DISEASES OF AQUATIC ORGANISMSDis Aquat Org

Vol 122 195ndash203 2017doi 103354dao03069

Published January 24

INTRODUCTION

Inhabiting the same inshore ecosystems used byhumans marine mammals as long-lived top-tierconsumers are sentinel species for both human andocean health (Bossart et al 2011) Knowledge of thediversity and distribution of marine mammal patho-gens is expanding rapidly (Maness et al 2011 Riveraet al 2012) Recent studies have underscored theimportance of domesticated and feral animal pop -ulations in both the emergence of novel and there-emergence of existing human pathogens (Wool-house amp Gowtage-Sequeria 2005) Streptococcus

iniae was first isolated in 1976 from skin lesions ofAmazon freshwater dolphins Inia geoffrensis in theUSA (Pier amp Madin 1976) and is considered a majoraquatic animal pathogen in many regions of theworld Since then many outbreaks of S iniae infec-tion with high mortality rates in aquaculture farmshave been reported with numerous fish species be -ing found to be susceptible to infection (Adams ampThompson 2006) including a variety of ornamentalfish (Hartman et al 2006) These bacteria are alsozoonotic with infections in humans associated withthe handling and preparation of infected fish Basedon the data from the Centers for Disease Control and

copy Inter-Research 2017 middot wwwint-rescomCorresponding author zhaodmcaueducn

Fatal bacterial septicemia in a bottlenose dolphinTursiops truncatus caused by Streptococcus iniae

Zhiqi Song1 Ruichao Yue1 Yanming Sun2 Chunfa Liu1 Sher Hayat Khan1 Chaosi Li1 Ying Zhao1 Xiangmei Zhou1 Lifeng Yang1 Deming Zhao1

1The State Key Laboratories for Agrobiotechnology Key Lab of Animal Epidemiology and Zoonosis Ministry of Agriculture National Animal Transmissible Spongiform Encephalopathy Laboratory Department of Veterinary

Pathology College of Veterinary Medicine China Agricultural University Beijing 100193 PR China2Beijing Aquarium Beijing 100081 PR China

ABSTRACT A captive 8 yr old male bottlenose dolphin Tursiops truncatus succumbed to septi -cemia with multisystemic inflammation including suppurative enteritis encephalitis and pneu-monia with chronic pancreatitis A pure culture of beta-hemolytic catalase- and oxidase-negativeGram-positive cocci was isolated from the hilar lymph nodes and pancreas The isolate was iden-tified by 16S rDNA sequencing as Streptococcus iniae Histological examination of the digestivesystem revealed a mixed infection of both bacteria and fungus Recognized as a pathogen in fishdolphins and humans this is the first report of S iniae in a dolphin in mainland China As thenumber of managed animals in oceanariums is increasing so is the frequency of contact with fishused as food for marine mammals and humans highlighting the importance of education andappropriate personal protective protocols to minimize the risk of transmission An understandingof marine mammal infectious disease organisms is essential to ensuring the health of marine mammals and humans coming into contact with such animals and their food This study illustratesa systematic clinical microbiological and pathological investigation into a septicemic bottlenosedolphin infected with S iniae Our findings provide useful information for those involved in thediagnosis and control of infectious diseases in marine mammals and offer insight into an importantzoonotic pathogen

KEY WORDS Streptococcus iniae middot Bottlenose dolphin middot Zoonosis

Resale or republication not permitted without written consent of the publisher

Dis Aquat Org 122 195ndash203 2017

Prevention Microbiology Laboratory the bacteriumwas first recognized to cause human infection in 1991in North America (Centers for Disease Control 1996)

S iniae tends to cause different disease states de -pending on the type of host it infects In dolphins thisbacterium was only associated with dermatitis andcellulitis and caused multifocal subcutaneous ab -scesses in 3 I geoffrensis (Pier amp Madin 1976 Pier etal 1978 Bonar amp Wagner 2003) In fish infectionwith S iniae generally results in meningitis andpanophthalmitis and consequently high levels ofmorbidity and mortality (Bromage amp Owens 2002)Humans tend to develop a septic cellulitis with oc -casional endocarditis meningitis arthritis sepsispneumonia osteomyelitis and toxic shock (Wein-stein et al 1997 Facklam et al 2005) Sun et al(2007) described a case report of an invasive S iniaeinfection in a 51 yr old woman with diabetes mellitusand hepatitis C-related liver cirrhosis in Taiwan in2007 In dolphins few cases were associated with Siniae infection (Bonar amp Wagner 2003) However Siniae has not received much attention or been re -ported previously in mainland China While thereare vaccines available for fish and antibiotics that cansuccessfully treat this bacterium the risk of transmis-sion is of significant concern for individuals handlingfish or marine mammal carcasses since an untreatedinfection could lead to permanent tissue damage ordeath Moreover there are no effective bacterins foruse in dolphins Here we report a systematic clinicalmicrobiological and pathological investigation of Siniae infection in a deceased 8 yr old male bottlenosedolphin Tursiops truncatus This Tursiops case showsthe first report of S iniae being related to a dissemi-nated suppurative inflammatory process Our find-ings highlight the need for clinicians to be vigilant forcases of S iniae infection and for microbiology labo-ratory staff to pay particular attention to specimensfrom patients with a history of fish handling

MATERIALS AND METHODS

Clinical history and therapeutic measures

A 100 kg 225 cm 8 yr old male Tursiops truncatusoriginally from Japan died on 2 April 2015 followingan extended illness and was necropsied within 24 hThe animal had been housed at Beijing aquariumsince 15 September 2010 when it was 3 yr old Theclinical history indicated occasional traumatic skinlesions as a result of fighting with another male dol-phin housed in the same pool from April to October

2013 The dolphin recovered from a caudal vertebralmuscle abscess with secondary vestibular sac pro-lapse and spondylarthritis after the administration oflong-term antibiotic treatment from December 2013to May 2014 using mainly clindamycine and cefdi -nir No bacteriology was undertaken on initial clinical presentation Following this treatment thedolphin had been in good health However ap -proximately 11 mo later slow weak swimming wasnoted on 5 March 2015 Increased white blood cellcount (15 times 103 mmminus3) and a marked left shift of neu-trophils in the blood indicated infection Alkalinephosphatase (60 IU lminus1) was markedly decreased andalanine aminotransferase (85 IU lminus1) was moderatelyincreased Creatine kinase values creatinine biliru-bin aspartate aminotransferase other enzymes andelectrolytes were within reference ranges (Zappulliet al 2005)The clinical manifestations that followedand the therapeutic measures taken are presented inTable 1

Necropsy and histopathology

A full necropsy was performed with tissue samplestaken from all organs and fixed in 10 formalin forhistologic evaluation using hematoxylin and eosin(HampE) staining Aerobic bacterial cultures were ob -tained from the lung hilar lymph nodes liver pan-creas and digestive tract A gram-positive coccuswas isolated from the hilar lymph nodes and pan-creas on blood agar and trypticase soy agar incu-bated at 25degC for 24 h The genus of the bacteriumwas identified using a conventional rapid identifica-tion system (API 20 Strep) and the species was veri-fied using 16S rRNA gene partial sequencing (Zhanget al 2000 El Aamri et al 2010) with BLAST analysis

RESULTS

Gross and histopathological assessment

Gross necropsy findings included mild to moder-ate enlargement and hyperemia of the hilar lymphnodes A pale yellow thick exudate composed princi-pally of neutrophils admixed with cellular debrisedema fluid and fibrin was present in the subarach-noid space on the dorsal surface of the brain and alsoin sulci The gyri were flattened indicating brainswelling and secondary compression The liver andpancreas had moderate to severe swelling On thesurface of the lungs were multifocal yellow and white

