Abstract
Knowledge of baseline cutaneous bacterial microbiota may be useful in interpreting diagnostic cultures from captive sick frogs and as part of quarantine or pretranslocation disease screening. Bacteria may also be an important part of innate immunity against chytridiomycosis, a fungal skin disease caused by Batrachochytrium dendrobatidis (Bd). In February 2009, 92 distinct bacterial isolates from the ventral skin of 64 apparently healthy Leiopelma archeyi and Leiopelma hochstetteri native frogs from the Coromandel and Whareorino regions in New Zealand were identified using molecular techniques. The most-common isolates identified in L. archeyi were Pseudomonas spp. and the most common in L. hochstetteri were Flavobacterium spp. To investigate the possible role of bacteria in innate immunity, a New Zealand strain of Bd (Kaikorai Valley-Lewingii-2008-SDS1) was isolated and used in an in vitro challenge assay to test for inhibition by bacteria. One bacterial isolate, a Flavobacterium sp., inhibited growth of Bd. These results imply that diverse cutaneous bacteria are present and may play a role in the innate defense in Leiopelma against pathogens, including Bd, and are a starting point for further investigation.
INTRODUCTION
New Zealand native frog fauna is comprised of four species of extant Leiopelmatids with the following classifications: Leiopelma archeyi (Archey's frog; critically endangered), Leiopelma hamiltoni (Hamilton's frog; endangered), Leiopelma hochstetteri (Hochstetter's frog; vulnerable) and Leiopelma pakeka (Maud Island frog; vulnerable) (IUCN 2011). They are all also listed in the top 100 amphibian species of the most evolutionarily distinct and globally endangered list, with L. archeyi holding the top position (Zoological Society of London 2011). All are nocturnal, terrestrial frogs except L. hochstetteri, which is semiaquatic.
In 1996, one of the two known populations of L. archeyi underwent a severe population crash in the Coromandel region (Bell et al. 2004). The cause of the decline was thought to be chytridiomycosis, as has occurred in many amphibian populations worldwide (Berger et al. 1998; Lips 1999; Daszak et al. 2000; Skerratt et al. 2007; Vredenburg et al. 2010). This finding sparked the testing of populations of L. archeyi in the Whareorino region and the 22 known populations of L. hochstetteri (Baber et al. 2006) for Batrachochytrium dendrobatidis (Bd) (Shaw et al. 2013). The Whareorino population of L. archeyi was positive for Bd, but population monitoring that occurred every 6 mo since 2005 has shown that the population size is stable (Shaw et al. 2013). Monitoring of the L. hochstetteri populations has been sporadic, but Bd has not been detected and their populations also appear to be stable (Whitaker and Alspach 1999; Baber et al. 2006; Shaw et al. 2013).
Amphibians are frequently brought into captivity and transferred between institutions for captive reproduction and treatment for conservation purposes. Currently, routine bacterial skin cultures are not collected as part of quarantine procedures (Pessier and Mendelson 2010), and there are little data on the baseline cutaneous bacterial flora in free-living amphibians. Therefore, when skin cultures from sick animals are analyzed (Pessier 2002), it is difficult to tell what organisms are likely to be pathogens and which are part of the normal bacterial microbiota. Bacterial cultures have been performed before from the dorsal skin surface on both captive and free-living L. archeyi from both the Coromandel and Whareorino populations (Potter and Norman 2006). Those authors identified 41 bacterial isolates using standard morphologic and biochemical tests and found that the bacterial skin flora differed between captive and free-living frogs and between locations of free-living frogs. However, as the bacterial swabs were taken only from the dorsal skin surface, the results may not be a true indication of the full spectrum of bacterial species present (Culp et al. 2007).
Amphibian species vary in their ability to resist Bd infection and their susceptibility to population declines. For example, in the case of New Zealand frogs, when L. archeyi and L. pakeka were experimentally infected with Bd they rapidly self-cured and did not show clinical signs (Shaw et al. 2010; Ohmer et al. 2013). Similar experiments in L. hochstetteri have shown equivocal results and indicate they are likely resistant to infection (Ohmer et al. 2013). Adaptive (acquired) immunity has not been found to play a role in Bd defense (Rosenblum et al. 2009; Stice and Briggs 2010).
