Isolation of culturable microorganisms from free-ranging bottlenose dolphins (Tursiops truncatus) from the southeastern United States

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lation of culturable microorganisms from free-ranging bottlenoselphins (Tursiops truncatus) from the southeastern United States

mela J. Morris a,b,*, Wesley R. Johnson b, John Pisani c, Gregory D. Bossart d,e, Jeff Adams f,n S. Reif g, Patricia A. Fair f

le W. Baruch Institute for Marine & Coastal Sciences, University of South Carolina, Charleston, SC 29412, United States

llings Marine Laboratory, Charleston, SC 29412, United States

crim Laboratory, 800 NE 62nd Street 202, Fort Lauderdale, FL 33334, United States

orgia Aquarium, 225 Baker Street, NW Atlanta, GA 30313, United States

rbor Branch Oceanographic Institute at Florida Atlantic University, 5600 U.S. 1 North, Ft. Pierce, FL 34946, United States

ional Oceanic and Atmospheric Administration/National Ocean Service/Center for Coastal Environmental Health and Biomolecular Research,

leston, SC 29412, United States

partment of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO 80524, United States

ntroduction

Marine mammals, such as cetaceans, are ideal sentinelshuman health as many reside in near-coastal habitats

and are exposed to a wide variety of infectious biologicalagents and chemical pollutants (Bossart, 2006; Moore,2008). While the number of reported marine mammaldiseases is increasing (Bossart, 2007), our knowledgeregarding the potential for the marine environment toserve as a reservoir for known and emerging pathogens islimited. Examination of a subset of stranded Atlanticbottlenose dolphins (Tursiops truncatus) indicated that 31%died of infectious disease with bacterial infectionsaccounting for the majority of mortality (McFee and

T I C L E I N F O

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A B S T R A C T

Reports of diseases in marine mammals are increasing worldwide, however our

understanding of the microorganisms associated with marine mammals is still limited.

In this study, we cultured bacteria and fungi isolated from the upper respiratory tract

(blowhole), gastric fluid and anus of 180 wild bottlenose dolphins (Tursiops truncatus)

from two estuarine locations along the southeastern Atlantic Coast of the United States. A

total of 339 and 491 isolates from Charleston, SC (CHS) and Indian River Lagoon, FL (IRL)

dolphins, respectively, were cultured from gastric (70 CHS/82 IRL), fecal (141 CHS/184

IRL), and blowhole (128 CHS/225 IRL) swabs on selective media used for routine clinical

microorganisms of human concern. The most frequently cultured Gram-negative bacteria

from all sample and study types were Plesiomonas shigelloides, Aeromonas hydrophila,

Escherichia coli, and Pseudomonas fluorescens. Among the Gram-positive bacteria,

Clostridium perfringens, Bacillus sp., and Staphylococcus Coag. Neg were the predominant

organisms. For fungi, the most abundant species were Candida glabrata, budding yeasts, and

Candida tropicalis. Of concern, the MRSA strain of Staphylococcus aureus was detected in the

blowhole and gastric swabs from CHS dolphins. In general, a greater prevalence of bacteria

and fungi (four-fold increase) were cultured from IRL than CHS animals. Together, these

culture-dependent studies, coupled to on-going culture-independent approaches, should

help establish a baseline of microorganisms associated with bottlenose dolphins and aid in

the identification of organisms responsible for infectious diseases(s) in these animals.

� 2010 Published by Elsevier B.V.

Corresponding author at: Hollings Marine Laboratory, 331 Ft Johnson

d, Charleston, SC 29412, United States. Tel.: +1 843 991 8355;

+1 843 762 8737.

E-mail address: [email protected] (P.J. Morris).

Contents lists available at ScienceDirect

Veterinary Microbiology

journa l homepage: www.e lsev ier .com/ locate /vetmic

ease cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenoseolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-ic.2010.08.025

8-1135/$ – see front matter � 2010 Published by Elsevier B.V.