196

Song et al Streptococcus iniae-induced septicemia in a dolphin

nodules ranging in the size from 1 to 15 cm in dia -meter The lung parenchyma was variably inflatedconsolidated and mottled dark red to mahoganyThe lung cut surface and major bronchus containedan exudate The bronchial lymph node was moder-ately enlarged and diffusely hyperemic Numerouspetechiae were present on the right ventricular myo -cardium In addition there were multiple congestiongastric erosions and diffuse hyperemia of the intes-tinal mucosa

Histopathology revealed multisystemic suppura-tive inflammation characterized by a predominantneutrophilic infiltration with acute bacterial sepsisfungal infection and chronic inflammation The pre-dominant lesions in the brain included suppurativeencephalitis (Fig 1) and hemorrhagic meningitisalong with abscess formation characterized by theinfiltration of inflammatory cells multifocal infiltra-tion of macrophages and necrotic cellular debris andmononuclear cell perivascular cuffing Neuronal de -generation microgliosis satellitosis neuronophagiaand occasional presence of multinucleate syncytiawere observed with these changes mostly affectingthe cortical and subcortical gray matter The lungsshowed acute suppurative bronchopneumonia(Fig 2AB) with hyperemia abundant neutrophilsmacrophages and cellular debris within the lumen ofbronchi bronchioles and alveoli Bile duct prolifera-

tion was present in the liver accompanied by focalinflammation (Fig 2CD) while the pancreas exhib-ited chronic inflammation histologically (Fig 3) Theintestinal tract showed suppurative gastritis (mainlythe second gastric chamber) and enteritis (Fig 4A)Numerous multifocal to coalescing basophilic fungalorganisms were pigmented on HE-stained sections inthe digestive tract Tight clusters of fungal hyphaewere diffusely arranged with a radial or coralloidappearance (Fig 4C) Large amorphous aggregateswere characterized by necrotic cell debris containinghyphae with regularly septate parallel basophilicwalls and dichotomous acute angle branching(Fig 4D) surrounded by Langhans multinucleatedgiant cells and variable numbers of neutrophils lym-phocytes epithelioid macrophages and fibroblasts(Fig 4BminusD) The cause of death for this dolphin wasmultifactorial associated with bacterial and fungalpathogens

Phenotypic characterization and PCR results

A catalase and oxidase negative β-hemolytic Gram-positive coccus was isolated from the hilar lymphnodes and pancreas The API 20 Strep test (Zhou etal 2008 El Aamri et al 2010) result was unable toidentify the Streptococcus species (Table 2) Molecu-

197

Duration of treatment Clinical manifestations Therapeutic measures

5minus12 March Anepithymia slow weak swimming Clindamycin 10 mg kgminus1 po bidleukocytosis neutrophilia with left shift

13minus19 March Depression anepithymia occasional sinking Levofloxacin hydrochloride 5 mg kgminus1 po bidslight high temperature leukocytosis Metronidazole 7 mg kgminus1 po bidneutrophilia with left shift

20minus23 March Depression occasional anepithymia Ceftriaxone sodium 20 mg kgminus1 2 g each time high temperature leukocytosis neutrophilia im sidwith left shift Sucralfate 1 g each time po tid

24minus27 March Vomiting high temperature slow and weak Cefdinir capsules 6 mg kgminus1 po bidswimming sometimes just floating Ivermectin tablets 02 mg kgminus1 po sid

28 March minus 1 April Vomiting slow and weak swimming Ceftriaxone sodium 20 mg kgminus1 2 g each time sometimes erratic behavior increased im sidfloating time left lateral tilt observed Imipenem and cilastatin sodium 10 mg kgminus1 iv

bidPraziquantel 3 mg kgminus1 po sid

2 April Respiratory frequency increased abnormal All rescue measures proved ineffectual and breathing patterns loss of orientation death occurreddisplayed swimming disorders compatible with neurological syndromes died at 0246 h

Table 1 Clinical manifestations observed and the therapeutic measures instituted during the illness of a captive bottlenosedolphin Tursiops truncatus in 2015 po by mouth bid twice dminus1 sid once dminus1 tid 3 times dminus1 im intramuscular in-

jection iv intravenous injection

Dis Aquat Org 122 195ndash203 2017

lar identification based on sequencing of the 16S rRNAgene was performed to confirm the bio chemicalidentification A polymerase chain reaction-ampli-fied product of the 16S rRNA gene was sequencedand the sequence obtained was subjected to BLASTanalysis against the National Center for Biotechno -logy Information (NCBI) database The 1011 bp fragment showed 100 similarity with sequenceAF335572 (type strain NR 025148 El Aamri et al2010) identifying the isolated strain as S iniae

DISCUSSION

Streptococcosis is classically associated with acuteand chronic mortality in many aquaculture specieswith Streptococcus iniae causing meningoencephali-tis systemic septicemia and subsequent mortality infish At least 27 species of fish have been documen -ted to be infected by S iniae worldwide (Agnew ampBarnes 2007) with the exception of mainland ChinaThe risk of zoonotic transmission of S iniae from

198

Fig 1 Histopathological analysis of the brain of a diseased Tursiops truncatus (HampE stain) (A) Suppurative encephalitis In thecortex and sub-cortex areas multifoci are evident with abscesses that vary in size (black arrows) Perivascular inflammatorycell infiltration is also indicated (red arrowheads) (B) Hemorrhagic meningitis Hemorrhaging and edema are evident underthe meninges (black arrowheads) along with diffuse inflammatory cells (C) Perivascular cuffing Monocytes (red arrow-heads) in the perivascular space have been recruited from the vasculature and form several layers of cuffing Higher magnifi-cation of a small abscess (black asterisk) that contains neutrophils other mononuclear inflammatory cells and cellular debris(D) Satellitosis and neuronophagia This necrotic neuron cell body is surrounded by macrophages or microglia that will phago-

cytose the cell debris (blue arrowheads) Scale bars = (AB) 100 microm (CD) 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

cetaceans to humans is unknown however it isknown to be a zoonotic pathogen leading to diseasein those directly handling infected fish For examplefresh whole fish are often purchased from retail wetmarkets followed by scaling cutting and removal offins and internal organs with bare hands Many pre-disposing or environmental factors have been identi-fied that enhance the virulence associated with thisinfection (Lau et al 2007)

In the case of the deceased dolphin described inthis report the cause of death was complex with a

number of potential contributing factors The grossnecropsy histopathology results and the microbio -logy evaluation indicated the cause of death to beassociated with a systemic S iniae infection Theinvolvement of the fungi in the intestinal tract sug-gested further immune compromise with this individ-ual prior to death

The clinical and medication history of this dolphincannot be ruled out as predisposing cofactors forsepti cemia and antibiotic resistance The long-termuse of antibiotics may have suppressed the immunity

199

Fig 2 Histopathological analysis of the lung and liver of a diseased Tursiops truncatus (HampE stain) (A) Chronic suppurativepneumonia The trachea (black dotted line) and alveoli (green dotted line) contain numerous neutrophils and macrophagesAlveoli are edematous and contain similar inflammatory cells (B) Higher magnification of pulmonary alveoli Alveolar septaare widened by inflammation Some of the neutrophils along with edema fluid and fibrinous exudate appear to be migratingfrom the capillaries of the lamina propria of the bronchiole into the alveoli (C) Chronic active fibrosing cholangiohepatitis withbiliary hyperplasia Proliferation of biliary ducts (black arrows) within the portal areas and periportal regions (D) Higher mag-nification of biliary hyperplasia The hypertrophied and hyperplastic bile ducts are surrounded by apparently randomlyarranged bands of fibrous connective tissue and focal inflammatory cells (black asterisk) Scale bars = (AC) 100 microm (BD) 20 microm

