Serologic Evaluation of New Zealand Sea Lions for Exposure to Brucella and Leptospira spp.

Serologic Evaluation of New Zealand Sea Lions for Exposure to Brucella and Leptospira spp. Author(s): Wendi D. Roe, Lynn E. Rogers, Brett D. Gartrell, B. Louise Chilvers, and Pádraig J. Duignan Source: Journal of Wildlife Diseases, 46(4):1295-1299. Published By: Wildlife Disease Association https://doi.org/10.7589/0090-3558-46.4.1295 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-46.4.1295 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne s Terms of Use, available at www.bioone.org/page/ terms_of_use. Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.

Journal of Wildlife Diseases, 46(4), 2010, pp. 1295 1299 # Wildlife Disease Association 2010 Serologic Evaluation of New Zealand Sea Lions for Exposure to Brucella and Leptospira spp. Wendi D. Roe, 1,4 Lynn E. Rogers, 1 Brett D. Gartrell, 1 B. Louise Chilvers, 2 and Pádraig J. Duignan 31 Institute of Veterinary Animal and Biomedical Sciences, Massey University, Palmerston North, PN4442, NewZealand; 2 Department of Conservation, Manners St., Te Aro 6011, Wellington, NewZealand; 3 Faculty of Veterinary Science, University of Melbourne, Melbourne, Werribee, Victoria 3030, Australia; 4 Corresponding author (email: w.d.roe@massey.ac.nz) ABSTRACT: A serologic survey of anti-brucella and antileptospiral antibodies was conducted on 147 adult, female New Zealand sea lions (Phocarctos hookeri). Most sea lions (n5138) were sampled at Sandy Bay, Enderby Island, Auckland Islands (50u309S, 166u179E), January 2000 March 2005. Nine were sampled at Otago, New Zealand (46u09S, 170u409E); four in April 2008 and five in March 2009. Serum from one of the Enderby Island females was weakly positive for antibodies to Brucella abortus using the competitive enzyme-linked immunosorbent assay, and one female had a low titer for Leptospira interrogans serovar pomona using the microscope agglutination test. All serum samples from Otago animals were negative. Brucellosis and leptospirosis are therefore considered unlikely to play a major role in population dynamics of these populations, and the low antibody prevalence of these agents suggests that they are an unlikely source of infection for humans, wildlife, or domestic species on mainland New Zealand. Key words: Brucella, leptospirosis, New Zealand sea lion, Phocarctos hookeri, pinniped, serology. The New Zealand sea lion (Phocarctos hookeri), New Zealand s only endemic pinniped, is a threatened species classified as vulnerable and in decline by the International Union for the Conservation of Nature (Gales, 2008). The total population is estimated to be 11,100 14,200 individuals, and over 85% of pup production is limited to three breeding sites in the Auckland Islands (Fig. 1; 50uS, 166uE; Chilvers et al., 2007). Recently a small number of New Zealand sea lion females began to breed in Otago (46u09S, 170u409E), in the South Island of New Zealand, possibly reflecting the beginning of a return to their historic breeding range (Lalas and Bradshaw, 2003), which covered a much broader area. Although this recolonization is a positive trend for the population as a whole, it increases the possibility that these individuals could be exposed to novel pathogens. In addition, as the mainland population increases, so does the potential for transmission of infectious agents from sea lions to other species, both human and nonhuman. Although the causes of mortality in New Zealand sea lion neonates have been investigated (Castinel et al., 2007), little is known about mortality in adults. Similarly, although 4% of pups are stillborn (Castinel et al., 2007), no investigations have been conducted on the cause of these stillbirths or on other possible causes of reproductive loss. In order to better manage this threatened species, it is important that we have a thorough understanding of their status with respect to infectious agents that are known to cause significant mortality or decreased reproductive success, as well as those that may pose a zoonotic or anthroponotic threat. Leptospirosis and brucellosis are potential causes of mortality and reproductive failure, and occur in a wide variety of terrestrial and marine mammals (Thorne, 2001). Both are also zoonotic. Leptospirosis has been reported as a cause of periodic mass mortality events in California sea lions (Zalophus californianus; Gulland et al., 1996), and has been suggested to play a role in abortion in both California sea lions (Gilmartin et al., 1976) and northern fur seals (Callorhinus ursinus; Smith et al., 1977). Six serovars of Leptospira are endemic to New Zealand. Recently, Mackereth et al. (2005) found serologic evidence of exposure to two of 1295