10.1016/j.vetmic.2010.08.025

P.J. Morris et al. / Veterinary Microbiology xxx (2010) xxx–xxx2

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Lipscomb, 2009). In a study of wild dolphins from the Gulfof Mexico and two Atlantic Ocean locations, numerousaerobic microorganisms of clinical significance wereisolated from fecal and blowhole samples, with Vibrio

alginolyticus and Vibrio damsela being the most commonlyrecovered (Buck et al., 2006). Establishing a baseline ofmicroorganisms associated with bottlenose dolphins willshed light on bottlenose dolphins as sentinel species forthe health of the coastal zone, and whether they mightserve as potential ecological reservoirs for known andemerging human and marine mammal pathogens (MarineMammal Commission, 2004).

Prior studies of diseases in humans and other mammalshave relied primarily on traditional clinical isolationapproaches, with culturing the critical first step inidentifying pathogenic microorganisms. In this study, wecultured and identified microorganisms of known humansignificance from the upper respiratory tracts (blowhole),gastric and fecal environments of 180 bottlenose dolphinsinhabiting two southeast coastal sites. These sites, theCharleston Harbor, SC (CHS) and the Indian River Lagoon,FL (IRL) are impacted by different environmental stressors(Fair et al., 2007). Our aim in this study was to characterizethe bacteria and fungi isolated from the upper respiratorytract (blowhole), gastric fluid and anus of wild bottlenosedolphins and investigate associations in microbial florabetween healthy and diseased status in dolphins. Wecompared these results to recent complementary studiesusing molecular approaches in samples collected fromdolphins in these same two areas.

2. Materials and methods

2.1. Study locations

Samples were collected from bottlenose dolphinsduring health assessment studies in the estuarine watersof the Indian River Lagoon, FL (IRL) and Charleston, SC(CHS). The health assessments in the IRL were conductednear Titusville, FL (2883604300N, 8084802700W) and Stuart, FL(2781105100N, 8081501000W) and included portions of theMosquito Lagoon, Indian River, Banana River, north andsouth forks of the St. Lucie River, and Sebastian Inlet. TheCHS study site (3284603500N, 7985505100W) consists of theCharleston Harbor Estuary, which includes the CharlestonHarbor, as well as the main channels and creeks of theAshley, Cooper, and Wando Rivers.

2.2. Study populations

During the summers of 2003, 2004 and 2005, 180 free-ranging bottlenose dolphins (T. truncatus) were captured,examined, sampled, marked and safely released (con-ducted under National Marine Fisheries Permit No. 998-1678; Harbor Branch Oceanographic Institutional AnimalCare and Use Committee) in CHS (n = 76) and IRL (n = 104)by the Dolphin Health and Risk Assessment (HERA) Project(Fair et al., 2006). Based on a suite of health data (i.e.,physical and ultrasound examinations, hematology andserum chemistry, cytologic and microbiologic evaluationof gastric, fecal and blowhole cytology, and urinalysis),

dolphins were classified by a veterinary panel as normal(i.e., free of disease), possibly diseased or definitelydiseased (Reif et al., 2008). In that paper the health ofapproximately 46% of the dolphins (50% CHS dolphins; 44%IRL dolphins) examined were classified as normal whiledolphins from both regions had a high prevalence ofdefinite disease (CHS 21% vs. 34%).

2.3. Sample collection

Swab samples were collected from the blowhole,gastric fluid and anus of each dolphin for bacterial andfungal evaluation (Fair et al., 2006). Although sampleswere collected from a total of 180 dolphins, variationsoccurred in the number of samples collected from each ofthe three sites (i.e., blowhole, gastric or anus) per eachindividual dolphin. Briefly, a sterile swab was inserted intothe blowhole during a breath, gently moved along the wallof the blowhole, and removed during the next breath.Gastric fluid was collected by inserting a well-lubricatedsoft, flexible, plastic foal stomach tube past the oropharynxto the first stomach and then a swab sample obtained formicrobiological evaluation. An anal swab was collected byinserting a sterile swab into the anal orifice, and gentlyswabbing the area. Collection of samples for aerobic,anaerobic and fungal cultures and targeted identificationwere made using Aimes culturettes (MML Diagnostics,Troutdale, OR). These were stored in coolers and shippedusing cold packs to Micrim Labs, Inc. (Fort Lauderdale, FL)within 24 h of sampling.