Dis Aquat Org 122 195ndash203 2017

of this animal as well as increasing the risk of bac -terial drug resistance during therapy and alteredmicroflora with increased susceptibility to infectionStreptococci are normally highly sensitive to peni-cillin which was not selected for therapy in this caseThe infection spread hematogenously and crossedthe bloodminusbrain barrier The suppurative encephali-tis observed suggested infection with a more virulentstrain that was able to evade intracellular killing andthis warrants further investigation Pseudohyphaeand multinuclear giant cells were observed in thedigestive system indicative of a long-term pseudo-

hyphal and yeast infection however antifungaldrugs were not used during the course of treatmentand the species of fungus was not determined Yeastinfection with pseudohyphae cannot be conclusivelyexcluded as a contributor in the disease course and inthe ultimate demise of the animal

Mycotic diseases in delphinoids have been seen un-der a number of circumstances and pathogenic fungalspecies have been isolated from their exhalation (Bucket al 2006) Some of these species are environmentalcontaminants whereas others such as Candida albicansand other Candida spp are part of the normal micro -

200

Fig 3 Histopathological analysis of the pancreas of a diseased Tursiops truncatus (HampE stain) (AB) Chronic active pancreati-tis with varying degrees of interlobular and dissecting intralobar fibrosis with parenchymal atrophy Note the severe widenedinterstitial fibrosis and edema of the pancreas (double-headed arrow) Scale bars = 100 microm (CD) Higher magnification ofchronic pancreatitis The remaining exocrine pancreatic cells appear separated into small lobules (black arrows) by abundantfibrous connective tissue Note the infiltration of plentiful neutrophils (black asterisk) around the residual pancreatic acini and

the necrotic cell debris (red asterisk) Scale bars = 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Dis Aquat Org 122 195ndash203 2017

Prevention Microbiology Laboratory the bacteriumwas first recognized to cause human infection in 1991in North America (Centers for Disease Control 1996)

S iniae tends to cause different disease states de -pending on the type of host it infects In dolphins thisbacterium was only associated with dermatitis andcellulitis and caused multifocal subcutaneous ab -scesses in 3 I geoffrensis (Pier amp Madin 1976 Pier etal 1978 Bonar amp Wagner 2003) In fish infectionwith S iniae generally results in meningitis andpanophthalmitis and consequently high levels ofmorbidity and mortality (Bromage amp Owens 2002)Humans tend to develop a septic cellulitis with oc -casional endocarditis meningitis arthritis sepsispneumonia osteomyelitis and toxic shock (Wein-stein et al 1997 Facklam et al 2005) Sun et al(2007) described a case report of an invasive S iniaeinfection in a 51 yr old woman with diabetes mellitusand hepatitis C-related liver cirrhosis in Taiwan in2007 In dolphins few cases were associated with Siniae infection (Bonar amp Wagner 2003) However Siniae has not received much attention or been re -ported previously in mainland China While thereare vaccines available for fish and antibiotics that cansuccessfully treat this bacterium the risk of transmis-sion is of significant concern for individuals handlingfish or marine mammal carcasses since an untreatedinfection could lead to permanent tissue damage ordeath Moreover there are no effective bacterins foruse in dolphins Here we report a systematic clinicalmicrobiological and pathological investigation of Siniae infection in a deceased 8 yr old male bottlenosedolphin Tursiops truncatus This Tursiops case showsthe first report of S iniae being related to a dissemi-nated suppurative inflammatory process Our find-ings highlight the need for clinicians to be vigilant forcases of S iniae infection and for microbiology labo-ratory staff to pay particular attention to specimensfrom patients with a history of fish handling

MATERIALS AND METHODS

Clinical history and therapeutic measures

A 100 kg 225 cm 8 yr old male Tursiops truncatusoriginally from Japan died on 2 April 2015 followingan extended illness and was necropsied within 24 hThe animal had been housed at Beijing aquariumsince 15 September 2010 when it was 3 yr old Theclinical history indicated occasional traumatic skinlesions as a result of fighting with another male dol-phin housed in the same pool from April to October

2013 The dolphin recovered from a caudal vertebralmuscle abscess with secondary vestibular sac pro-lapse and spondylarthritis after the administration oflong-term antibiotic treatment from December 2013to May 2014 using mainly clindamycine and cefdi -nir No bacteriology was undertaken on initial clinical presentation Following this treatment thedolphin had been in good health However ap -proximately 11 mo later slow weak swimming wasnoted on 5 March 2015 Increased white blood cellcount (15 times 103 mmminus3) and a marked left shift of neu-trophils in the blood indicated infection Alkalinephosphatase (60 IU lminus1) was markedly decreased andalanine aminotransferase (85 IU lminus1) was moderatelyincreased Creatine kinase values creatinine biliru-bin aspartate aminotransferase other enzymes andelectrolytes were within reference ranges (Zappulliet al 2005)The clinical manifestations that followedand the therapeutic measures taken are presented inTable 1

Necropsy and histopathology

A full necropsy was performed with tissue samplestaken from all organs and fixed in 10 formalin forhistologic evaluation using hematoxylin and eosin(HampE) staining Aerobic bacterial cultures were ob -tained from the lung hilar lymph nodes liver pan-creas and digestive tract A gram-positive coccuswas isolated from the hilar lymph nodes and pan-creas on blood agar and trypticase soy agar incu-bated at 25degC for 24 h The genus of the bacteriumwas identified using a conventional rapid identifica-tion system (API 20 Strep) and the species was veri-fied using 16S rRNA gene partial sequencing (Zhanget al 2000 El Aamri et al 2010) with BLAST analysis

RESULTS

Gross and histopathological assessment

Gross necropsy findings included mild to moder-ate enlargement and hyperemia of the hilar lymphnodes A pale yellow thick exudate composed princi-pally of neutrophils admixed with cellular debrisedema fluid and fibrin was present in the subarach-noid space on the dorsal surface of the brain and alsoin sulci The gyri were flattened indicating brainswelling and secondary compression The liver andpancreas had moderate to severe swelling On thesurface of the lungs were multifocal yellow and white

196

Song et al Streptococcus iniae-induced septicemia in a dolphin

nodules ranging in the size from 1 to 15 cm in dia -meter The lung parenchyma was variably inflatedconsolidated and mottled dark red to mahoganyThe lung cut surface and major bronchus containedan exudate The bronchial lymph node was moder-ately enlarged and diffusely hyperemic Numerouspetechiae were present on the right ventricular myo -cardium In addition there were multiple congestiongastric erosions and diffuse hyperemia of the intes-tinal mucosa

Histopathology revealed multisystemic suppura-tive inflammation characterized by a predominantneutrophilic infiltration with acute bacterial sepsisfungal infection and chronic inflammation The pre-dominant lesions in the brain included suppurativeencephalitis (Fig 1) and hemorrhagic meningitisalong with abscess formation characterized by theinfiltration of inflammatory cells multifocal infiltra-tion of macrophages and necrotic cellular debris andmononuclear cell perivascular cuffing Neuronal de -generation microgliosis satellitosis neuronophagiaand occasional presence of multinucleate syncytiawere observed with these changes mostly affectingthe cortical and subcortical gray matter The lungsshowed acute suppurative bronchopneumonia(Fig 2AB) with hyperemia abundant neutrophilsmacrophages and cellular debris within the lumen ofbronchi bronchioles and alveoli Bile duct prolifera-

tion was present in the liver accompanied by focalinflammation (Fig 2CD) while the pancreas exhib-ited chronic inflammation histologically (Fig 3) Theintestinal tract showed suppurative gastritis (mainlythe second gastric chamber) and enteritis (Fig 4A)Numerous multifocal to coalescing basophilic fungalorganisms were pigmented on HE-stained sections inthe digestive tract Tight clusters of fungal hyphaewere diffusely arranged with a radial or coralloidappearance (Fig 4C) Large amorphous aggregateswere characterized by necrotic cell debris containinghyphae with regularly septate parallel basophilicwalls and dichotomous acute angle branching(Fig 4D) surrounded by Langhans multinucleatedgiant cells and variable numbers of neutrophils lym-phocytes epithelioid macrophages and fibroblasts(Fig 4BminusD) The cause of death for this dolphin wasmultifactorial associated with bacterial and fungalpathogens