Many factors can contribute to host vulnerability, such as Bd strain, climate, and habitat, as well as innate immunity (Berger et al. 2004, 2005; Rollins-Smith et al. 2006; Ramsey et al. 2010; Puschendorf et al. 2011). Antimicrobial skin peptides from L. archeyi have higher in vitro activity against Bd than do those from L. hochstetteri and L. pakeka, and these may be vital in their initial defense (Melzer and Bishop 2010). Another aspect of innate defense is the cutaneous bacterial flora, and many bacterial species produce metabolites that inhibit growth of Bd on nutrient agar (Harris et al. 2009). In some frog species, individuals with inhibitory bacteria resist Bd while those individuals without these beneficial bacteria may become infected (Harris et al. 2009). Using probiotic symbiotic bacteria as a treatment to protect amphibians against chytridiomycosis has had mixed success (Harris et al. 2009; Becker et al. 2011; Woodhams et al. 2011).
Our objectives were to obtain baseline cutaneous bacterial flora data from the ventral skin of L. archeyi and L. hochstetteri and to test the bacteria against a New Zealand isolate of Bd in vitro to see if bacterial metabolites were produced that prevented Bd growth. We hoped to gain insight into the apparent immunity to Bd in leiopelmatid frogs and aid further development of bacteria as a bioaugmentation tool in amphibian species susceptible to chytridiomycosis.
MATERIALS AND METHODS
Sample collection for cutaneous bacteria
In February 2009, New Zealand Department of Conservation staff collected swab samples from 33 L. archeyi and 20 L. hochstetteri in the Whareorino forest (38°23′59.9″S, 174°48′0″E) and 11 L. archeyi from the Coromandel Peninsula (38°23′59.9″S, 174°48′0″E) of New Zealand. To remove surface dirt and transient, environmental bacteria from the skin, the entire ventral surface of all frogs was washed twice with 10 mL of sterile water (10-mL plastic vials; Astra Zeneca Ltd., North Ryde, Australia) in the Coromandel, or with rainwater in the Whareorino. Frogs were swabbed to collect skin bacteria using a sterile transport swab (Copan 157C, Copan, Via F., Perotti, Brecia, Italy) which was placed into sterile collection media, transported to the lab in a chilled container, and stored at 4 C until plated on nutrient agar within 48 hr of collection.
Bacterial culture and identification
Bacteria were transferred from the swabs onto Difco™ R2A agar plates (Bd New Zealand, Mt. Wellington, Auckland, New Zealand; Harris et al. 2006) within a laminar flow cabinet at Landcare Research (Auckland, New Zealand). Swabs were wiped over the surface of the agar in the plate while rotating the tip of the swab to ensure complete transfer. Agar plates were incubated in the dark at 18 C to simulate normal growth conditions of the ventral surface of L. archeyi. Plates were checked daily and obvious single colonies of bacteria were transferred to a fresh agar plate and isolated to pure culture. Each pure culture was numbered and stored on Difco R2A agar slants at 4 C. Pure cultures were compared and, for each frog species and site, those bacteria that had similar morphology were grouped. Given financial constraints, only one representative from each of the morphologically distinct groups was identified by 16S rRNA sequencing (Landcare Research). DNA was extracted using a Sigma REDExtract-N-Amp™ tissue kit following the manufacturer's instructions (Sigma-Aldrich, Castle Hill, New South Wales, Australia). The extracted DNA samples were amplified using the bacterial 16S rRNA primers 1F and 1509R and the following PCR conditions: 95 C for 4 min; 95 C for 30 sec, 53 C for 30 sec, and 72 C for 1 min for 25 cycles; and 72 C for 10 min. Successful amplifications were confirmed by running the PCR products on a 1.5% (wt/vol) agarose gel at 150V for 30 min, staining with ethidium bromide, and visualizing under ultraviolet light. The PCR products were sequenced using an ABI Genetic Analyzer 3130xL sequencing machine (Applied Biosystems, Mulgrave, Victoria, Australia). Sequence data were analyzed using the Sequencher software v. 5.0 (Gene Codes Corp., Ann Arbor, Michigan, USA) and identities confirmed using the Basic Local Alignment Search Tool (NCBI 2014) using the program Geneious (v.5.65) (Biomatters, Ltd., Auckland, New Zealand).
