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BRUCELLA PINNIPEDIALIS INFECTIONS IN PACIFIC HARBOR SEALS

(PHOCA VITULINA RICHARDSI) FROM WASHINGTON STATE, USA

Dyanna M. Lambourn,1,9 Michael Garner,2 Darla Ewalt,3 Stephen Raverty,4 Inga Sidor,5,8

Steven J. Jeffries,1 Jack Rhyan,6 and Joseph K. Gaydos7

1 Washington Department of Fish and Wildlife, Marine Mammal Investigations, 7801 Phillips Rd. S.W., Lakewood,Washington 98498, USA2 Northwest ZooPath, 654 West Main, Monroe, Washington 98272, USA3 National Veterinary Services Laboratories, 1920 Dayton Ave, Ames, Iowa 50010, USA4 Animal Health Centre, British Columbia Ministry of Agriculture, 1767 Angus Campbell Road, Abbotsford, BritishColumbia V3G 2M3, Canada5 Mystic Aquarium & Institute for Exploration, 55 Coogan Blvd., Mystic, Connecticut 06355, USA6 National Wildlife Research, 4101 Laporte Ave., Fort Collins, Colorado 80521, USA7 Wildlife Health Center–Orcas Island Office, UC Davis School of Veterinary Medicine, 942 Deer Harbor Rd., Eastsound,Washington 98245, USA8 Current address: New Hampshire Veterinary Diagnostic Laboratory, University of New Hampshire, 129 Main St.,Durham, New Hampshire 03824, USA9 Corresponding author (email: [email protected])

ABSTRACT: In 1994 a novel Brucella sp., later named B. pinnipedialis, was identified in strandedharbor seals (Phoca vitulina). This Brucella sp. is a potential zoonotic pathogen and is capable ofcausing disease in domestic animals. Serologic, microbiologic, and pathologic data collected fromlive captured and stranded harbor seals were used to better describe the epizootiology ofB. pinnipedialis in harbor seals from Washington State, USA, in 1994 through 2006. We found nosex predilection in harbor seal exposure or infection with B. pinnipedialis but noted a significantdifference in prevalence among age classes, with weaned pups, yearlings, and subadults havinghighest exposure and infection. The most common postmortem finding in 26 Brucella-positiveanimals (culture and/or PCR) was verminous pneumonia due to Parafilaroides spp. orOtostrongulus circumlitus. Our data are consistent with exposure to B. pinnipedialis post-weaning,and it is likely that fish or invertebrates and possibly lungworms are involved in the transmission toharbor seals. Brucella pinnipedialis was cultured or detected by PCR from seal salivary gland, lung,urinary bladder, and feces, suggesting that wildlife professionals working with live, infected sealscould be exposed to the bacterium via exposure to oral secretions, urine, or feces. Endangeredsympatric wildlife species could be exposed to B. pinnipedialis via predation on infected seals orthrough a common marine fish or invertebrate prey item involved in its transmission. More work isrequired to elucidate further potential fish or invertebrates that could be involved in thetransmission of B. pinnipedialis to harbor seals and better understand the potential risk they couldpose to humans or sympatric endangered species who also consume these prey items.

Key words: Brucella pinnipedialis, brucellosis, disease screening, harbor seal, marine, Phocavitulina richardsi, zoonosis.

INTRODUCTION

In 1994 a novel Brucella was reported inharbor seals (Phoca vitulina), harborporpoise (Phocoena phocoena), and acommon dolphin (Delphinus delphis);(Ross et al., 1994) and from an abortedbottlenose dolphin (Tursiops truncatus)fetus (Ewalt et al., 1994). Subsequentlyantibodies to and isolates of this marineBrucella were detected in multiple marinemammal species (Foster et al., 1996;Nielson et al., 2001). Marine Brucellaspp. have been classified as two species,

Brucella ceti and Brucella pinnipedialis,for isolates from cetaceans and seals,respectively (Cloeckaert et al., 2001;Foster et al., 2007; Banai and Corbel,2010) with subgroups identified withineach (Maquart et al., 2009).

Like terrestrial isolates, B. ceti andpotentially B. pinnipedialis, are zoonotic(Cloeckaert et al., 2011). In 1999 a case oflaboratory-acquired brucellosis of marineorigin occurred (Brew et al., 1999).Subsequently, three cases of naturallyacquired brucellosis of marine origin werereported from people having no known

DOI: 10.7589/2012-05-137 Journal of Wildlife Diseases, 49(4), 2013, pp. 802–815# Wildlife Disease Association 2013

802

history of exposure to marine mammals(Sohn et al., 2003; McDonald et al., 2006).Consumption of raw seafood is a potentialroute of human exposure (Whatmoreet al., 2008); however, the lack of under-standing of the epizootiology of marineBrucella infection makes it difficult toassess how people were exposed in thecases of naturally acquired infections.