Phenotypic characterization and PCR results

A catalase and oxidase negative β-hemolytic Gram-positive coccus was isolated from the hilar lymphnodes and pancreas The API 20 Strep test (Zhou etal 2008 El Aamri et al 2010) result was unable toidentify the Streptococcus species (Table 2) Molecu-

197

Duration of treatment Clinical manifestations Therapeutic measures

5minus12 March Anepithymia slow weak swimming Clindamycin 10 mg kgminus1 po bidleukocytosis neutrophilia with left shift

13minus19 March Depression anepithymia occasional sinking Levofloxacin hydrochloride 5 mg kgminus1 po bidslight high temperature leukocytosis Metronidazole 7 mg kgminus1 po bidneutrophilia with left shift

20minus23 March Depression occasional anepithymia Ceftriaxone sodium 20 mg kgminus1 2 g each time high temperature leukocytosis neutrophilia im sidwith left shift Sucralfate 1 g each time po tid

24minus27 March Vomiting high temperature slow and weak Cefdinir capsules 6 mg kgminus1 po bidswimming sometimes just floating Ivermectin tablets 02 mg kgminus1 po sid

28 March minus 1 April Vomiting slow and weak swimming Ceftriaxone sodium 20 mg kgminus1 2 g each time sometimes erratic behavior increased im sidfloating time left lateral tilt observed Imipenem and cilastatin sodium 10 mg kgminus1 iv

bidPraziquantel 3 mg kgminus1 po sid

2 April Respiratory frequency increased abnormal All rescue measures proved ineffectual and breathing patterns loss of orientation death occurreddisplayed swimming disorders compatible with neurological syndromes died at 0246 h

Table 1 Clinical manifestations observed and the therapeutic measures instituted during the illness of a captive bottlenosedolphin Tursiops truncatus in 2015 po by mouth bid twice dminus1 sid once dminus1 tid 3 times dminus1 im intramuscular in-

jection iv intravenous injection

Dis Aquat Org 122 195ndash203 2017

lar identification based on sequencing of the 16S rRNAgene was performed to confirm the bio chemicalidentification A polymerase chain reaction-ampli-fied product of the 16S rRNA gene was sequencedand the sequence obtained was subjected to BLASTanalysis against the National Center for Biotechno -logy Information (NCBI) database The 1011 bp fragment showed 100 similarity with sequenceAF335572 (type strain NR 025148 El Aamri et al2010) identifying the isolated strain as S iniae

DISCUSSION

Streptococcosis is classically associated with acuteand chronic mortality in many aquaculture specieswith Streptococcus iniae causing meningoencephali-tis systemic septicemia and subsequent mortality infish At least 27 species of fish have been documen -ted to be infected by S iniae worldwide (Agnew ampBarnes 2007) with the exception of mainland ChinaThe risk of zoonotic transmission of S iniae from

198

Fig 1 Histopathological analysis of the brain of a diseased Tursiops truncatus (HampE stain) (A) Suppurative encephalitis In thecortex and sub-cortex areas multifoci are evident with abscesses that vary in size (black arrows) Perivascular inflammatorycell infiltration is also indicated (red arrowheads) (B) Hemorrhagic meningitis Hemorrhaging and edema are evident underthe meninges (black arrowheads) along with diffuse inflammatory cells (C) Perivascular cuffing Monocytes (red arrow-heads) in the perivascular space have been recruited from the vasculature and form several layers of cuffing Higher magnifi-cation of a small abscess (black asterisk) that contains neutrophils other mononuclear inflammatory cells and cellular debris(D) Satellitosis and neuronophagia This necrotic neuron cell body is surrounded by macrophages or microglia that will phago-

cytose the cell debris (blue arrowheads) Scale bars = (AB) 100 microm (CD) 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

cetaceans to humans is unknown however it isknown to be a zoonotic pathogen leading to diseasein those directly handling infected fish For examplefresh whole fish are often purchased from retail wetmarkets followed by scaling cutting and removal offins and internal organs with bare hands Many pre-disposing or environmental factors have been identi-fied that enhance the virulence associated with thisinfection (Lau et al 2007)

In the case of the deceased dolphin described inthis report the cause of death was complex with a

number of potential contributing factors The grossnecropsy histopathology results and the microbio -logy evaluation indicated the cause of death to beassociated with a systemic S iniae infection Theinvolvement of the fungi in the intestinal tract sug-gested further immune compromise with this individ-ual prior to death

The clinical and medication history of this dolphincannot be ruled out as predisposing cofactors forsepti cemia and antibiotic resistance The long-termuse of antibiotics may have suppressed the immunity

199

Fig 2 Histopathological analysis of the lung and liver of a diseased Tursiops truncatus (HampE stain) (A) Chronic suppurativepneumonia The trachea (black dotted line) and alveoli (green dotted line) contain numerous neutrophils and macrophagesAlveoli are edematous and contain similar inflammatory cells (B) Higher magnification of pulmonary alveoli Alveolar septaare widened by inflammation Some of the neutrophils along with edema fluid and fibrinous exudate appear to be migratingfrom the capillaries of the lamina propria of the bronchiole into the alveoli (C) Chronic active fibrosing cholangiohepatitis withbiliary hyperplasia Proliferation of biliary ducts (black arrows) within the portal areas and periportal regions (D) Higher mag-nification of biliary hyperplasia The hypertrophied and hyperplastic bile ducts are surrounded by apparently randomlyarranged bands of fibrous connective tissue and focal inflammatory cells (black asterisk) Scale bars = (AC) 100 microm (BD) 20 microm

Dis Aquat Org 122 195ndash203 2017

of this animal as well as increasing the risk of bac -terial drug resistance during therapy and alteredmicroflora with increased susceptibility to infectionStreptococci are normally highly sensitive to peni-cillin which was not selected for therapy in this caseThe infection spread hematogenously and crossedthe bloodminusbrain barrier The suppurative encephali-tis observed suggested infection with a more virulentstrain that was able to evade intracellular killing andthis warrants further investigation Pseudohyphaeand multinuclear giant cells were observed in thedigestive system indicative of a long-term pseudo-

hyphal and yeast infection however antifungaldrugs were not used during the course of treatmentand the species of fungus was not determined Yeastinfection with pseudohyphae cannot be conclusivelyexcluded as a contributor in the disease course and inthe ultimate demise of the animal

Mycotic diseases in delphinoids have been seen un-der a number of circumstances and pathogenic fungalspecies have been isolated from their exhalation (Bucket al 2006) Some of these species are environmentalcontaminants whereas others such as Candida albicansand other Candida spp are part of the normal micro -