To assess if location or species affected the presence or frequency of bacterial genera identified, the data were analyzed using Fisher's exact tests with the WINPEPI statistical program, v. 11.20 (Brixton Health 2013).
Nucleotide sequence accession number
All 16S rRNA gene sequences of the bacterial species isolated in this study were deposited in the NCBI GenBank database under accessions KC306404–KC306502. The bacterial isolates have been cryopreserved at −70 C at the Auckland Zoo and the Bd isolate has been cryopreserved at −70 C at Landcare Research (Boyle et al. 2003).
In vitro Bd-bacterial challenge assay
Thirty-one bacterial isolates from the Coromandel population of L. archeyi were challenged against Bd using the technique described by Harris et al. (2006). All procedures were performed in a class-two biosafety cabinet. A New Zealand isolate of Bd was cultured by standard methods (Berger et al. 2005) and identified as a unique genotype (Kaikorai Valley-Lewingii-2008-SDS1) using methods described by James et al. (2009; Fig. 1). Actively growing Bd cultures in TGhL broth were passaged to TGhL agar plates (Berger et al. 2009) and incubated at 15 C. After 3 days, zoospores were collected by flushing plates with 6 mL sterile distilled water. Zoospores were counted using a Neubauer hemocytometer and resuspended using sterile distilled water to a concentration of 4×106 zoospores/mL. One milliliter of the zoospore suspension was spread evenly on a new TGhL plate and air-dried in a sterile biohazard cabinet until the plate appeared dry, but still glistening. One streak of freshly cultured and identified challenge bacterium was made on the left side of the plate while a sterile loop with no bacteria was streaked on the right side of the plate as a negative control. This process was repeated until a bacterium that did not inhibit Bd was found (Chryseobacterium sp. 3A blue). This was used as a negative bacterial control.
The plates were incubated at 15 C and inspected 24, 48, and 72 hr after inoculation. They were scored as 1) positive inhibition if there was Bd growth and a zone of inhibition around the bacterial streak, 2) negative inhibition if there was Bd growth up to the bacterial streak, or 3) indeterminate if Bd did not grow at all or if the bacterial streak overtook the whole plate. If an indeterminate result was obtained, the experiment was repeated up to two more times before being recorded as indeterminate.
RESULTS
Bacterial culture and identification
Thirty-one of the bacterial isolates obtained from the 11 L. archeyi at the Coromandel site were distinct isolates and 21 of the 31 isolates (68%) were Pseudomonas spp. (Table 1) Thirty-four of the 62 isolates from the 33 L. archeyi at the Whareorino site were distinct and 24 of the 34 (71%) were Pseudomonas spp. (Table 1). Thirty-one of the 50 bacterial isolates from the 20 L. hochstetteri at the Whareorino site were distinct. Flavobacterium spp. was the most common genera identified and comprised 12 of the 31 bacterial isolates (39%) (Table 1).
Three isolates of Pseudomonas were found in more than one location (Pseudomonas putida isolate PSB31, Pseudomonas sp. BR6-10, and Pseudomonas sp. 29H), which made the total distinct isolates identified 92 (Table 1). Flavobacterium species were significantly more prevalent in the Whareorino L. hochstetteri compared with the Whareorino L. archeyi (Fisher's exact test, P = 0.02 [odds ratio 6.3 with 95% confidence interval [CI] 1.3–33.1]) and when compared with all L. archeyi at both the Whareorino and Coromandel locations together (P = 0.01 [odds ratio 6.4 with 95% CI 1.5–29.2]).
In vitro Bd-bacterial challenge assay
The Bd-bacterial challenge assay was only performed for bacterial species from L. archeyi from the Coromandel because it was difficult to obtain consistent results using the technique developed by Harris et al. (2006). From 31 bacterial challenges, one was positive, (Flavobacterium sp. XAS590; Fig. 2), 20 were negative, and 10 were indeterminate despite repeated attempts to get a definitive result. The reasons for a test being indeterminate were 1) the Bd agar plate was too dry, thus killing the zoospores, or 2) the plate was not dry enough, so some mucoid bacteria (e.g., Pseudomonas) took over the entire plate within 24 hr so that a 24-hr reading could not be obtained.