Marine Brucella spp. also can causedisease in domestic animals. Experimentsusing marine Brucella of different originsand different inoculation routes causedabortion and seroconversion in cattle(Rhyan et al., 2001), infection and sero-conversion without abortion in sheep, andfulminant infection in guinea pigs (Perrettet al., 2003). Detailed information on howmarine Brucella is transmitted is neededto assess the risk that it presents todomestic animals.

We used serologic, microbiologic, andpathologic data collected from live-captured,stranded, and rehabilitated animals to eluci-date the epizootiology of B. pinnipedialis inharbor seals and hypothesized potentialroutes of infection for humans, domesticanimals, and sympatric endangered species.

MATERIALS AND METHODS

Harbor seal capture, handling, and samplecollection

Harbor seals were captured at 24 sites inWashington State, USA (Fig. 1), using a beachseine (Jeffries et al., 1993) or beach rush.Blood was drawn from the extradural intra-vertebral vein using 18 gauge 3.81–8.89 cmneedles (Bossart et al., 2001). Whole bloodwas stored chilled; serum was separated assoon as possible and frozen (220 C). Bloodcollected in heparinized containers was frozenfor culture.

Using known pupping dates from a well-studied harbor seal haul-out site (GertrudeIsland, Washington) and weight/age correla-tions for harbor seals from British Columbia(Bigg, 1969, Cottrell et al., 2002), harbor sealswere classified as pups (,2 mo), weaned pups(2–12 mo), yearlings (12–24 mo), subadults(24–48 mo), or adults (.48 mo).

Stranded harbor seal carcasses in goodpostmortem condition (carcass code 2 or 3;

Geraci and Lounsbury, 2005) were necropsied,as were seals that died or were euthanizedduring rehabilitation. Fresh placentas wererecovered at harbor seal haul-out sites. Tissuescollected for histopathology were stored in 10%neutral buffered formalin. Tissues collected forbacterial isolation and other tests were storedfrozen (230 or 240 C). Using methodsdescribed above, blood also was collected fromlive and dead stranded animals.

Serology

Serum was tested at the WashingtonDepartment of Agriculture (Olympia, Wash-ington) for antibodies to Brucella using apreviously described method (Garner et al.,1997), identified as suitable for detectingantibodies to marine Brucella (Nielsen,2002). Animals were considered suspect pos-itive if the buffered plate agglutination testantigen (BAPA) or brucellosis card test usingbuffered Brucella antigen (BBA) detected

FIGURE 1. Map of 24 harbor seal haulout loca-tions from four regions in Washington State, USA,where harbor seals (Phoca vitulina) were capturedand sampled. Regional divisions are based on knownharbor seal genetics and or contaminant levelsand signatures.

LAMBOURN ET AL.—BRUCELLA IN HARBOR SEALS 803

antibodies. They were considered positive ifthey were positive on BAPA or BBA and alsopositive on either or both the complementfixation (CF) and the Rivanol (RIV; +50 to200) precipitation tests.

To evaluate serology by location, age-class-adjusted serologic results from all sites sam-pled were lumped as a reference population.Serologic results from harbor seal haul-outsites were grouped based on harbor sealgenetics (Huber et al., 2010; Lamont et al.,1996) and contaminant levels (Ross et al.,2004). Samples were divided into four loca-tions (Fig. 1): South Puget Sound (SPS; 3sites), Hood Canal (HC; 4 sites), WashingtonOuter Coast (WOC; 13 sites), and theNorthwest Straits (NWS; 4 sites).