2.4. Microbiological culture and isolations

Clinical isolation approaches were used to isolate andidentify known opportunistic or pathogenic organisms.One blowhole swab from each animal and sample type wascollected for selective culture and identification of bacteriaand fungi. Media used for bacterial isolations includedtryptic soy agar (TSA) with 5% sheep blood, MacConkeyagar, Hektoen enteric, thiosulfate-citrate bile sucrose(TCBS) agar, and thioglycollate broth (for isolation ofanaerobic bacteria). For fungal isolations, Sabouraud-Dextrose agar (Emmons) and Sabouraud-Dextrose agar(both amended with 0.05 g/L chloramphenicol), TSA with5% sheep blood, malt extract, and mycobiotic agar (0.05 g/Lchloramphenicol) and 0.4 g/L cyclohexamide. Anaerobiccultures were placed in anaerobic jars treated withAnaeroPacks (Mitsubishi Gas Chemical, New York, NY)in order to obtain a reduced oxygen environment (0.01%O2, 15% CO2, with the balance being N2). All media wereobtained from Remel Inc. (Lenexa, KS).

Aerobic and anaerobic bacterial cultures were incubatedat 37 8C while fungal cultures were incubated at both 24 and37 8C. Aerobic incubations were observed for growth after24 h, while anaerobic cultures were incubated for 48 hbefore examination. All cultures were screened daily for thefirst week, and then twice a week for the next 3 weeks. Allnegative cultures were held a full 4 weeks before reportingas a negative response, and all positive cultures werereported when identification was complete. When growthwas observed, isolates with different colony morphologies

Please cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenosedolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-mic.2010.08.025

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P.J. Morris et al. / Veterinary Microbiology xxx (2010) xxx–xxx 3

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re further streaked to purity and subsequently identifiedg the appropriate identification system.

For characterization of microbial isolates, the followingntification systems were used: API 20E (bioMerieux,., Durham, NC) for Gram-negative fermenters (controlanisms used: Klebsiella pneumoniae, ATCC-35657;erobacter cloacae, ATCC-13047; Proteus mirablis, ATCC59; Stenotrophomonas maltophila, ATCC-51331), API 20for Gram-negative non-fermentors (control organismsd: Pseudomonas aeruginosa, ATCC-27853; Aeromonas

rophila, ATCC-35654; Alcaligenes fecalis, ATCC-35655;ingobacterium multivorum, ATCC-35656), Analytabducts API 20 C AUX for yeast (control organisms used:ptococcus laurentii, ATCC-18803; Candida glabrata,C-15126), Remel-Rapid ANA System for anaerobes

ntrol organisms used: Clostridium sordellii, ATCC-9714;teroides distasonis, ATCC-8503, Bacilus uniformis, ATCC-02); Remel-Staph Aurex for determination of coagulase

ction (Control organisms used: Staphylococcus aureus,C-25923; Staphylococcus epidermidis, ATCC-12228).

Statistical analysis

Mantel–Haenszel odds ratios were calculated for theuency of each microorganism among the dolphinspled using the FREQ procedure in SAS (Statisticallysis Software, Cary, NC). The frequency estimates ands ratio estimates were controlled for sample type (i.e.,tric, blowhole, fecal). Principal component analysisA) was performed on clinical isolation data toestigate patterns of bacterial species among healthy

diseased dolphins from the two estuaries. A binaryre was determined for the presence (1) or absence (0) ofh of the 49 bacterial and fungal species and strainntified by clinical isolation for each sample typestric, blowhole, or fecal) from each animal. The analysiss performed using the principal component algorithmhin the MultiVariate Statistical Package (MVSP)vach Computing Services, Wales, UK) using theransformed data.

esults

Microbiological culturing

A total of 339 isolates from CHS dolphins and 491ates from IRL dolphins were cultured from the gastricCHS/82 IRL), fecal (141 CHS/184 IRL), and blowhole

