DOI: 10.7589/2012-09-226 Journal of Wildlife Diseases, 49(2), 2013, pp. 441–446# Wildlife Disease Association 2013

Coxiella burnetii in Northern Fur Seals and Steller Sea Lions of Alaska

Cody Minor,1 Gilbert J. Kersh,2 Tom Gelatt,3 Ashley V. Kondas,2 Kristy L. Pabilonia,1 Christina B. Weller,1

Bobette R. Dickerson,3 and Colleen G. Duncan1,4 1Colorado State University, College of Veterinary Medicineand Biomedical Sciences, 300 West Drake Ave., Fort Collins, Colorado 80523, USA; 2Rickettsial ZoonosesBranch, Centers for Disease Control and Prevention, Atlanta, Georgia, USA; 3National Marine MammalLaboratory, Alaska Fisheries Science Center, National Marine Fisheries Service, National Oceanic andAtmospheric Administration, Seattle, Washington, USA; 4Corresponding author (email: colleen.duncan@colostate.edu)

ABSTRACT: Coxiella burnetii, a zoonotic bac-terium, has recently been identified in severalmarine mammal species on the Pacific Coast ofNorth America, but little is known about theepidemiology, transmission, and pathogenesisin these species. We tested sera archived fromnorthern fur seals (NFS, Callorhinus ursinus;n5236) and Steller sea lions (SSL, Eumetopiasjubatus; n572) sampled in Alaska for C.burnetii antibodies, and vaginal swabs fromNFS (n540) for C. burnetii by qPCR. Theantibody prevalence in NFS samples from 2009and 2011 (69%) was significantly higher than in1994 (49%). The antibody prevalence of SSLsamples from 2007 to 2011 was 59%. All NFSvaginal swabs were negative for C. burnetii,despite an 80% antibody prevalence in thematched sera. The significant increase inantibody prevalence in NFS from 1994 to2011 suggests that the pathogen may beincreasingly common or that there is markedtemporal variation within the vulnerable NFSpopulation. The high antibody prevalence inSSL suggests that this pathogen may also besignificant in the endangered SSL population.These results confirm that C. burnetii is moreprevalent within these populations than previ-ously known. More research is needed todetermine how this bacterium may affectindividual, population, and reproductive healthof marine mammals.

Key words: Antibody prevalence, Coxiellaburnetii, marine mammals, northern fur seal,Steller sea lion.

Coxiella burnetii, causative agent ofhuman ‘‘Q fever,’’ is an obligate intracel-lular Gram-negative bacterium that isshed in reproductive tissues of infectedfemales during parturition and transmittedvia aerosolization and inhalation (Maurinand Raoult, 1999). Although infection isoften subclinical in many species, it canpresent clinically as infertility and abortionin ruminants (McQuiston et al., 2002). Thebulk of knowledge of coxiellosis pertains to

terrestrial species, but case reports andstudies of C. burnetii infection in marinemammals increasingly suggest that thepathogen may be common and clinicallysignificant in marine mammals. Coxiellaburnetii placentitis was documented in anailing, and later euthanized, pregnantPacific harbor seal (Phoca vitulina ri-chardsi) found on a beach in NorthernCalifornia in 1998 (Lapointe et al., 1999).Coxiella burnetii was also found in theplacenta of a dead pregnant Steller sea lion(SSL, Eumetopias jubatus) on the coast ofWashington in 2008 (Kersh et al., 2010). A2010 cross-sectional survey of Pacific har-bor seals, harbor porpoises (Phocoenaphocoena), and SSL collected in the PacificNorthwest suggested that C. burnetiiinfection is common among multiple spe-cies of marine mammals in this region(Kersh et al., 2012).

The significance of this reproductivepathogen in more northern regions isunclear. The northern fur seal (NFS;Callorhinus ursinus) is listed as vulnerableby the International Union for Conserva-tion of Nature (IUCN) and, in 2010, 75%

of 146 sampled placentas from Saint PaulIsland, Alaska, were positive for C. burne-tii by quantitative polymerase chain reac-tion (qPCR) (Duncan et al., 2012a). Thewestern stock of SSL, occupying much ofthe same range as the NFS, is listed asendangered (IUCN), but nothing is knownabout the prevalence of C. burnetiiinfection within this population. To bettercharacterize the epidemiology of C. bur-netii in a subset of Alaskan pinnipeds, weconducted a serosurvey using archivedsamples from NFS and SSL sampled atvarying time points between 1994 and

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2011. To examine shedding in thesespecies, we tested a subset of vaginalswabs from these animals for the IS1111and com1 genomic regions of C. burnetiiby qPCR.