Bacterial culture and identification

Bacterial isolation and identification wereperformed at the National Veterinary ServicesLaboratory (Ames, Iowa) with the addition ofColumbia agar with 5% blood (Alton et al.,1988; Ewalt et al., 1983; Ewalt 1989). Tissueswere dissected, mixed with approximately2 mL of sterile phosphate buffered saline(pH 7.2), macerated, and inoculated ontotryptose agar with 5% bovine serum andantibiotics (7.5 U/mL bacitracin, 30 mg/mLcycloheximide, and 1.8 U/mL polymyxin B);tryptose agar with 5% bovine serum, antibiot-ics, and ethyl violet; Ewalt’s media; Farrell’smedia; and Columbia agar with 5% blood.Plates were incubated for 14 days in 10% CO2

at 37 C and observed for growth at 7 and14 days. Bacterial isolates were circular,convex, smooth, translucent, off-white/honeycolored. After 7 days colonies of average sizeconsistent with Brucella (1.5–2 mm diameter)were counted and recorded, and representa-tive colonies were transferred for identifica-tion (Mayfield et al., 1990).

Marine Brucella isolates were confirmedwith the following tests: growth in thepresence of basic fuchsin (1:25,000 and1:100,000), thionin (1:25,000 and 1:100,000),and thionin blue (1:500,000); growth onmedium containing penicillin (5 units/mL) orerythritol (1 mg/mL and 2 mg/mL plus 5%bovine serum); urease and catalase activity;H2S production; and CO2 dependence. Bio-typing was conducted as previously described(Alton et al., 1988). The dominant antigen wasdetermined with an agglutination test using A-and M- monospecific antisera (1:50-1:200) andR antiserum (1:25-1:100). Isolates were testedfor susceptibility to lysis by the followingphages: Tbilisi (Tb), Firenze (Fi), Weybridge(Wb), S708, Me/75, D, BK2, R, R/C, and R/O.

Polymerase chain reaction

Polymerase chain reaction (PCR) was con-ducted at Animal Health Center (AHC) andMystic Aquarium & Institute for Exploration(MAIE) using previously described PCRtechniques for Brucella (AHC; Bricker et al.,2000) and real-time PCR (qPCR) analysisusing primers, probe, and adapted protocolstargeting the gene for a 31 kDa outermembrane protein bcsp31 specific to thegenus Brucella (MAIE; Probert et al., 2004,Sidor et al., 2013).

Histopathology and immunohistochemistry

Formalin-fixed, paraffin-embedded tissueswere stained with hematoxylin and eosin andselect sections were also stained with Giemsaand with Brown and Brenn. Immunohisto-chemistry was performed on a subset ofculture-positive cases. Tissue sections weremounted on charged slides, deparaffinized,hydrated with a buffer (PBS), treated with 3%H2O2 (5 min) to quench endogenous peroxi-dase, incubated for 5 min at 37 C withnonimmune goat serum, rinsed, and incubatedfor 30 min at 37 C with a polyclonal antibody(1:10,000) prepared against B. abortus. Am-plification was conducted with biotinylated,goat origin, anti-rabbit immunoglobulin (Ig),and peroxidase-labeled streptavidin; the chro-magen was 3-amino-9 ethylcarbazole in N, N-dimethylformamide. Sections were counter-stained with Gill II hematoxylin. Nonimmu-nized rabbit Ig fraction was substituted forprimary antibody as a negative control (Garneret al., 1997).

Statistical analysis

The chi-square test of independence wasused to evaluate sex predilection for animalsthat were serologically positive for Brucellaantibodies, and to evaluate sex predilection foranimals that were culture-positive or negativefor marine Brucella. Chi-square tests also wereused to evaluate serologic and culture-positiveanimals by age class. For comparing serologicresults among the four locations sampled(SPS, HC, WOC, and NWS), direct standard-ization was used to remove the biasing effectof age structure. Using chi-square tests, thenumber of serologically positive animals ineach subpopulation was compared to thepredicted number of positive animals basedon the single standard population, which wasdeveloped by grouping the four populations(Jeckel et al., 2001).

804 JOURNAL OF WILDLIFE DISEASES, VOL. 49, NO. 4, OCTOBER 2013

RESULTS

Serology

Between 1993 and 2006, 1,314 serumsamples were collected from live healthyharbor seals captured at 24 haul-out sitesfrom four discrete locations (Fig. 1): SPS(n5826), HC (n5212), WOC (n5116),and NWS (n5160). One hundred wereantibody-positive, 101 suspect, and 1,113negative animals. When suspects werelumped with negative animals, no sexpredilection was detected with 7.4% ofmales (n550) and 7.8% of females (n550)positive (x250.0813, df53, P50.994). Thedistribution of animals with Brucellaantibodies was significantly differentamong age classes (x25229.078, df54,P,0.001) with 0.4% of pups, 17.2% ofweaned pups, 37.7% of yearlings, 21.5%

of subadults, and 2.4% of adults positive(Fig. 2).