200

Fig 3 Histopathological analysis of the pancreas of a diseased Tursiops truncatus (HampE stain) (AB) Chronic active pancreati-tis with varying degrees of interlobular and dissecting intralobar fibrosis with parenchymal atrophy Note the severe widenedinterstitial fibrosis and edema of the pancreas (double-headed arrow) Scale bars = 100 microm (CD) Higher magnification ofchronic pancreatitis The remaining exocrine pancreatic cells appear separated into small lobules (black arrows) by abundantfibrous connective tissue Note the infiltration of plentiful neutrophils (black asterisk) around the residual pancreatic acini and

the necrotic cell debris (red asterisk) Scale bars = 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Song et al Streptococcus iniae-induced septicemia in a dolphin

nodules ranging in the size from 1 to 15 cm in dia -meter The lung parenchyma was variably inflatedconsolidated and mottled dark red to mahoganyThe lung cut surface and major bronchus containedan exudate The bronchial lymph node was moder-ately enlarged and diffusely hyperemic Numerouspetechiae were present on the right ventricular myo -cardium In addition there were multiple congestiongastric erosions and diffuse hyperemia of the intes-tinal mucosa

Histopathology revealed multisystemic suppura-tive inflammation characterized by a predominantneutrophilic infiltration with acute bacterial sepsisfungal infection and chronic inflammation The pre-dominant lesions in the brain included suppurativeencephalitis (Fig 1) and hemorrhagic meningitisalong with abscess formation characterized by theinfiltration of inflammatory cells multifocal infiltra-tion of macrophages and necrotic cellular debris andmononuclear cell perivascular cuffing Neuronal de -generation microgliosis satellitosis neuronophagiaand occasional presence of multinucleate syncytiawere observed with these changes mostly affectingthe cortical and subcortical gray matter The lungsshowed acute suppurative bronchopneumonia(Fig 2AB) with hyperemia abundant neutrophilsmacrophages and cellular debris within the lumen ofbronchi bronchioles and alveoli Bile duct prolifera-

tion was present in the liver accompanied by focalinflammation (Fig 2CD) while the pancreas exhib-ited chronic inflammation histologically (Fig 3) Theintestinal tract showed suppurative gastritis (mainlythe second gastric chamber) and enteritis (Fig 4A)Numerous multifocal to coalescing basophilic fungalorganisms were pigmented on HE-stained sections inthe digestive tract Tight clusters of fungal hyphaewere diffusely arranged with a radial or coralloidappearance (Fig 4C) Large amorphous aggregateswere characterized by necrotic cell debris containinghyphae with regularly septate parallel basophilicwalls and dichotomous acute angle branching(Fig 4D) surrounded by Langhans multinucleatedgiant cells and variable numbers of neutrophils lym-phocytes epithelioid macrophages and fibroblasts(Fig 4BminusD) The cause of death for this dolphin wasmultifactorial associated with bacterial and fungalpathogens

Phenotypic characterization and PCR results

A catalase and oxidase negative β-hemolytic Gram-positive coccus was isolated from the hilar lymphnodes and pancreas The API 20 Strep test (Zhou etal 2008 El Aamri et al 2010) result was unable toidentify the Streptococcus species (Table 2) Molecu-

197

Duration of treatment Clinical manifestations Therapeutic measures

5minus12 March Anepithymia slow weak swimming Clindamycin 10 mg kgminus1 po bidleukocytosis neutrophilia with left shift

13minus19 March Depression anepithymia occasional sinking Levofloxacin hydrochloride 5 mg kgminus1 po bidslight high temperature leukocytosis Metronidazole 7 mg kgminus1 po bidneutrophilia with left shift

20minus23 March Depression occasional anepithymia Ceftriaxone sodium 20 mg kgminus1 2 g each time high temperature leukocytosis neutrophilia im sidwith left shift Sucralfate 1 g each time po tid

24minus27 March Vomiting high temperature slow and weak Cefdinir capsules 6 mg kgminus1 po bidswimming sometimes just floating Ivermectin tablets 02 mg kgminus1 po sid

28 March minus 1 April Vomiting slow and weak swimming Ceftriaxone sodium 20 mg kgminus1 2 g each time sometimes erratic behavior increased im sidfloating time left lateral tilt observed Imipenem and cilastatin sodium 10 mg kgminus1 iv

bidPraziquantel 3 mg kgminus1 po sid

2 April Respiratory frequency increased abnormal All rescue measures proved ineffectual and breathing patterns loss of orientation death occurreddisplayed swimming disorders compatible with neurological syndromes died at 0246 h

Table 1 Clinical manifestations observed and the therapeutic measures instituted during the illness of a captive bottlenosedolphin Tursiops truncatus in 2015 po by mouth bid twice dminus1 sid once dminus1 tid 3 times dminus1 im intramuscular in-

jection iv intravenous injection

Dis Aquat Org 122 195ndash203 2017

lar identification based on sequencing of the 16S rRNAgene was performed to confirm the bio chemicalidentification A polymerase chain reaction-ampli-fied product of the 16S rRNA gene was sequencedand the sequence obtained was subjected to BLASTanalysis against the National Center for Biotechno -logy Information (NCBI) database The 1011 bp fragment showed 100 similarity with sequenceAF335572 (type strain NR 025148 El Aamri et al2010) identifying the isolated strain as S iniae

DISCUSSION

Streptococcosis is classically associated with acuteand chronic mortality in many aquaculture specieswith Streptococcus iniae causing meningoencephali-tis systemic septicemia and subsequent mortality infish At least 27 species of fish have been documen -ted to be infected by S iniae worldwide (Agnew ampBarnes 2007) with the exception of mainland ChinaThe risk of zoonotic transmission of S iniae from

198

Fig 1 Histopathological analysis of the brain of a diseased Tursiops truncatus (HampE stain) (A) Suppurative encephalitis In thecortex and sub-cortex areas multifoci are evident with abscesses that vary in size (black arrows) Perivascular inflammatorycell infiltration is also indicated (red arrowheads) (B) Hemorrhagic meningitis Hemorrhaging and edema are evident underthe meninges (black arrowheads) along with diffuse inflammatory cells (C) Perivascular cuffing Monocytes (red arrow-heads) in the perivascular space have been recruited from the vasculature and form several layers of cuffing Higher magnifi-cation of a small abscess (black asterisk) that contains neutrophils other mononuclear inflammatory cells and cellular debris(D) Satellitosis and neuronophagia This necrotic neuron cell body is surrounded by macrophages or microglia that will phago-

cytose the cell debris (blue arrowheads) Scale bars = (AB) 100 microm (CD) 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

cetaceans to humans is unknown however it isknown to be a zoonotic pathogen leading to diseasein those directly handling infected fish For examplefresh whole fish are often purchased from retail wetmarkets followed by scaling cutting and removal offins and internal organs with bare hands Many pre-disposing or environmental factors have been identi-fied that enhance the virulence associated with thisinfection (Lau et al 2007)

In the case of the deceased dolphin described inthis report the cause of death was complex with a

number of potential contributing factors The grossnecropsy histopathology results and the microbio -logy evaluation indicated the cause of death to beassociated with a systemic S iniae infection Theinvolvement of the fungi in the intestinal tract sug-gested further immune compromise with this individ-ual prior to death

The clinical and medication history of this dolphincannot be ruled out as predisposing cofactors forsepti cemia and antibiotic resistance The long-termuse of antibiotics may have suppressed the immunity

199

Fig 2 Histopathological analysis of the lung and liver of a diseased Tursiops truncatus (HampE stain) (A) Chronic suppurativepneumonia The trachea (black dotted line) and alveoli (green dotted line) contain numerous neutrophils and macrophagesAlveoli are edematous and contain similar inflammatory cells (B) Higher magnification of pulmonary alveoli Alveolar septaare widened by inflammation Some of the neutrophils along with edema fluid and fibrinous exudate appear to be migratingfrom the capillaries of the lamina propria of the bronchiole into the alveoli (C) Chronic active fibrosing cholangiohepatitis withbiliary hyperplasia Proliferation of biliary ducts (black arrows) within the portal areas and periportal regions (D) Higher mag-nification of biliary hyperplasia The hypertrophied and hyperplastic bile ducts are surrounded by apparently randomlyarranged bands of fibrous connective tissue and focal inflammatory cells (black asterisk) Scale bars = (AC) 100 microm (BD) 20 microm