8 CHS/225 IRL) swabs (Tables 1 and 2). Overall, therere 11 Gram-negative and 28 Gram-positive bacterialcies or groups (Table 1). The most frequently culturedm-negative organisms from all swab types and bothdy sites in descending order were Plesiomonas shigel-

es, A. hydrophila, Escherichia coli, and Pseudomonas

rescens. Distributions patterns of both P. shigelloides

E. coli were similar for both CHS and IRL dolphins withhest frequencies in fecal swabs followed by blowholebs and most rarely occurring in gastric samples. AmongGram-positive bacteria cultured, Clostridium perfrin-

s, Bacillus sp., and Staphylococcus Coag. Neg were thest predominant (Table 1), with Bacillus sp. occurring in

both blowhole and gastric samples, but not in fecal swabs.Interestingly, the methicillin-resistant S. aureus (MRSA)strain of S. aureus was only detected in the blowhole andgastric swabs from CHS dolphins. For fungi, the mostabundant species were C. glabrata, budding yeasts, andCandida tropicalis (Table 2). In general, a greater prevalenceof bacteria and fungi (four-fold increase) were culturedfrom IRL than CHS animals.

3.2. Frequency of cultured isolates

Twelve of the cultured organisms exhibited significantdifferences in isolation frequency between CHS and IRLdolphins (a = 0.05), with budding yeast showing thegreatest difference at more than 9.5 times more likely tobe cultured from IRL samples (Table 3). Torulopsis candida

and S. aureus are estimated to be 5.7 and 6.4 times morecommon, respectively, from IRL dolphins than those fromCHS. The budding yeast and T. candida were all culturedexclusively from IRL samples. IRL dolphins also hadsignificantly higher frequencies of Acinetobacter bauman-

nii, Edwardsiella tarda, enteric flora, Morganella morganii, P.

fluorescens and S. aureus. While the frequency of S. aureus

was higher in IRL dolphins, MRSA was found only in threeCHS dolphins. CHS dolphins had significantly higheroccurrence of Bacillus sp. and E. coli. Similarly, testsdemonstrated growth of P. fluorescens significantly morefrequently in IRL cultures than in those from CHS. P.

fluorescens and A. hydrophila were more common inblowhole samples than in the fecal or gastric swabs.

3.3. Distribution of cultured isolates

The distribution pattern of isolates from blowhole andfecal swabs showed no differences among normal, possiblydiseased, and diseased dolphins. Principle componentanalysis (PCA) of all samples revealed four clearly distinctgroups, and loadings of the isolate types in the eigenanalysis demonstrated these groups were the determinedlargely by the presence or absence of the four dominantGram-negative bacteria. These groupings were not differ-entiated by site, swab type (gastric, fecal, blowhole), orhealth status (Fig. 1). Differences were observed in isolatesobtained from gastric swabs among the dolphins’ healthclassification, most notable of which was greater frequen-cies of budding yeast and Candida spp. in diseased culturesthan in either the normal or concerned categories.However, these differences were not statistically signifi-cant (a = 0.05).

4. Discussion

Over 20 different bacterial and 10 different fungalspecies were isolated from wild dolphins sampled from thetwo estuaries in this study. The most abundant bacteriaincluded P. shigelloides, followed by A. hydrophila, E. coli,and P. fluorescens. In contrast, V. alginolyticus and V.

damsela were the most commonly recovered bacteria fromboth anal/fecal and blowhole dolphin samples in a surveyby Buck et al. (2006). In our study, V. alginolyticus was alsofrequently isolated, particularly from blowholes. This

ease cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenoseolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-ic.2010.08.025

Table 1

Frequency of bacterial blowhole, fecal, and gastric isolates cultured from CHS and IRL bottlenose dolphins.

CHS IRL Grand total

Blowhole Fecal Gastric Total Blowhole Fecal Gastric Total

Gram (+) bacteria

Bacillus sp 5 (3.9) 16 (22.9) 21 (6.1) 3 (1.3) 8 (9.8) 11 (2.2) 32Clostridium bifermentans 1 (0.7) 1 (0.3) 1 (0.5) 3 (3.7) 4 (0.8) 5Clostridium perfringens 3 (2.3) 3 (2.0) 15 (21.4) 21 (6.1) 4 (2.1) 12 (14.6) 16 (3.2) 37Diptheroids 1 (1.4) 1 (0.3) 1 (0.4) 1 (0.2) 2Enterococcus sp 2 (1.6) 1 (1.4) 3 (0.9) 1 (0.4) 1 (1.2) 2 (0.4) 5Staph aureus 1 (0.7) 1 (0.3) 4 (1.8) 1 (0.5) 3 (3.7) 8 (1.6) 9Staph aureus (MRSA) 2 (1.6) 1 (1.4) 3 (0.9) 3Staph Coag. Neg. 9 (7) 1 (0.7) 4 (5.7) 14 (4.1) 8 (9.8) 8 (1.6) 22Staph Coag. Neg. subsp.#1 1 (1.2) 1 (0.2) 1Staph Coag. Neg. subsp.#2 1 (1.2) 1 (0.2) 1Strep viridans 1 (1.4) 1 (0.3) 1 (1.2) 1 (0.2) 2