Compared to the overall referencepopulation, antibody prevalence was dif-ferent by subregion (x257.259, df53,P50.064; Table 1). The SPS had a higher

number of positive animals than predict-ed, while HC, WOC, and NWS animalshad fewer.

Of 1,314 harbor seals sampled, 79 samplescame from 39 animals that were capturedand sampled more than once. Twenty-eight(72%) of these animals were negative forantibodies to Brucella at first capture andagain on subsequent captures. Two pups(5%; one newly weaned, SPENO904, theother nursing, SPENO1873) were negativeon initial capture but then suspect 2 wks and4 yr later, respectively. Six animals (15%)were suspect or positive initially and negativeat a later capture. One animal (SPENO902)was suspect when captured as a subadult andagain 4 yr later, and negative when recap-tured the following year as an adult. A sub-adult (SPENO933) and adult (SPENO938)were suspect on initial capture and nega-tive subsequently 3 and 1 yr later, respec-tively. Three animals were positive initiallyand then negative, including a weaned pup(SPENO987) recaptured as a subadult 2 yrlater, another weaned pup (SPENO1630)recaptured as an adult 5 yr later, and anadult (SPENO1010) estimated to be 4 yr oldat initial capture and recaptured 2 yr later.Three animals (8%) were suspect or positiveinitially and remained so at recaptureincluding an adult (SPENO277) and yearling(SPENO899) with suspect titers initially,and then again when recaptured 4 yr and2 wks later, respectively, and a yearling(SPENO854) that was positive when cap-tured and again as an adult 4 yr later.

Bacterial culture

Between 1996 and 2004 Brucella wascultured from 18 of 102 harbor seals tested(Table 2; Fig. 3). All isolates were CO2-r-equired, H2S-negative, urease-positive,catalase-positive, basic fuchsin-positive,thionin blue-positive, thionin-positive, pen-icillin-positive, erythritol-positive, domi-nant antigen A, no to partial lysis ofBrucella phage, rifampacin-negative, Wb-negative, Fi-negative, delta-negative, BK2-positive, R/O-negative, S708-negative, Me/75-negative, and R/C-negative. Based on

FIGURE 2. Brucella serology results as percent-age positive by age class for 1,314 harbor seals (Phocavitulina) live captured in Washington State, USA,between 1993 and 2007. Numbers correspond to thenumbers of positive and negative animals by age class.


biochemical tests and origin (seals), thespecies was identified as B. pinnipedialis(Foster et al., 2007). Of the seals tested,14% of males (8/57) and 24% of females(10/42) were positive. Both animals ofunknown sex were negative. No sex predi-lection was detected (x253.948, df53,P50.267). Distribution of culture-positiveanimals by age class was significantlydifferent (x2519.018, df53, no yearlingssampled, P,0.001; Fig. 3), with B. pinni-pedialis cultured from 37% of weaned pups(n546) and 25% of subadults (n54).Culture attempts were negative for allplacentas (n524), fetuses/stillborn (n53)pups (n526), and adults (n523) sampled.B. pinnipedialis was cultured from 36 of 45different tissues, organs, and parasites. Thehighest number of colonies cultured andthe highest percentage of positive sampleswere from lung, mediastinal and pulmo-nary lymph nodes, vitreous humor, andlungworms (Table 2).

Polymerase chain reaction

As tested at the AHC, four of the 336harbor seals and none of 16 harbor sealplacentas were positive for Brucella sp. byPCR on pooled tissues (typically brain,lung, liver, spleen and lymph node). AtMAIE, four of 83 seals and none of eightplacentas were positive for Brucella sp. byPCR. All four were weaned pups. Three ofthe four seals were confirmed positive bybacterial culture as well. The PCR-positivetissues included an abscess, brain, feces,kidney, liver, lung, lungworm (Otostron-gulus circumlitus), lymph node (inguinal,mediastinal, mesenteric, and suprascapu-

lar), a reproductive tract, and spleen(Table 3; Fig. 4). The PCR failed toamplify Brucella nucleic acid from urinarybladder, vitreous humor, and whole blood(Table 3).

