudomonas sp. BR6-10c EU853194 100.0 1 2 —Pseudomonas sp. CBZ-4 JQ782892 99.9 — — 1Pseudomonas sp. Ch313c AB289615 99.9 1 — —Pseudomonas sp. Ch313 AB289615 99.8 — 1 —Pseudomonas sp. Cmc27 JQ917993 99.5 — 1 —Pseudomonas sp. DPs-27 JQ074038 99.8 — 1 —Pseudomonas sp. G1-21-2 EU781539 98.0 — — 1Pseudomonas sp. G52 FN547408 98.0 — — 1Pseudomonas sp. JSPB2 JQ308615 100.0 — 2 —Pseudomonas sp. KA-26 HE979862 99.5 — — 1Pseudomonas sp. KBOS 17c AY653222 99.9 1 — —Pseudomonas sp. LD002c HQ713573 99.1 1 — —Pseudomonas sp. PDD-32b-42 HQ256842 90.0 — 1 —Pseudomonas sp. RPBP7 JN411670 100.0 — — 1Pseudomonas sp. SaCS18 JQ806427 99.9 — 1 —Pseudomonas sp. SGb14 HQ224617 99.8 1 — —Pseudomonas sp. SGb149 HQ224651 99.8 1 — —Pseudomonas sp. SGb149 HQ224651 99.9 1 — —Pseudomonas sp. SY7 EU073118 99.4 1 — —Pseudomonas sp. SY7 EU073118 99.5 — 1 —Pseudomonas sp. TB2-1-II AY599711 100.0 — 1 —Pseudomonas sp. VS-1 JF699698 99.9 — — 1Pseudomonas sp. VTAE174c JN886726 99.9 1 — —Pseudomonas sp. 6A DQ417331 99.9 — 1 —Pseudomonas sp. 29Hc EU057890 100.0 1 1 —Rahnella aquatilisc GU171376 98.7 1 — —Rahnella aquatilis strain 2B-CDF FJ811859 99.4 — 1 —Rahnella sp. WMR15c AM167519 99.0 1 — —Rahnella sp. WMR58c AM160791 99.0 1 — —Serratia sp. AC-CS-1Bc FJ231172 99.9 1 — —Serratia sp. A7 DQ103507 100.0 — 1 —Serratia sp. D1 DQ103511 100.0 — 1 —Serratia sp. D5 EU100389 96.7 — — 1Serratia sp. ORC3c JQ236628 99.9 1 — —Serratia sp. 136-2 EU557341 100.0 — 2 —Stenotrophomonas rhizophila strain AB11 JQ410475 99.8 — — 1Stenotrophomonas rhizophila strain Bacteria_188 JQ800450 98.7 — — 1

Total unique isolates 31 34 31

a n5number of frogs that had bacterial isolates cultured. Some had more than one isolate.b Negative control.c Negative isolates in Bd-bacterial challenge.d Positive isolate in Bd-bacterial challenge.

TABLE 1. Continued.

SHAW ET AL.—CUTANEOUS BACTERIA OF NEW ZEALAND NATIVE FROGS 729

in L. archeyi, we would expect a higherprevalence than 2/10 (Coromandel) and 5/24 (Whareorino) because previous studieshave shown that if a high proportion ofsusceptible frogs have at least one anti-Bdbacterial species present, the populationcan persist despite the presence of Bd(Woodhams et al. 2007; Lam et al. 2010).Therefore, L. archeyi may not use bacte-rial inhibition as a principle means ofdefense against Bd, unless other uniden-tified species are inhibitory. We recom-mend that bacteria are tested furtherusing the new broth challenge assaydeveloped by Bell et al. (2013). Thistechnique avoids the issues of the agarplate method and may provide more-reliable results. Another potential methodto test to assess Bd viability after bacterialexposure uses a combination of ethidiummonazide with quantitative PCR (Blooi

et al. 2013). Flavobacterium should beinvestigated further for its role in hostresistance to Bd and added to the growinglist of bacteria that can be used inpotential bioaugmentation trials.

Species of Pseudomonas were the mostcommon isolates found in L. archeyi inboth locations. Although these mucoidbacteria were difficult to screen in ourBd-bacterial challenge, they have beensuccessfully challenged in other studiesand some species had anti-Bd properties(Lauer et al. 2007, 2008; Woodhams et al.2007; Lam et al. 2010). We suggest thatthe Pseudomonas isolates from New Zea-land should be investigated further for Bdinhibition.

ACKNOWLEDGMENTS

Funding for this project was provided bythe Auckland Zoo Charitable Trust Conserva-tion Fund. Many thanks to the staff at Land-care Auckland who were instrumental in thebacterial culturing, identification, and storageof chytrid samples: Stanley Bellgard, MaureenFletcher, Karen Hoksbergen, Daniel Than,Bevan Weir, and Paula Wilke. Thanks alsoto Lisa Daglish and Amanda Haigh fromthe Department of Conservation who col-lected the bacterial samples. Many thanks toReid Harris, Brianna Lam, and Jennifer Walkefor technical advice. Thanks also to the NewZealand Maori iwi for supporting native frogresearch.

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Submitted for publication 25 July 2013.Accepted 4 April 2014.

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