Gram (�) bacteria

Acinetobacter baumannii 3 (2.0) 2 (2.9) 5 (1.4) 16 (7.1) 4 (2.1) 1 (1.2) 21 (4.2) 26Acinetobacter hemolyticus 3 (1.3) 1 (1.2) 4 (0.8) 4Aeromonas hydrophila 26 (20.3) 24 (16.3) 6 (8.6) 56 (16.2) 47 (20.9) 35 (18.6) 8 (9.8) 90 (18.2) 146Aeromonas sobria 1 (0.5) 1 (0.2) 1Bacteroides fragilis 4 (5.7) 4 (1.2) 1 (1.2) 1 (0.2) 5Chryseomonas luteola 1 (1.2) 1 (0.2) 1Corynebacterium sp. 2 (1.6) 2 (0.6) 2E. coli 10 (7.8) 32 (21.8) 4 (5.7) 46 (13.3) 8 (3.6) 30 (16) 1 (1.2) 39 (7.9) 85E. coli subsp.#1 1 (0.4) 1 (0.5) 2 (0.4) 2E. coli subsp.#2 1 (0.5) 1 (0.2) 1Edwardsiella tarda 5 (3.4) 1 (1.4) 6 (1.7) 14 (6.2) 8 (4.3) 22 (4.4) 28Enteric flora 5 (2.7) 5 (1.0) 5Enterobacter agglomerans 1 (1.2) 1 (0.2) 1Enterobacter cloacae 1 (0.8) 1 (0.7) 2 (0.6) 1 (0.4) 1 (0.5) 1 (1.2) 3 (0.6) 5Klebsiella pneumoniae 2 (1.6) 3 (2.0) 1 (1.4) 6 (1.7) 5 (2.2) 10 (5.3) 15 (3) 21Marine vibrio 4 (3.1) 2 (1.4) 6 (1.7) 2 (0.9) 2 (1.1) 2 (2.4) 6 (1.2) 12Morganella morganii 1 (0.4) 4 (2.1) 5 (1) 5Pantoea agglomerans 1 (0.4) 1 (0.2) 1Plesiomonas shigelloides 27 (21.1) 43 (29.3) 8 (11.4) 78 (22.6) 28 (12.4) 42 (22.3) 15 (18.3) 85 (17.2) 163Proteus mirabilis 3 (2.3) 5 (3.4) 8 (2.3) 3 (1.3) 3 (1.6) 6 (1.2) 14Pseudomonas aeruginosa 2 (1.6) 1 (0.7) 3 (0.9) 9 (4) 1 (0.5) 2 (2.4) 12 (2.4) 15Pseudomonas alcaligenes 1 (1.4) 1 (0.3) 1Pseudomonas fluorescens 8 (6.3) 9 (6.1) 2 (2.9) 19 (5.5) 39 (17.3) 20 (10.6) 4 (4.9) 63 (12.7) 82Pseudomonas stutzeri 3 (2.3) 1 (0.7) 4 (1.2) 6 (2.7) 1 (0.5) 1 (1.2) 8 (1.6) 12Serratia rubidaea 1 (0.8) 1 (0.3) 1Shewanella putrefaciens 8 (6.3) 6 (4.1) 14 (4.1) 10 (4.4) 9 (4.8) 2 (2.4) 21 (4.2) 35Steno maltophilia 1 (0.4) 1 (0.2) 1Vibrio alginolyticus 10 (7.8) 6 (4.1) 2 (2.9) 18 (5.2) 21 (9.3) 4 (2.1) 3 (3.7) 28 (5.7) 46

Grand total 128 147 70 345 225 188 82 495 840

Numbers in parentheses represent the percent of total bacterial isolates from each swab type.