Pathology and immunohistochemistry

Between 1996 and 2007, necropsieswere performed on 24 weaned pups andtwo subadult harbor seals (Table 4; Fig. 2)that were Brucella-positive by bacterialculture (n518) or PCR (n58). Commonfindings included verminous pneumonia(22/26) due to Parafilaroides spp., O.circumlitus, or a combination of the two,lymph node lymphoid hyperplasia (16/26),emaciation (12/26), enterocolitis (9/26),and gastritis (6/26) with gastrointestinalparasitism (acanthocephalans, unidenti-fied nematodes, and trematodes; 5/26).Of the 21 culture- or PCR-positive ani-mals that also were tested for Brucellaantibodies, only 52% (n511) were posi-tive, 33% (n57) were suspect positive,and 14% (n53) were negative (Table 4).

Immunohistochemistry exhibited positivestaining in four of seven culture-positivecases (Table 4). Tissues staining positiveincluded lung, lungworm (Parafilaroidesspp.), lung abscess, spleen, and lymphnodes.

Rehabilitation cases

Of the 205 harbor seals (177 pups and28 weaned pups) admitted into rehabilita-tion in Washington State between 1996and 2004 and tested for Brucella antibod-ies, five of 28 weaned pups (18%) werepositive. Four of five animals (WDFW97-4

TABLE 1. Actual number of antibody-positive harbor seals (Phoca vitulina) by region (Washington State,USA) compared to the predicted number of positive animals based on the reference population and correctedfor age class of animals sampled.

LocationActual no. of

antibody-positive animalsPredicted no. of

antibody-positive animals

Hood Canal 8 16.2NW Straits 6 7.1Outer Coast 5 7.6South Puget Sound 81 69.1


TABLE 2. Tissues cultured and bacteriologic results from Brucella-positive harbor seals (Phoca vitulina;n518) from Washington State, USA. Tissue or sample cultured is listed in descending order from highestnumber of colonies cultured.

Tissue or samplecultured No. positive No. tested % positive No. of colonies cultured

Lymph nodes

Mediastinal 9 14 64 1–.1,000Pulmonary 12 15 80 2–1,000Sublingual 7 14 50 1–750Supra scapular 9 15 60 1–705Inguinal 6 15 40 2–600Pancreatic 3 4 75 3–420Sub scapular 7 15 47 1–395Submandibular 5 14 36 3–306Mesenteric 10 16 63 1–147Reproductive 3 4 75 1–72Hepatic 5 7 71 1–67Iliac 2 10 20 1–38Cardiac 5 7 71 12–37Splenic 4 6 67 1–23Renal 4 9 44 1–16Gastric 3 7 43 1–14Ovarian 1 2 50 12Pooled sample 1 1 100 1–10

Samples

Lung 9 18 50 65–pure growthVitreous humor 1 1 100 .1,000Eye 3 11 27 4–400Feces 3 9 33 6–300Pancreas 2 16 13 1–85Reproductive tract 2 16 13 1–64Urinary bladder 1 11 9 60Salivary gland 4 13 31 1–58Kidney 2 18 11 1–52Spleen 3 17 18 2–11Spinal cord 1 11 9 11Brain 2 14 14 11Tonsil 4 13 31 1–10Liver 2 17 12 1–7Adrenal gland 1 15 7 6Synovial tissue 1 5 20 3Thymus 1 10 10 1Stomach 0 16 0 0Heart 0 17 0 0Blood 0 5 0 0Urine 0 4 0 0Abscess 0 1 0 0Gall bladder 0 4 0 0

Parasites

Lungworm 3 6 50 1–.1,000Flipper lice 0 3 0 0Heartworm 0 1 0 0Stomach worms 0 3 0 0


reported in Garner et al., 1997, WDFW98-7, WDFW0404-08, and WH03-742; Ta-ble 4) were euthanized and necropsied.The fifth animal (WH02-687) was releasedafter being surgically implanted with aVHF transmitter. Three of the four eutha-nized animals were positive for antibodiesto Brucella on admission. The fourth(WH03-742) was negative for Brucella(BAPA and BBA) on 27 November 2003at admission and seroconverted on 15January 2004 (BAPA, RIV +50). It re-mained positive when retested on 5 Feb-ruary (BAPA, BBA, RIV and CF) and onfour additional tests between February andApril. Feces collected on 26 February 2004were culture-positive for B. pinnipedialis,and the seal was euthanized (Table 4). Theseal that was ultimately released (WH02-687) tested negative upon admission(BAPA negative) on 18 September 2002and seroconverted on 5 December 2002(BAPA, BBA, and RIV +200). It remainedpositive on five additional tests between

FIGURE 3. Brucella culture results as percentpositive by age class for 102 harbor seals (Phocavitulina) sampled in Washington State between 1996and 2004. Numbers correspond to the numbers ofpositive and negative animals by age class.