Serum samples were collected by veni-puncture from live NFS and SSL duringroutine animal capture activities focusedon animal behavior and physiology. Sub-adult male NFS were sampled immedi-ately after being killed for subsistenceharvest by native Alaskans. Vaginal swabs(sterile polyester, Puritan Medical, Guil-ford, Maine, USA) were collected fromadult female NFS only during the sameoperations. All samples were frozen at280 C until thawed for analysis. Indirectimmunofluorescence assay was conductedon serum samples at the Centers for DiseaseControl and Prevention Q Fever Laboratory(Atlanta, Georgia, USA), as previouslyreported (Kersh et al., 2012). Serial dilutionsof sera from 1:128 to 1:16,384 were platedon slides coated with acetone-fixed C.burnetii strains Nine Mile Phase 1 andPhase 2. Binding was visualized using afluorescein isothiocyanate–conjugated goat,anti-dog secondary antibody (Kirkegaardand Perry Laboratories, Gaithersburg,Maryland, USA). To eliminate reporting offalse positives from cross-reactivity of anti-bodies, a positive cutoff was set at 1:128similar to previous marine mammal studies(Kersh et al., 2012).

Genomic DNA was extracted from adultNFS vaginal swabs from 2009 and 2011using the QIAamp DNA Blood Mini Kit(Qiagen, Valencia, California, USA) ac-cording to the manufacturer’s directions.Quantitative PCR was used to test theextracted DNA for C. burnetii com1 andIS1111 gene targets as described by Kershet al. (2010). Quantitative PCR was con-ducted utilizing the Applied BiosystemsTaqManH Universal PCR Master Mix Kit,No AmpEraseH UNG (Applied Biosys-tems, Foster City, California, USA) andan Applied Biosystems 7500 96-well platethermocycler. Descriptive and comparativestatistics were conducted using commer-cially available software (SPSS 20, IBM,Inc., Chicago, Illinois, USA). The frequen-cy of positive titers was compared betweenyears, sexes, and age classes using the chi-square test.

Serology was conducted on 236 NFSsamples collected from Saint Paul Island,Alaska (Fig. 1). Samples included werefrom 72 subadult males from 1994, 30adult females from 2009, 80 subadultmales from 2011, 24 adult females from2011, and 30 3-mo-old female pups from2011. The antibody prevalences in NFSper year and stratified by sex and age arepresented in Table 1. Overall, 63% ofsamples had a titer $1:128. The frequencyof positive titers varied significantly be-tween years (P,0.001), with an apparent

FIGURE 1. Sites where northern fur seal (Callorhinus ursinus) and Steller sea lion (Eumetopias jubatus)serum and vaginal swab samples were collected in Alaska, 1994–2011.

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increase from 49% in 1994 to 69% in 2009and 2011. Similar percentages of antibody-positive animals were females (65%) andmales (61%; P50.514). Only 2009 and2011 samples were included in the age-class analysis to eliminate the effect of anapparent temporal increase; 69% of thesesamples had a positive titer, and thefrequency of antibody-positive animalsvaried significantly between age groups(P50.046), with positive titers in 50% ofpups, 73% of subadults, and 74% ofadults.

The frequency and range of titers tophase 1 and phase 2 antibodies arepresented in Figure 2. There was a widerange of titers, from 1:128 to 1:16,384, forboth phase 1 and phase 2, with 64 animalshaving a titer of 1:256 for phase 1 and 51animals having a titer of 1:512 for phase 2.Positive ($1:128) phase 2 titers were morecommon (n5145) than positive phase 1

titers (n5129), but the majority of positiveNFS (85%) had both phase 1 and phase 2titers.

Serology was conducted on 72 SSLsamples, including seven samples from2007 (three female and four male), 18from 2008 (nine female and nine male), 13from 2010 (11 female and two male), and34 from 2011 (19 female and 15 male). Allsamples were from pups under 2 mo oldat sampling. Steller sea lion sera werecollected from eight islands in the Gulf ofAlaska and Aleutian archipelago (Fig. 1),including Agattu (n510), Akun (n57),Bogoslof (n59), Akutan (n58), Marmot(n55), Amak (n510), Sugarloaf (n59),and Ugamak (n514). Overall, 59% ofsamples were positive (Table 2), and therewas an increasing trend in antibodyprevalence over time; however, there wasno statistical difference in the frequency ofpositive animals by year (P50.16). There

TABLE 1. Temporal antibody prevalence by sex and age in Saint Paul Island, Alaska, USA northern fur seal(Callorhinus ursinus) population, 1994–2011.

Year Sex Age Positive Total Prevalence (%)

1994 Male Subadult 35 72 492009 Female Adult 27 30 902011 Female Adult 13 24 54

Male Subadult 58 80 73Female Pup 15 30 50Total 2011 86 134 64

All years 148 236 63

FIGURE 2. Frequency distribution of anti-Coxiella burnetii Phase 1 and Phase 2 antibody titers in SaintPaul Island, Alaska, northern fur seal (Callorhinus ursinus) serum samples, 1994–2011.

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was no difference in antibody prevalencebetween females (60%) and males (59%,P50.721). As with NFS, there was a widerange of titers (Fig. 3), with positive phase1 titers more common (n544) thanpositive phase 2 titers (n539). Mostsamples were positive for both phase 1and phase 2 (86%).