Dis Aquat Org 122 195ndash203 2017

of this animal as well as increasing the risk of bac -terial drug resistance during therapy and alteredmicroflora with increased susceptibility to infectionStreptococci are normally highly sensitive to peni-cillin which was not selected for therapy in this caseThe infection spread hematogenously and crossedthe bloodminusbrain barrier The suppurative encephali-tis observed suggested infection with a more virulentstrain that was able to evade intracellular killing andthis warrants further investigation Pseudohyphaeand multinuclear giant cells were observed in thedigestive system indicative of a long-term pseudo-

hyphal and yeast infection however antifungaldrugs were not used during the course of treatmentand the species of fungus was not determined Yeastinfection with pseudohyphae cannot be conclusivelyexcluded as a contributor in the disease course and inthe ultimate demise of the animal

Mycotic diseases in delphinoids have been seen un-der a number of circumstances and pathogenic fungalspecies have been isolated from their exhalation (Bucket al 2006) Some of these species are environmentalcontaminants whereas others such as Candida albicansand other Candida spp are part of the normal micro -

200

Fig 3 Histopathological analysis of the pancreas of a diseased Tursiops truncatus (HampE stain) (AB) Chronic active pancreati-tis with varying degrees of interlobular and dissecting intralobar fibrosis with parenchymal atrophy Note the severe widenedinterstitial fibrosis and edema of the pancreas (double-headed arrow) Scale bars = 100 microm (CD) Higher magnification ofchronic pancreatitis The remaining exocrine pancreatic cells appear separated into small lobules (black arrows) by abundantfibrous connective tissue Note the infiltration of plentiful neutrophils (black asterisk) around the residual pancreatic acini and

the necrotic cell debris (red asterisk) Scale bars = 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Dis Aquat Org 122 195ndash203 2017

lar identification based on sequencing of the 16S rRNAgene was performed to confirm the bio chemicalidentification A polymerase chain reaction-ampli-fied product of the 16S rRNA gene was sequencedand the sequence obtained was subjected to BLASTanalysis against the National Center for Biotechno -logy Information (NCBI) database The 1011 bp fragment showed 100 similarity with sequenceAF335572 (type strain NR 025148 El Aamri et al2010) identifying the isolated strain as S iniae

DISCUSSION

Streptococcosis is classically associated with acuteand chronic mortality in many aquaculture specieswith Streptococcus iniae causing meningoencephali-tis systemic septicemia and subsequent mortality infish At least 27 species of fish have been documen -ted to be infected by S iniae worldwide (Agnew ampBarnes 2007) with the exception of mainland ChinaThe risk of zoonotic transmission of S iniae from

198

Fig 1 Histopathological analysis of the brain of a diseased Tursiops truncatus (HampE stain) (A) Suppurative encephalitis In thecortex and sub-cortex areas multifoci are evident with abscesses that vary in size (black arrows) Perivascular inflammatorycell infiltration is also indicated (red arrowheads) (B) Hemorrhagic meningitis Hemorrhaging and edema are evident underthe meninges (black arrowheads) along with diffuse inflammatory cells (C) Perivascular cuffing Monocytes (red arrow-heads) in the perivascular space have been recruited from the vasculature and form several layers of cuffing Higher magnifi-cation of a small abscess (black asterisk) that contains neutrophils other mononuclear inflammatory cells and cellular debris(D) Satellitosis and neuronophagia This necrotic neuron cell body is surrounded by macrophages or microglia that will phago-

cytose the cell debris (blue arrowheads) Scale bars = (AB) 100 microm (CD) 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

cetaceans to humans is unknown however it isknown to be a zoonotic pathogen leading to diseasein those directly handling infected fish For examplefresh whole fish are often purchased from retail wetmarkets followed by scaling cutting and removal offins and internal organs with bare hands Many pre-disposing or environmental factors have been identi-fied that enhance the virulence associated with thisinfection (Lau et al 2007)

In the case of the deceased dolphin described inthis report the cause of death was complex with a

number of potential contributing factors The grossnecropsy histopathology results and the microbio -logy evaluation indicated the cause of death to beassociated with a systemic S iniae infection Theinvolvement of the fungi in the intestinal tract sug-gested further immune compromise with this individ-ual prior to death

The clinical and medication history of this dolphincannot be ruled out as predisposing cofactors forsepti cemia and antibiotic resistance The long-termuse of antibiotics may have suppressed the immunity

199

Fig 2 Histopathological analysis of the lung and liver of a diseased Tursiops truncatus (HampE stain) (A) Chronic suppurativepneumonia The trachea (black dotted line) and alveoli (green dotted line) contain numerous neutrophils and macrophagesAlveoli are edematous and contain similar inflammatory cells (B) Higher magnification of pulmonary alveoli Alveolar septaare widened by inflammation Some of the neutrophils along with edema fluid and fibrinous exudate appear to be migratingfrom the capillaries of the lamina propria of the bronchiole into the alveoli (C) Chronic active fibrosing cholangiohepatitis withbiliary hyperplasia Proliferation of biliary ducts (black arrows) within the portal areas and periportal regions (D) Higher mag-nification of biliary hyperplasia The hypertrophied and hyperplastic bile ducts are surrounded by apparently randomlyarranged bands of fibrous connective tissue and focal inflammatory cells (black asterisk) Scale bars = (AC) 100 microm (BD) 20 microm

Dis Aquat Org 122 195ndash203 2017

of this animal as well as increasing the risk of bac -terial drug resistance during therapy and alteredmicroflora with increased susceptibility to infectionStreptococci are normally highly sensitive to peni-cillin which was not selected for therapy in this caseThe infection spread hematogenously and crossedthe bloodminusbrain barrier The suppurative encephali-tis observed suggested infection with a more virulentstrain that was able to evade intracellular killing andthis warrants further investigation Pseudohyphaeand multinuclear giant cells were observed in thedigestive system indicative of a long-term pseudo-

hyphal and yeast infection however antifungaldrugs were not used during the course of treatmentand the species of fungus was not determined Yeastinfection with pseudohyphae cannot be conclusivelyexcluded as a contributor in the disease course and inthe ultimate demise of the animal

Mycotic diseases in delphinoids have been seen un-der a number of circumstances and pathogenic fungalspecies have been isolated from their exhalation (Bucket al 2006) Some of these species are environmentalcontaminants whereas others such as Candida albicansand other Candida spp are part of the normal micro -

200

Fig 3 Histopathological analysis of the pancreas of a diseased Tursiops truncatus (HampE stain) (AB) Chronic active pancreati-tis with varying degrees of interlobular and dissecting intralobar fibrosis with parenchymal atrophy Note the severe widenedinterstitial fibrosis and edema of the pancreas (double-headed arrow) Scale bars = 100 microm (CD) Higher magnification ofchronic pancreatitis The remaining exocrine pancreatic cells appear separated into small lobules (black arrows) by abundantfibrous connective tissue Note the infiltration of plentiful neutrophils (black asterisk) around the residual pancreatic acini and

the necrotic cell debris (red asterisk) Scale bars = 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Song et al Streptococcus iniae-induced septicemia in a dolphin

cetaceans to humans is unknown however it isknown to be a zoonotic pathogen leading to diseasein those directly handling infected fish For examplefresh whole fish are often purchased from retail wetmarkets followed by scaling cutting and removal offins and internal organs with bare hands Many pre-disposing or environmental factors have been identi-fied that enhance the virulence associated with thisinfection (Lau et al 2007)