1296 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 4, OCTOBER 2010 FIGURE 1. Map showing location of Auckland Islands relative to New Zealand. New Zealand sea lions breed mainly at Sandy Bay on Enderby Island, on Dundas Island, and on Figure of Eight Island in the Auckland Islands. A small breeding group is also located at Otago Peninsula on the New Zealand mainland. these (serovars hardjobovis and pomona) in preweaned New Zealand fur seal (Arctocephalus forsteri) pups in Otago, suggesting that transmission may be occurring from a reservoir species in this area. Numerous potential reservoirs of these serovars are present in domestic animals on the New Zealand mainland, including cattle (Bos taurus), sheep (Ovis aries), deer (Cervus elaphus and Cervus canadensis), and pigs (Sus scrofa). New Zealand sea lions share haul-out sites with New Zealand fur seals, both on the mainland and in the Auckland Islands, and could theoretically be infected via contact with this species, or by contact with other reservoir species. Enderby Island is currently free of all introduced terrestrial mammals, but is a haul-out site for New Zealand fur seals, sub-antarctic fur seals (Arctocephalus tropicalis), and Southern elephant seals (Mirounga leonina) during the summer. Although the leptospire status of these populations is unknown, it is possible that these animals could be a source of infection. Marine strains of Brucella spp. are widespread throughout the world and, although in most cases disease associated with these organisms tends to be mild or asymptomatic, the possibility of a role in chronic reproductive losses, as occurs in terrestrial species, cannot be ruled out (Foster et al., 2002). Serologic studies of Antarctic pinnipeds have demonstrated exposure of Antarctic fur seals (Arctocephalus gazella; Abalos et al., 2009) and Weddell seals (Leptonychotes weddellii, Retamal et al., 2000), and the isolation of a marine-origin strain of Brucella spp. from a naturally occurring case of osteomyelitis in a human patient in New Zealand (McDonald et al., 2006) indicates that marine Brucella species are present in this region. Each summer adult female New Zealand sea lions are captured at Sandy Bay, Enderby Island (50u309S, 166u179E), a breeding site in the Auckland Island group, as part of an ongoing study into foraging ecology (Chilvers et al., 2005). Captured animals are anesthetized using inhaled isoflurane (Baxter Healthcare, Mt Wellington, Auckland, New Zealand) and blood is collected from the caudal gluteal vein. After centrifugation, serum is removed and frozen in liquid nitrogen. On return to the mainland, frozen serum samples are transferred to a 280 C freezer for storage. Between January 2000 and March 2005, 138 serum samples were available for analysis. In 2008 and 2009 a small number of females (four and five, respectively) from the Otago breeding group of 11 were caught and blood was sampled as described above. All sampling was conducted under permit from the New Zealand Department of Conservation, with the approval of the Department of Conservation Ethics Committee.

SHORT COMMUNICATIONS 1297 A competitive enzyme-linked immunosorbent assay (celisa) kit was used, according to the manufacturer s instructions, to detect antibodies against Brucella abortus (Brucella-Ab C-ELISA; Svanova Biotech AB, Uppsala, Sweden). Samples with a percent inhibition (PI) value.30% were considered positive (Retamal et al., 2000). A standard microscopic agglutination test (MAT; Cole et al., 1973) was used to test sera against Leptospira interrogans serovar pomona and Leptospira borgpetersenii serovar hardjobovis, using twofold dilutions starting at 1:24. The highest dilution that showed 50% agglutination was considered to be the titer. Of the 138 adult female Enderby Island sea lions, one (sampled in 2001) had a PI of 35.85 for B. abortus using the celisa. The remainder had PI values,28. One sea lion (sampled in 2001) had a titer of 1:192 for L. interrogans serovar pomona using the MAT. The remainder had titers of,1:24 for this serovar. All 138 were negative for antibodies against serovar hardjobovis. All nine Otago samples were negative for antibodies against leptospiral serovars hardjobovis and pomona, and for Brucella abortus antibodies. Based on these findings, the antibody prevalence of leptospiral serovars pomona and hardjobovis in adult female New Zealand sea lions at Sandy Bay appears to be low, making it unlikely that these serovars are being maintained within this group. Prolonged survival of leptospires in the sub-antarctic environment is unlikely, as these organisms do not tolerate low temperatures or high salinity (Leighton and Kuiken, 2001). Persistence at this location would therefore require the presence of a suitable maintenance host. Furthermore, because acutely infected animals tend to have a high antibody titer (Heath and Johnson, 1994), our results suggest that active transmission of leptospiral infection to adult female New Zealand sea lions during the summer breeding period is not common. There was no evidence of exposure of Otago females, but the population and sample sizes are too small for us to be confident that this group is truly free from exposure. Furthermore, we investigated exposure to serovars that are endemic to New Zealand and have been found to be present in another New Zealand pinniped species. It is possible that overall exposure to leptospirosis has been underestimated because other serovars were not evaluated. Diagnostic testing of marine mammals for Brucella spp. can be problematic. The organism is fastidious and can be difficult to grow, particularly from tissues that are not fresh, as is often the case in marine mammal necropsy material. Although validation studies are not available for marine species, the celisa for detection of antibody against B. abortus has been used as a screening test in pinniped studies (Retamal et al., 2000; Mackereth et al., 2005; Nielsen et al., 2005). Although high rates of Brucella antibody prevalence in a population suggest endemic infection and could be associated with ongoing reproductive losses, low or zero prevalence may indicate a naïve and therefore highly susceptible population. Based on our findings, few female sea lions at Sandy Bay have been exposed to brucellosis, suggesting that the majority of this group is likely to be highly susceptible to infection. Continued monitoring of the New Zealand sea lion population is necessary in order to determine whether exposure of the population is increasing. There were several limitations to this study. Firstly, because sensitivity and specificity of the described celisa and MAT for the agents being investigated are unknown for New Zealand sea lions, no definitive comments can be made about antibody prevalence within the populations examined. Serologic testing can result in false positive results due to group cross reactions to other organisms with similar antigens, nonspecific inhibitors in serum, and nonspecific agglutinins that can mimic the effects of agglutinating antibodies (Thrusfield, 2005). False nega-