We conducted qPCR on 40 vaginalswabs from adult female NFS collectedin October 2009 (n529) and October 2011(n511). Thirty-two (80%) were antibodypositive. Seven were positive for onlyphase 2; 25 were positive for both phase1 and phase 2; and 35% had titers greaterthan 1:1,000. All swabs were PCR negativefor C. burnetii.

Our results suggest that C. burnetii is acommon pathogen in western stock SSLand NFS in Alaska. Previously, C. burnetiiin SSL was only identified by PCR in oneof two SSL placental samples from thePacific Northwest (Kersh et al., 2012).Although our sample sizes from some

years were small, there appeared to bean increase in antibody prevalence from2007 to 2011. Placentitis was evident in C.burnetii-infected SSL in a previous casereport from 2008 (Kersh et al., 2010) aswell as the Pacific Northwest (Kersh et al.,2012), suggesting that infection with thispathogen could have a significant impact onreproductive health and, therefore, popu-lation health.

Our results show that C. burnetii waspresent in NFS as early as 1994. When thehigh prevalence of infection was identifiedin 2010 (Duncan et al., 2012a), it wasunknown if the pathogen was new to thepopulation or simply newly identified.Results of the present study suggest thatthe pathogen has been present in thepopulation since the early 1990s; however,the prevalence became significantlyhigher in the 2000s. The 20% increase inantibody prevalence from 1994 to 2009and 2011 is suggestive of an increasingtrend of exposure within the NFS popu-lation, but it is also possible that there iscyclical variation in population antibodyprevalence that could influence the ob-served difference over time. The highantibody prevalence is consistent with thehigh placental prevalence identified in thisNFS population by qPCR (Duncan et al.,2012a, b).

In humans, phase 2 titers are presentduring acute infection and can remainelevated for years, whereas phase 1 titers

TABLE 2. Temporal antibody prevalence in westernstock Alaska, USA, Steller sea lion (Eumetopiasjubatus) pup samples, 2007–2011.

Year Age Positive Total Prevalence (%)

2007 Pup 2 7 292008 Pup 10 18 562010 Pup 8 13 622011 Pup 24 36 67Total Pup 44 74 59

FIGURE 3. Frequency distribution of anti-Coxiella burnetii Phase 1 and Phase 2 antibody titers in westernstock Alaska Steller sea lion (Eumetopias jubatus) serum samples, 2007–2011.

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are typically only elevated during chronicinfection (Peacock et al., 1983). If thisantibody–antigen relationship holds truefor marine mammals, the frequency oftiters to phase 1 and phase 2 C. burnetiiantigens suggests that NFS and SSL mayexperience both acute and chronic infec-tions. Given the aggregation of animals atthe time of parturition, it is hypothesizedthat animals could be re-exposed annuallyduring pupping season.

Vaginal C. burnetii was not detectedusing the archived NFS vaginal swabsamples. Given that 80% of the animalstested were antibody positive and the highplacental prevalence in this population(Duncan et al., 2012a, b), this negativefinding is interesting. In iatrogenicallyinfected domestic goats, C. burnetii wasexcreted in vaginal discharge up to 14 daysafter abortion (Bouvery et al., 2003).However, given that the NFS swabs werecollected in October, 3–4 mo after partu-rition and when the majority of adultfemales are pregnant (Gentry, 1998), it islikely the organism had been cleared fromthe vaginal vault. To better characterizeshedding, sample collection would ideallytake place closer to time of parturition.

This study provides increasing epidemi-ologic information regarding C. burnetii inAlaskan marine mammals. Reproductivehealth is a critical component of popula-tion stability in any animal cohort. Coxiellaburnetii infection may significantly altercellular apoptosis in NFS placentas(Myers et al., 2012), but the impact ofthis cellular alteration is difficult toextrapolate to the population level. Sinceboth of these species have shown recentpopulation declines, we recommend thatfurther investigation into the role ofreproductive pathogens, including C. bur-netii, be continued for both species.

C.M. was supported by an appointmentto the Merial Veterinary Scholars Programand funded by the US Department ofAgriculture, National Institute of Foodand Agriculture, Animal Health and Dis-ease Program. A.V.K. was supported by an

appointment to the Emerging InfectiousDiseases Fellowship program adminis-tered by the Association of Public HealthLaboratories and funded by the Centersfor Disease Control and Prevention(CDC). Serum samples from NFS sub-adult males were collected with coopera-tion from the Aleut Community of SaintPaul Island Tribal Government. Thefindings and conclusions in this reportare those of the authors and do notnecessarily represent the views of theCDC or the Department of Health andHuman Services. All tissue samples werecollected under authority of US MarineMammal Permit 782-1708 issued to theNational Marine Mammal Lab, Seattle,Washington, USA. The findings and con-clusions in this paper are those of theauthors and do not necessarily representthe views of the National Marine FisheriesService, National Oceanic and Atmospher-ic Administration (NMFS, NOAA). Refer-ence to trade names does not implyendorsement by NMFS, NOAA. We thankKatie Sweeney for creating Figure 1.

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Submitted for publication 21 September 2012.Accepted 26 November 2012.

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