In the case of the deceased dolphin described inthis report the cause of death was complex with a

number of potential contributing factors The grossnecropsy histopathology results and the microbio -logy evaluation indicated the cause of death to beassociated with a systemic S iniae infection Theinvolvement of the fungi in the intestinal tract sug-gested further immune compromise with this individ-ual prior to death

The clinical and medication history of this dolphincannot be ruled out as predisposing cofactors forsepti cemia and antibiotic resistance The long-termuse of antibiotics may have suppressed the immunity

199

Fig 2 Histopathological analysis of the lung and liver of a diseased Tursiops truncatus (HampE stain) (A) Chronic suppurativepneumonia The trachea (black dotted line) and alveoli (green dotted line) contain numerous neutrophils and macrophagesAlveoli are edematous and contain similar inflammatory cells (B) Higher magnification of pulmonary alveoli Alveolar septaare widened by inflammation Some of the neutrophils along with edema fluid and fibrinous exudate appear to be migratingfrom the capillaries of the lamina propria of the bronchiole into the alveoli (C) Chronic active fibrosing cholangiohepatitis withbiliary hyperplasia Proliferation of biliary ducts (black arrows) within the portal areas and periportal regions (D) Higher mag-nification of biliary hyperplasia The hypertrophied and hyperplastic bile ducts are surrounded by apparently randomlyarranged bands of fibrous connective tissue and focal inflammatory cells (black asterisk) Scale bars = (AC) 100 microm (BD) 20 microm

Dis Aquat Org 122 195ndash203 2017

of this animal as well as increasing the risk of bac -terial drug resistance during therapy and alteredmicroflora with increased susceptibility to infectionStreptococci are normally highly sensitive to peni-cillin which was not selected for therapy in this caseThe infection spread hematogenously and crossedthe bloodminusbrain barrier The suppurative encephali-tis observed suggested infection with a more virulentstrain that was able to evade intracellular killing andthis warrants further investigation Pseudohyphaeand multinuclear giant cells were observed in thedigestive system indicative of a long-term pseudo-

hyphal and yeast infection however antifungaldrugs were not used during the course of treatmentand the species of fungus was not determined Yeastinfection with pseudohyphae cannot be conclusivelyexcluded as a contributor in the disease course and inthe ultimate demise of the animal

Mycotic diseases in delphinoids have been seen un-der a number of circumstances and pathogenic fungalspecies have been isolated from their exhalation (Bucket al 2006) Some of these species are environmentalcontaminants whereas others such as Candida albicansand other Candida spp are part of the normal micro -

200

Fig 3 Histopathological analysis of the pancreas of a diseased Tursiops truncatus (HampE stain) (AB) Chronic active pancreati-tis with varying degrees of interlobular and dissecting intralobar fibrosis with parenchymal atrophy Note the severe widenedinterstitial fibrosis and edema of the pancreas (double-headed arrow) Scale bars = 100 microm (CD) Higher magnification ofchronic pancreatitis The remaining exocrine pancreatic cells appear separated into small lobules (black arrows) by abundantfibrous connective tissue Note the infiltration of plentiful neutrophils (black asterisk) around the residual pancreatic acini and

the necrotic cell debris (red asterisk) Scale bars = 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Dis Aquat Org 122 195ndash203 2017

of this animal as well as increasing the risk of bac -terial drug resistance during therapy and alteredmicroflora with increased susceptibility to infectionStreptococci are normally highly sensitive to peni-cillin which was not selected for therapy in this caseThe infection spread hematogenously and crossedthe bloodminusbrain barrier The suppurative encephali-tis observed suggested infection with a more virulentstrain that was able to evade intracellular killing andthis warrants further investigation Pseudohyphaeand multinuclear giant cells were observed in thedigestive system indicative of a long-term pseudo-

hyphal and yeast infection however antifungaldrugs were not used during the course of treatmentand the species of fungus was not determined Yeastinfection with pseudohyphae cannot be conclusivelyexcluded as a contributor in the disease course and inthe ultimate demise of the animal

Mycotic diseases in delphinoids have been seen un-der a number of circumstances and pathogenic fungalspecies have been isolated from their exhalation (Bucket al 2006) Some of these species are environmentalcontaminants whereas others such as Candida albicansand other Candida spp are part of the normal micro -

200

Fig 3 Histopathological analysis of the pancreas of a diseased Tursiops truncatus (HampE stain) (AB) Chronic active pancreati-tis with varying degrees of interlobular and dissecting intralobar fibrosis with parenchymal atrophy Note the severe widenedinterstitial fibrosis and edema of the pancreas (double-headed arrow) Scale bars = 100 microm (CD) Higher magnification ofchronic pancreatitis The remaining exocrine pancreatic cells appear separated into small lobules (black arrows) by abundantfibrous connective tissue Note the infiltration of plentiful neutrophils (black asterisk) around the residual pancreatic acini and

the necrotic cell debris (red asterisk) Scale bars = 20 microm

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Song et al Streptococcus iniae-induced septicemia in a dolphin

flora of mucous membranes (Buck et al 2006 Taka-hashi et al 2010) Additionally a correlation between theoccur rence of lobomycosis and the immune status offree-ranging dolphins was reported in bottlenose dol-phins in Florida USA (Murdoch et al 2008 Durden etal 2009 Reif et al 2009) Human pathogenic yeast spe-cies were isolated from more than 70 of captive dol-phins and environmental water samples which werehigher rates than in wild dolphins (Buck 1980) Admin-istration of antibiotics and steroids among captive dol-phins might be a contributing factor with environmentaland social stress for this observation (Bagg et al 2003

Melo et al 2004) However further studies are neededto investigate correlations be tween stress and immuno-suppression host defense mechanisms and infectionrates and drug meta bolism in these animals

This dolphin was likely in a long-term weakenedstate (chronically ill) compounded by a chronic my -cotic infection and succumbed to an acute strepto-coccosis septicemia Metabolic disorders associatedwith pancreatic destruction coupled with suppressedimmunity were therefore considered confoundingfactors in the causes of death High-risk factors forS iniae infection in humans also include compro-

201

Fig 4 Histopathological analysis of the digestive tract of a diseased Tursiops truncatus (HampE stain) (A) Suppurative gastritis(the second gastric chamber) Multifocal inflammatory cells containing neutrophils other mononuclear inflammatory cellsand cellular debris in the muscular layer of the stomach (black arrows) (B) Granulomatous enteritis Mononuclear inflamma-tory cells (macrophages lymphocytes) and multinucleated giant cells (red arrows) are present in the lamina propria and submucosa (C) Pseudohyphae The epithelia of the intestine appear markedly compressed by pseudohyphae and necrotic cell de-bris resulting in their exfoliation and loss (black asterisk) (D) Higher magnification of the pseudohyphae (blue arrows) Scale

bars = (AminusC) 20 microm (D) 10 microm

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Dis Aquat Org 122 195ndash203 2017

mised immunity underlying medical conditions oldage and Asian ethnicity (Koh et al 2004 Lau et al2007 Sun et al 2007) With the number of animalsmanaged in oceanariums rehabilitation facilitiesand research facilities the contact between marinemammals and humans is increasing Furthermore ascoastal communities expand opportunities for en -counters with marine wildlife increase the potentialfor zoonotic disease transmission Marine mammalresearchers rehabilitators trainers veterinarians vo -lunteers and subsistence hunters have an in creasedrisk of injury and exposure to zoonotic diseasesthrough extended occupational exposure Informa-tion on marine mammal pathology is scarce in thepublished literature the information presented herewill therefore be useful for public health profession-als pathologists veterinarians and wildlife biolo-gists involved in handling cetaceans