Table 2

Frequency of fungal isolates cultured from CHS and IRL bottlenose dolphins.

Fungi CHS (%) IRL (%) Grand total

Blowhole Fecal Gastric Total Blowhole Fecal Gastric Total

Aspergillus fumigatus GRP 1 (14.3) 1 (7.1) 1 (4.2) 1 (3.7) 2 (3.2) 3Aspergillus niger GRP 2 (28.6) 2 (14.3) 2Budding yeast 7 (29.2) 3 (27.3) 9 (33.3) 19 (30.6) 19Candida albicans 1 (4.2) 1 (3.7) 2 (3.2) 2Candida glabrata 1 (50.0) 1 (20.0) 2 (14.3) 8 (33.3) 3 (27.3) 7 (25.9) 18 (29) 20Candida rugosa 1 (14.3) 1 (20.0) 2 (14.3) 2Candida tropicalis 1 (14.3) 1 (20.0) 2 (14.3) 5 (20.8) 2 (18.2) 5 (18.5) 12 (19.4) 14Cunninghamella bertholletiae 1 (14.3) 1 (20.0) 2 (14.3) 2Torulopsis candida 2 (8.3) 3 (27.3) 4 (14.8) 9 (14.5) 9Trichosporon beigelii 1 (14.3) 1 (50.0) 1 (20.0) 3 (21.4) 3Total fungal isolates 7 2 5 14 24 11 27 62 76

Numbers in parentheses represent the percent of total fungal isolates from each swab type.

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Please cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenosedolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-mic.2010.08.025

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terium is commonly found in association with marinena, and rarely causes infections in humans.S. aureus was found in dolphins from both sitesluding the identification of MRSA from two blowhole

ples and one gastric sample in three individual CHSphins as described previously (Schaefer et al., 2009).ough most MRSA infections have been hospital-

ociated, recent increases in community-acquired infec-s have been reported (Klevens et al., 2007). S. aureus

s identified as the highest risk bacterial pathogen in the. Navy Marine Mammal Program dolphin populationnn-Watson et al., 2008).

Antibiotic resistance is a rising concern in the marineenvironment and limited information exists on antibioticresistance in free-ranging marine mammals. Greig et al.(2007) analyzed resistance patterns in E. coli fecal samplesfrom CHS and IRL dolphins and found that 47% of dolphinssampled harbored E. coli resistant to one or moreantibiotics, and 25% were resistant to antibiotics com-monly used in humans and animals (i.e., penicillin,cephalothin, ampicillin and amoxicillin). In a recentcompanion paper to our study assessing microorganismsfound in CHS and IRL dolphins, Schaefer et al. (2009) founda high prevalence of antibiotic resistance, with resistance

le 3

mated odds ratios, controlled for swab type, for organisms showing significant differences in frequency between IRL and CHS dolphins.

late Culture type Odds ratio (IRL/CHS) LCI UCI p value

inetobacter baumannii Bacterium 3.39 1.27 9.03 0.0092

cillus sp. Bacterium 0.39 0.18 0.87 0.0189

coli Bacterium 0.57 0.34 0.96 0.0286

wardsiella tarda Bacterium 1.61 0.58 4.49 0.0268

teric flora Bacterium 9.32 0.51 171.55 0.0428

organella morganii Bacterium 4.38 0.50 38.32 0.0452

eudomonas fluorescens Bacterium 3.14 1.78 5.53 <0.0001

aph aureus Bacterium 6.43 0.81 51.28 0.0436

dding yeast Fungus 9.54 1.77 51.54 0.0002

ndida glabrata Fungus 5.10 1.29 20.07 0.0035

ndida tropicalis Fungus 4.18 1.06 16.51 0.021

rulopsis candida Fungus 5.73 1.02 32.20 0.0064

and UCI are lower and upper confidence intervals, respectively.

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Fig. 1. PCA biplots of clinical isolate frequencies from combined blowhole, fecal, and gastric swabs.

ease cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenoseolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-ic.2010.08.025

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most commonly found to erythromycin (91%), followed indecreasing frequency by clindamycin, ampicillin, cepha-lothin, piperacillin and amoxicillin.