TABLE 3. Tissues or harbor seals (Phoca vitulina) from Washington State, USA, tested by PCR and resultsfrom Brucella-positive animals (n58). Samples are listed in descending order from the highestpercent positive.

Tissue or sample tested by PCR No. positive No. tested % positive

Lymph nodes

Mediastinal 3 3 100Inguinal 2 4 50Mesenteric 1 2 50Suprascapular 1 3 30

Organ or sample

Lung 3 3 100Brain 2 2 100Flipper (cutaneous) abscess 1 1 100Spleen 3 4 80Tonsil 1 2 50Reproductive tract 1 2 50Feces 1 3 30Kidney 1 3 30Liver 1 3 30Whole blood 0 0 0Vitreous humor 0 1 0Urinary bladder 0 1 0Pooled tissue 4 8 50

Parasites

Lungworm 2 2 100


January and May with no change in RIVtiter. Attempts to culture Brucella fromnasal and oral swabs were negative, and theseal was implanted with a subcutaneousVHF transmitter (Lander et al., 2005) andreleased. The seal’s VHF radio signal wasnever detected post-release.

DISCUSSION

In Washington State, harbor seal expo-sure to Brucella pinnipedialis is wide-spread. Animals seroconvert after weaningand positive animals typically present withfew pathologic lesions. Our data suggestexposure through foraging with the high-est number of culture- and antibody-positive animals occurring after seals areweaned. The 0.4% of nursing pups withdetectable Brucella antibodies likely rep-resented passively acquired antibodies or

exposure secondary to ingestion of infect-ed prey items late in the preweaning stage.Low antibody prevalence in pups and lackof infection in placentas fail to supporttransplacental or transmammary transmis-sion.

Adult harbor seals are primarily pisciv-orous, feeding on fish that are seasonallyabundant (Olesiuk et al. 1990; Lance et al.,2012), suggesting fish as a possible routeof B. pinnipedialis exposure. Weanedpups also consume shrimp (Pandalusspp.), crabs (Cancer spp.), and otherinvertebrates (Lambourn, unpubl. data),suggesting invertebrate consumption alsomight play a role in B. pinnipedialistransmission. Serologic data (Fig. 2) areconsistent with Brucella seroconversionbeginning at weaning, with a highernumber of yearlings than weaned pupsshowing exposure due to an increasedopportunity for encountering the bacteri-um through feeding over time. Usingcompetitive enzyme-linked immunosor-bent assays (cELISA), Zarnke et al.(2006) found similar serologic results withBrucella antibody prevalence in harborseals from Alaska increasing from 11% inpups to 70% in yearlings, 68% forsubadults, and dropping to 43% in adults.Our age-class–dependent culture resultsmimic our serologic results with theexception of culture data from yearlingharbor seals due to lack of samples.

The marine origin competitive ELISAthat is more sensitive than the serologyused in this study (Meegan et al., 2010)was not available when this work com-menced. Evaluating serology from culture-and PCR-positive animals necropsied inthis study corroborates the work of Meeganet al. (2010) and suggests that the serologyused for B. abortus underestimates thenumber of infected animals by about 50%.Consequently infection in weaned pups,yearlings, and subadults is likely higherthan indicated by our serologic results(Fig. 2). Future efforts should use morespecific independent competitive immuno-assays such as the competitive and indirect

FIGURE 4. Locations of origin for harbor seals(Phoca vitulina) found positive for marine Brucellaby culture or PCR. Numbers correspond to individ-ual detail provided in Table 4.


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ELISA used to test harbor seals fromAlaska (Zarnke et al., 2006) and Hawaiianmonk seals (Monachus schauinslandi)(Nielsen et al., 2005).

Two of five Brucella-infected harborseals in rehabilitation seroconverted al-most 7 wk post-admission. Because theserologic tests used underestimate positiveanimals by 50%, this could represent atype-2 testing error. Alternately, it couldsuggest that infection occurred very closeto the time of admission and could not bedetected for several months, that it wasinfected by a fish fed while in rehabilita-tion, or that alternate modes of transmis-sion for B. pinnipedialis exist.