DISCUSSION
We isolated and identified 92 unique bacterial isolates from 44 L. archeyi and 20 L. hochstetteri in the Coromandel and Whareorino regions. One of 31 isolates challenged, Flavobacterium sp. XAS590 inhibited the growth of Bd in vitro. Flavobacterium spp. occurred more frequently in L. hochstetteri than in L. archeyi.
Baseline data on the cutaneous bacteria in healthy, free-ranging L. archeyi and L. hochstetteri could be used to interpret bacterial culture results as part of a diagnostic work-up in sick frogs. The data also may be useful when interpreting bacterial skin cultures from pretranslocation or quarantine disease screening, where abnormal results can jeopardize an entire movement of frogs. When comparing our results to those of Potter and Norman (2006), only Serratia spp. were found in both studies. This difference in bacterial isolates found could reflect the more-precise molecular DNA identification techniques used in our study (Ludwig 2008) or differences between the bacterial flora on the dorsal and ventral skin surfaces (Culp et al. 2007). For bacterial culture, we used Difco R2A agar plates and lower incubation temperatures to simulate conditions in wild frogs and also those favorable to Bd growth (Berger et al. 2004); thus, our methods could have selected for different bacteria. Another difference between this study and Potter and Norman (2006) is that we did not identify all the bacterial isolates. By grouping morphologically similar isolates we expected to identify most of the flora. However, as bacteria are difficult to distinguish solely by gross morphology, or were unculturable, we likely missed species that would only be detected by molecular screening (e.g., with next-generation sequencing; MacLean et al. 2009).
Flavobacterium spp. were isolated significantly more frequently in L. hochstetteri than in L. archeyi in both the Whareorino location and when combining both the Coromandel and Whareorino locations. Flavobacterium XAS590 from L. archeyi was the only bacterial isolate that showed anti-Bd properties in our experiments. Although the Bd-bacterial challenge was not complete for the Flavobacterium spp. isolated in L. hochstetteri, this is the first time that bacteria from Leiopelma spp. have been shown to exhibit in vitro anti-Bd properties.
If Flavobacterium spp. are important in innate immunity against chytridiomycosis in L. archeyi, we would expect a higher prevalence than 2/10 (Coromandel) and 5/24 (Whareorino) because previous studies have shown that if a high proportion of susceptible frogs have at least one anti-Bd bacterial species present, the population can persist despite the presence of Bd (Woodhams et al. 2007; Lam et al. 2010). Therefore, L. archeyi may not use bacterial inhibition as a principle means of defense against Bd, unless other unidentified species are inhibitory. We recommend that bacteria are tested further using the new broth challenge assay developed by Bell et al. (2013). This technique avoids the issues of the agar plate method and may provide more-reliable results. Another potential method to test to assess Bd viability after bacterial exposure uses a combination of ethidium monazide with quantitative PCR (Blooi et al. 2013). Flavobacterium should be investigated further for its role in host resistance to Bd and added to the growing list of bacteria that can be used in potential bioaugmentation trials.
Species of Pseudomonas were the most common isolates found in L. archeyi in both locations. Although these mucoid bacteria were difficult to screen in our Bd-bacterial challenge, they have been successfully challenged in other studies and some species had anti-Bd properties (Lauer et al. 2007, 2008; Woodhams et al. 2007; Lam et al. 2010). We suggest that the Pseudomonas isolates from New Zealand should be investigated further for Bd inhibition.
ACKNOWLEDGMENTS
Funding for this project was provided by the Auckland Zoo Charitable Trust Conservation Fund. Many thanks to the staff at Landcare Auckland who were instrumental in the bacterial culturing, identification, and storage of chytrid samples: Stanley Bellgard, Maureen Fletcher, Karen Hoksbergen, Daniel Than, Bevan Weir, and Paula Wilke. Thanks also to Lisa Daglish and Amanda Haigh from the Department of Conservation who collected the bacterial samples. Many thanks to Reid Harris, Brianna Lam, and Jennifer Walke for technical advice. Thanks also to the New Zealand Maori iwi for supporting native frog research.