Generally, IRL dolphins exhibited a greater prevalenceof bacteria and fungi compared to CHS dolphins. A numberof variables may influence the microbial communities offree-ranging dolphins including contamination sourcesand water temperatures. Water quality in the IRL hasdeteriorated due to fresh and storm water discharges(Scott et al., 2003) and specific observed pathologies havebeen observed in stranded IRL dolphins (Bossart et al.,2003). Since 1996, bottlenose dolphins in the IRL have hadhigh incidences of seasonal strandings (Stolen et al., 2006)

and in 2001 �30 bottlenose dolphins died over 2 monthsdue to unknown causes (Marine Mammal Commission,2002).

A list of the bacterial and fungal agents isolated in threeprevious investigations as well as this study that havespecifically examined the microflora of the blowhole,stomach, and/or feces/anus of bottlenose dolphins isprovided in Table 4. The most commonly isolatedorganisms include numerous Gammaproteobacteria suchas Vibrio spp., E. tarda, E. coli, P. shigelloides, Proteus

mirabilis, Pseudomonas spp., and Shewanella putrefaciens.Among the Firmicutes, S. aureus is the most frequentlyobserved isolate. While these studies indicate the presence

Table 4

Bacteria and fungi isolated from blowhole, fecal/anus, and gastric samples from (1) this study; (2) Buck et al. (2006); (3) Harper et al. (2000); (4) Buck et al.

(1987).

Taxon Organism Gastric Fecal/Anus Blowhole

Gammaproteobacteria Acinetobacter baumannii 1 1 1

Acinetobacter calcoaceticus – 2 2

Acinetobacter hemolyticus 1 1 1

Aeromonas hydrophila 1 1, 2 1,2

Aeromonas sobria – 1

Escherichia coli 1 1, 2 1,2

Edwardsiella tarda 1 1, 2, 4 1, 2

Enterobacter cloacae 1 1, 2 1, 2

Enterobacter agglomerans 1 2 2

Klebsiella pneumoniae 1 1 1

Morganella morganii – 1 1

Pantoea agglomerans – – 1

Plesiomonas shigelloides 1 1, 2 1, 2

Proteus mirabilis – 1, 4 1

Providencia sp. – 4 –

Pseudomonas aeruginosa 1 1 1

Pseudomonas alcaligenes 1 – –

Pseudomonas fluorescens 1 1 1

Pseudomonas stutzeri 1 1, 2 1, 2

Serratia rubidaea – – 1

Shewanella putrefaciens 1 1, 2 1, 2

Stenotrophomonas maltophilia – – 1

Vibrio alginolyticus – 2 1, 2

Vibrio damsela – 2 2

Vibrio fluvialis – 2 2

Vibrio furnissii – 2 2

Vibrio parahaemolyticus – 2 2

Epsilonproteobacteria Helicobacter spp. 3

Firmicutes Bacillus spp. – – 1

Clostridium bifermentans 1 1 –

Clostridium perfringens 1 1 1

Corynebacterium spp. 1 – 1, 2

Enterococcus spp. – – 1, 4

Staphylococcus aureus 1 1 1, 2, 4

Staphylococcus epidermidis – 4 –

Staphylococcus hyicus – 4 –

Streptococcus viridans 1 – –

Bacteroidetes Bacteroides fragilis 1

Chryseomonas luteola 1

Fungi Aspergillus spp. 1 – 1

Budding yeast 1 1 1

Candida albicans – 2 2

Candida glabrata 1 1 1

Candida rugosa 1 – 1

Candida tropicalis 1 1, 2 1, 2

Cunninghamella bertholletiae 1 1

Torulopsis candida 1 1 1

Trichosporon beigelii 1 1 1

Please cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenosedolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-mic.2010.08.025

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Pldm

umerous pathogens in association with dolphins, it isortant to note that the clinical isolation methodsen specifically for known pathogens, primarily ofan significance. In addition, animals sampled in these

dies (Table 4) include stranded and diseased dolphins asll as individuals presumed to be healthy. We did noterve any significant odds ratios for any microorganismong normal (i.e., healthy), possibly diseased, andnitely diseased animals in our present study (Tablesuggesting there is no measurable influence of healthus on the culturable microflora of bottlenose dolphins.