If harbor seals remain infected orproduce anti-Brucella antibodies for life,we would have expected to see highernumbers of culture- and antibody-positiveadults. Young harbor seals infected with B.pinnipedialis could have higher mortalityrates than those not infected, reducingantibody prevalence in older animals.Alternately, it is possible that younginfected animals cease to produce Brucellaantibodies, or clear infection and becomeserologically negative. Data from onerecaptured animal (SPENO854) that waspositive as a yearling and again as an adult4 yr later show that seals can maintainBrucella titers for at least 4 yr. However,in three other recaptured animals, Brucel-la antibodies had waned when theseals were retested 2 yr (SPENO987,SPENO1010) and 5 yr (SPENO1630)later. It is unknown if seals can clearinfection and be reinfected and what theconsequent antibody response could be.

Since harbor seals are exposed to B.pinnipedialis after they began to forage,fish or invertebrates could be involved inB. pinnipedialis transmission. There areno known sex-based dietary preferences inharbor seals. Our findings of no sexpredilection for Brucella exposure orinfection and similar findings by Nielsenet al. (2001) are consistent with this. Thenext step is to attempt to identify B.pinnipedialis in fish or invertebrate prey.

The lungworms O. circumlitus andParafilaroides spp., which are transmittedto seals via fish, could also be responsiblefor B. pinnipedialis transmission. Weidentified B. pinnipedialis in three of sixlungworms tested by culture (Table 2) andtwo of two tested by PCR (Table 3).Immunohistochemistry also exhibitedstaining in lung, lungworm (Parafilaroidesspp.) and lung abscesses associated withParafilaroides spp. Lungworm transmis-sion of Brucella has been suggested forharbor seals (Garner et al., 1997) and forharbor porpoise (Dawson et al., 2008);however, more work is required to deter-mine the role that lungworms play inBrucella transmission. Verminous pneu-monia was identified as the most commonpathologic finding in positive seals and isconsistent with findings from harbor sealsin Scotland (Foster et al., 2002); however,verminous pneumonia is a common find-ing in weaned and yearling strandedharbor seals, not all of which are infectedwith B. pinnipedialis. For example, be-tween 2000 and 2006, lungworms wereidentified in nine of 90 harbor seals thatstranded in San Juan County and werenecropsied (Gaydos, unpubl. data). Allseals were weaned pups or yearlingsexcept for one adult. Species identifiedincluded O. circumlitus (n52), Parafilar-oides gullundae (n51), Parafilaroidesgymnurus (n51), and unidentified Paraf-ilaroides (n57). One animal was coinfect-ed with O. circumlitus and P. gymnurusand another with O. circumlitus and anunidentified Parafilaroides. Of nine ani-mals infected with one or more species oflungworm, six were diagnosed with ver-minous pneumonia, but Brucella infectionwas identified by PCR or bacterial culturein only three animals, all of which hadlungworms and were diagnosed withverminous pneumonia, suggesting a 50%

coinfection rate for verminous pneumoniaand Brucella. Testing known or putativeintermediate fish hosts for Parafilaroidesspp. and O. circumlitus (Bergeron et al.,1997) for B. pinnipedialis is a good


starting point for better understanding therelationship between the fish, lungworms,and transmission of B. pinnipedialis. Thedefinitive life cycles for these metastron-gyloids are not well known, and testingshould include invertebrates, as they toocould be intermediate or paratenic hosts(Bergeron et al., 1997).

If harbor seals are exposed to Brucella viaingestion of prey, humans also could poten-tially be exposed to marine Brucella byconsuming uncooked marine fish or inver-tebrates. None of the three documentedcases of community-acquired marine Bru-cella infection in humans had prior exposureto marine mammals (Sohn et al., 2003;McDonald et al., 2006). People also could beexposed to marine Brucella via the environ-ment. We cultured B. pinnipedialis from thefeces of 33% of infected harbor seals testedand isolated marine Brucella nucleic acidfrom the feces of 33% of infected harborseals tested. Seals defecating on docks andbeaches could expose humans to B. pinni-pedialis without their being aware of theinfection source. The potential for humansto be exposed to B. pinnipedialis withoutapparent exposure to harbor seals warrantsadditional research to uncover the history ofinfected humans and to better understandthe putative role that marine fishes andinvertebrates could play in the transmissionof this potentially zoonotic bacterium to thepublic. Physicians should be made awarethat, where clinical signs warrant, marineBrucella should be considered a differentialdiagnosis, even in patients from countriesconsidered Brucella-free because of theabsence of Brucella of livestock or domesticanimal origin.