As the clinical manifestations of a chronic illnessor an acutely ill animal may not be outwardlyapparent appropriate personal protective precau-tions should be taken until the absence of zoonoticagents is confirmed in any suspected case Wehope that the information on animal diseaseencountered in managed and wild cetaceans willbe used by public health professionals veterinari-ans wildlife biologists and pathologists to betterunderstand diagnose and prevent marine mammalzoonoses and to improve the welfare of animals inChina and the rest of the world

Acknowledgements This work was supported by the Foun-dation of Chinese Ministry of Science and Technology (Pro-ject Nos 2015BAI09B01 and 2015BAI07B02)

LITERATURE CITED

Adams A Thompson KD (2006) Biotechnology offers revolu-tion to fish health management Trends Biotechnol 24 201minus205

Agnew W Barnes A (2007) Streptococcus iniae an aquaticpathogen of global veterinary significance and a chal-lenging candidate for reliable vaccination Vet Microbiol122 1minus15

Bagg J Sweeney MP Lewis MA Jackson MS and others(2003) High prevalence of non-albicans yeasts anddetection of anti-fungal resistance in the oral flora ofpatients with advanced cancer Palliat Med 17 477minus481

Bonar CJ Wagner RA (2003) A third report of lsquogolf ball dis-easersquo in an Amazon River dolphin (Inia geoffrensis) asso-ciated with Streptococcus iniae J Zoo Wildl Med 34 296minus301

Bossart GD Romano TA Peden-Adams MM Schaefer Aand others (2011) Clinicoimmunopathologic findings inAtlantic bottlenose dolphins Tursiops truncatus with pos-itive cetacean morbillivirus antibody titers Dis AquatOrg 97 103minus112

Bromage ES Owens L (2002) Infection of barramundi Latescalcarifer with Streptococcus iniae effects of differentroutes of exposure Dis Aquat Org 52 199minus205

Buck JD (1980) Occurrence of human-associated yeasts inthe feces and pool waters of captive bottlenosed dolphins(Tursiops truncatus) J Wildl Dis 16 141minus149

Buck JD Wells RS Rhinehart HL Hansen LJ (2006) Aerobicmicroorganisms associated with free-ranging bottlenosedolphins in coastal Gulf of Mexico and Atlantic Oceanwaters J Wildl Dis 42 536minus544

Centers for Disease Control and Prevention (CDC) (1996)Invasive infection with Streptococcus iniae mdash Ontario1995ndash1996 MMWR Morb Mortal Wkly Rep 45 650minus653

Durden WN St LJ Stolen M Mazza T Londono C (2009)Lacaziosis in bottlenose dolphins (Tursiops truncatus) inthe Indian River Lagoon Florida USA J Wildl Dis 45 849minus856

El Aamri F Padilla D Acosta F Caballero MJ and others(2010) First report of Streptococcus iniae in red porgy(Pagrus pagrus L) J Fish Dis 33 901minus905

Facklam R Elliott J Shewmaker L Reingold A (2005) Iden-tification and characterization of sporadic isolates ofStreptococcus iniae isolated from humans J Clin Micro-biol 43 933minus937

Hartman KH Yanong RP Harms CA Lewbart GA (2006)The future of training for aquatic animal health veteri-narians J Vet Med Educ 33 389minus393

Koh TH Kurup A Chen J (2004) Streptococcus iniae discitisin Singapore Emerg Infect Dis 10 1694minus1696

Lau SK Woo PC Fan RY Lee RC Teng JL Yuen KY (2007)Seasonal and tissue distribution of Laribacter hongkon-gensis a novel bacterium associated with gastroenteritisin retail freshwater fish in Hong Kong Int J Food Micro-biol 113 62minus66

Maness HT Nollens HH Jensen ED Goldstein T and others(2011) Phylogenetic analysis of marine mammal herpes -viruses Vet Microbiol 149 23minus29

Melo NR Taguchi H Jorge J Pedro RJ and others (2004)Oral Candida flora from Brazilian human immunodefi-ciency virus-infected patients in the highly active anti-retroviral therapy era Mem Inst Oswaldo Cruz 99 425minus431

Murdoch ME Reif JS Mazzoil M McCulloch SD Fair PA

202

API 20 STREP test Result API 20 STREP test Result

Hemolysis β Mannitol +Oxidase minus Sorbitol minusCatalase minus Lactose minusPyrrolidonyl arylamidase + Trehalose +Voges Proskauer minus Inulin minusHippurate hydrolysis minus Raffinose minusEsculine degradation + Amidon +α-galactosidase minus Glycogen +β-glucuronidase + Arginine +β-galactosidase minus Lysine minusAlkaline phosphatase + Ornithine minusLeucine aminopeptidase + Indol minusArginine dihydrolase + Kligler H2S production minusRibose + Urease minusArabinose minus Nitrate reduction minus

Table 2 Phenotypic characterization of Streptococcus iniae isola -ted from a diseased captive bottlenose dolphin Tursiops truncatus

Song et al Streptococcus iniae-induced septicemia in a dolphin

Bossart GD (2008) Lobomycosis in bottlenose dolphins(Tursiops truncatus) from the Indian River LagoonFlorida estimation of prevalence temporal trends andspatial distribution EcoHealth 5 289minus297

Pier GB Madin SH (1976) Streptococcus iniae sp nov abeta-hemolytic streptococcus isolated from an Amazonfreshwater dolphin Inia geoffrensis Int J Syst Bacteriol26 545minus553

Pier GB Madin SH Al-Nakeeb S (1978) Isolation and char-acterization of a second isolate of Streptococcus iniae IntJ Syst Bacteriol 28 311minus314

Reif JS Peden-Adams MM Romano TA Rice CD Fair PABossart GD (2009) Immune dysfunction in Atlantic bot-tlenose dolphins (Tursiops truncatus) with lobomycosisMed Mycol 47 125minus135

Rivera R Robles-Sikisaka R Hoffman EM Stacy BA JensenED Nollens HH Wellehan JJ (2012) Characterization ofa novel papillomavirus species (ZcPV1) from two Califor-nia sea lions (Zalophus californianus) Vet Microbiol 155 257minus266

Sun JR Yan JC Yeh CY Lee SY Lu JJ (2007) Invasive infec-tion with Streptococcus iniae in Taiwan J Med Microbiol56 1246minus1249

Takahashi H Ueda K Itano EN Yanagisawa M andothers (2010) Candida albicans and C tropicalis iso-lates from the expired breathes [sic] of captive dol -phins and their environments in an aquarium VetMed Int 2010 349364

Weinstein MR Litt M Kertesz DA Wyper P and others(1997) Invasive infections due to a fish pathogen Strep-tococcus iniae N Engl J Med 337 589minus594

Woolhouse ME Gowtage-Sequeria S (2005) Host range andemerging and reemerging pathogens Emerg Infect Dis11 1842minus1847

Zappulli V Mazzariol S Cavicchioli L Petterino C Bargel-loni L Castagnaro M (2005) Fatal necrotizing fasciitisand myositis in a captive common bottlenose dolphin(Tursiops truncatus) associated with Streptococcus agal -actiae J Vet Diagn Invest 17 617minus622

Zhang Z Schwartz S Wagner L Miller W (2000) A greedyalgorithm for aligning DNA sequences J Comput Biol 7 203minus214

Zhou SM Xie MQ Zhu XQ Ma Y Tan ZL Li AX (2008)Identification and genetic characterization of Streptococ-cus iniae strains isolated from diseased fish in ChinaJ Fish Dis 31 869minus875

203

Editorial responsibility Steven Raverty Abbotsford British Columbia Canada

Submitted March 22 2016 Accepted October 12 2016Proofs received from author(s) January 17 2017