The exposure of humans to bottlenose dolphinssents potential risks of zoonotic infection by micro-anisms that may be carried by the bottlenose dolphinser as components of their normal flora or as pathogens.notic transmission from dolphins to humans may occur

ough common use of coastal waters and exchange ofmal flora or pathogens in the water column. Bottlenosephins in managed-care facilities are known to sufferctions from numerous fungal agents, including Asper-

s fumigatus, Blastomyces dermatitidis, Cryptococcus spp.,Candida spp. (Reidarson et al., 2001), most of which

ur as severe lung infections. In some locations, wildphins are frequently observed with the severe mycotic

infection, lobomycosis, caused by the yeast-likegus (Reif et al., 2006). The fungi cultured in this study

blowhole fluids are known to cause pulmonary andtemic infections in immunocompromised humans.ergillus spp. and Candida spp. are responsible forsing the majority of fungal infections in humans (Ustal., 2004; Hernandez et al., 2004). In our study, C. albicans

s isolated only from IRL animals (e.g., blowhole andtric swabs) similar to findings observed by Buck et al.06) in which this organism was isolated only fromphins in Florida and not from those sampled in Texas

North Carolina.It is well known that most microorganisms are notturable using traditional isolation approaches (Deth-en et al., 2007). However, studies of diseases in humansother mammals have historically relied primarily on

ical isolation approaches, with culturing the first stepharacterizing potentially pathogenic microorganisms.

crepancies in the identification of bacteria have beenerved using routine microbial diagnostics, for example,atic Vibrio strains have been misidentified using API(bioMerieux) diagnostic systems (Israil et al., 2003).study has shown that the upper respiratory tracts of

tlenose dolphins contain over 20 different bacterialcies of clinical human relevance. In comparison, aplimentary study using a molecular, culture-indepen-

t approach has revealed a far more diverse bacterialmunity in bottlenose dolphin blowhole fluids than is

icated by culture-based studies, with estimates ofcies richness ranging from 50 to more than 200 species,ny of which appear to be novel taxa (Johnson et al.,9). Only a single organism, Bacteroides fragilis, wasected by both techniques, indicating a commonlyerved disparity between the two methodologies inich culture-based analysis selects for organisms thatw on specific media rather than those that are abundant

bacteria associated with human sputum samples usingculturing and molecular approaches, and observed agreater diversity of organisms using the molecularapproach. The combination of clinical and molecularapproaches to assess the diversity of the dolphin micro-biota should provide a more comprehensive view of thecommunities and their roles in health and disease (Gomez-Dıaz, 2009).

5. Conclusion

The relevance of bacterial and fungal organisms observedusing traditional clinical approaches to the health status ofbottlenose dolphin is unclear (Venn-Watson et al., 2008), asthese microbial isolates are often found in both healthy anddiseased animals and in different sample types (blowhole vs.gastric vs. fecal). However, this study and others (cited inTable 4) clearly demonstrate that pathogens of humanconcern are associated with bottlenose dolphins. With morein-depth studies focused on the microbiome of thebottlenose dolphin and other marine mammals, the roleof these known pathogens as well as the recent observationof other known and unknown organisms (Johnson et al.,2009) associated with bottlenose dolphins will shed furtherlight on the health of these animals, and their relevance as asentinel species.

Conflict of interest

The authors have no conflict of interest to declare.

Acknowledgements

We thank the numerous researchers and volunteerswho participated in the dolphin capture and releasestudies in South Carolina and Florida. We are especiallygrateful to Dr. Forrest Townsend, Mr. Larry Fulford, Mr.Larry Hansen, Mr. Eric Zolman, Mr. Steve McCulloch, theNOAA and HBOI staff, the collaborators and veterinarianswho provided their expertise. This study was supportedthrough NOAA/NCCOS/CCEHBR, NOAA Fisheries MarineMammal Health and Stranding Response Program and theFlorida Protect Wild Dolphins License Plate Fund.

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Please cite this article in press as: Morris, P.J., et al., Isolation of culturable microorganisms from free-ranging bottlenosedolphins (Tursiops truncatus) from the southeastern United States. Vet. Microbiol. (2010), doi:10.1016/j.vet-mic.2010.08.025