Based on our current understanding ofB. pinnipedialis epizootiology, laboratoryworkers, biologists, veterinarians, andwildlife rehabilitators who work withharbor seals or seal samples likely havethe highest risk for exposure, especiallywhen working with weaned harbor sealpups and yearlings. We cultured ordetected marine Brucella by PCR fromtonsil, salivary gland, lung, urinary blad-

der, and feces of infected animals, sug-gesting that oral secretions, bites, urine,and feces are potential sources for humanexposure from live seals.

South Puget Sound had a higher numberof animals serologically positive for Brucel-la than predicted (Table 1). This warrantsfurther investigation. Genetics and con-taminant work suggest the four regionssampled represent discrete harbor sealsubpopulations. One hypothesis for higherantibody prevalence in SPS could be thepossibility of polychlorinated biphenyl(PCB) mediated immunotoxicity increasingB. pinnipedialis infection rates. Harborseals in SPS are more heavily contaminatedwith high levels of PCBs than seals fromNWS and WOC (Ross et al., 2004). Harborseal PCB contaminant levels in SPSapproach the threshold for PCB immuno-toxicity, suggesting that young seals fromthis region could be more vulnerable todiseases, including infection with B. pinni-pedialis, because of reduced immunocom-petence (Ross et al., 2004).

It has been suggested that marineBrucella infection in endangered southernresident killer whales (Orcinus orca) couldreduce their fecundity and ultimately thepopulation’s recovery (Gaydos et al.,2004). Killer whales could carry or beexposed to B. ceti but could also beexposed to B. pinnipedialis through occa-sional predation of young harbor seals(Gaydos et al., 2005 and unpubl. data) ormore likely through consumption of fish(Ford et al., 1998), if fish are in factinvolved in B. pinnipedialis transmission.

Harbor seals are exposed to and infect-ed with B. pinnipedialis after beingweaned when they begin foraging for fishand invertebrates, supporting the hypoth-esis that marine fish or invertebrates areresponsible for transmitting this bacteriumbetween seals. This bacterium is consid-ered enzootic within the harbor sealpopulation, yet seals are currently atcarrying capacity (Jeffries et al., 2003),suggesting that B. pinnipedialis is likelynot affecting seal populations in Washing-


ton State. The presence of this pathogenin harbor seals, the potential transmissionthrough feces, urine, and oral secretions,and marine fish or invertebrates as vectorsof B. pinnipedialis indicate the importanceof understanding the epizootiology of thisbacterium and of evaluation of the risk itposes to humans, domestic animals, andsympatric endangered species.

ACKNOWLEDGMENTS

All samples were collected under permitsfrom the National Marine Fisheries Service(Scientific Research Permits 835, 782-1446,782-1702). Funding and support for this workwas provided by the Washington Departmentof Fish and Wildlife, Puget Sound AmbientMonitoring Program, National Marine Fisher-ies Service, National Marine Mammal Labo-ratory, and the John H. Prescott MarineMammal Rescue Assistance Grant Program.In-kind support was provided by the SeaDocSociety, a program of the U.C. Davis WildlifeHealth Center. We thank the agencies andindividuals that have helped with harbor sealcaptures and stranding response, includingpersonnel from Washington Department ofFish and Wildlife, National Marine MammalLaboratory, National Marine Fisheries Ser-vice; Oregon Department of Fish and Wildlife;Department of Fisheries and Oceans (Cana-da); and Cascadia Research, particularly:T. Cyra, J. Evenson, H. Huber, K. Hughes, M.Lance, B. Murphie, B. Norberg, D. Nysewan-der, and J. Pierce. We also thank the NorthwestMarine Mammal Stranding Network partici-pants including the Central Puget SoundMarine Mammal Stranding Network, the SanJuan County Marine Mammal Stranding Net-work, the Whale Museum and Cascadia Re-search, especially J. Huggins, S. Murphy S.Norman, R. Osborne, and A. Traxler. Therehabilitation staff at the Progressive AnimalWelfare Society, Wolf Hollow Wildlife Reha-bilitation Center, and Sarvey Wildlife Rehabil-itation Center helped collect samples and carefor seals in rehabilitation, especially D. De-ghetto, J. Huckabee, and S. Lockwood. S. Gaydosand I. Vilchis provided statistical consultation.A number of laboratories and researchersprovided expertise in disease screening andinterpretation of results, including the Washing-ton Department of Agriculture, the US Depart-ment of Agriculture, the National VeterinaryService Laboratory, Northwest ZooPath, PhoenixCentral Laboratory, Animal Health Centre, andMystic Aquarium.

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Submitted for publication 16 May 2012.Accepted 15 March 2013.


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