1298 JOURNAL OF WILDLIFE DISEASES, VOL. 46, NO. 4, OCTOBER 2010 tives may also occur, particularly with Brucella testing using the B. abortus celisa, as there may be antigenic differences between strains of terrestrial and marine origin. The lack of evaluation for other leptospiral serovars may have resulted in an underestimate of antibody prevalence for leptospirosis. Secondly, in addition to the limitations of serologic testing, it is not possible to confidently extrapolate our results to the entire population, as only adult females were tested and only two sampling sites were used. The latter limitation is minimized because significant movement of individuals has been shown to occur between breeding sites (Gales and Fletcher, 1999; Robertson et al., 2006); however, this may also increase the frequency of transfer of infectious agents between sites, and may prevent breeding groups from behaving as closed populations with respect to infectious disease. Because there is currently no information available about the serologic status of cohorts other than adult females, it is possible that this study may underestimate the true prevalence of these agents. Further clarification of the role played by these agents in population dynamics would require investigating the presence or absence of disease, as well as serologic evidence of exposure. Despite these limitations, the results of this study suggest that leptospirosis and brucellosis are unlikely to play a significant role in the population dynamics of the New Zealand sea lion. In addition, the low antibody prevalence of these agents within the groups studied suggests that they are an unlikely source of infection for humans, wildlife, or domestic species on mainland New Zealand. In order to evaluate more fully the threat posed by these infectious agents and to enhance population management strategies, continued surveillance is advisable, in conjunction with more detailed histologic, molecular, and microbiologic studies directed at detecting clinical disease associated with these bacteria. We thank Aurelie Castinel, Amelie Auge, Jacinda Amey, Laureline Meynier, Jim Fyfe, and other field workers for their help with sample collection. The fieldwork for this research was conducted and funded under permit from the New Zealand Department of Conservation (DOC) in parallel with field work undertaken for DOC Conservation Services Programme (www.csp.org.nz) project POP2007/01. Approval for all work was obtained from the DOC Animal Ethics Committee (Approval AEC 157 and 174, 1 Oct 2008). Serology was partly funded by the Lewis Fitch Veterinary Research Fund. LITERATURE CITED ABALOS, P., P. RETAMAL, O. BLANK, D. TORRES, AND V. VALDENEGRO. 2009. Brucella infection in marine mammals in Antarctica. Veterinary Record 164: 250 250. CASTINEL, A., P. J. DUIGNAN, W. E. POMROY, N. LOPEZ-VILLALOBOS, N.J.GIBBS, B.L.CHILVERS, AND I. S. WILKINSONS. 2007. Neonatal mortality in New Zealand sea lions (Phocarctos hookeri) at Sandy Bay, Enderby Island, Auckland Islands from 1998 to 2005. Journal of Wildlife Diseases 43: 461 474. CHILVERS, B. L., I. S. WILKINSON, P. J. DUIGNAN, AND N. J. GEMMELL. 2005. Summer foraging areas for lactating New Zealand sea lions Phocarctos hookeri. Marine Ecology Progress Series 304: 235 247.,, AND S. CHILDERHOUSE. 2007. New Zealand sea lion, Phocarctos hookeri, pup production 1995 to 2006. New Zealand Journal of Marine and Freshwater Research 41: 205 213. COLE, J. R. JR., C. R. SULZER, AND A. R. PURSELL. 1973. Improved microtechnique for the leptospiral microscopic agglutination test. Applied Environmental Microbiology 25: 976 980. FOSTER, G., A. P. MACMILLAN,J.GODFROID,F.HOWIE, H. M. ROSS, A.CLOECKAERT, R.J.REID, S.BREW, AND I. A. P. PATTERSON. 2002. A review of Brucella sp. infection of sea mammals with particular emphasis on isolates from Scotland. Veterinary Microbiology 90: 563 580. GALES, N. 2008. Phocarctos hookeri. IUCN Red List of Threatened Species. Version 2009. http://www. iucnredlist.org/. Accessed April 2010., AND D. J. FLETCHER. 1999. Abundance, distribution and status of the New Zealand sea lion, Phocarctos hookeri. Wildlife Research 26: 35 52. GILMARTIN, W. G., R. L. DELONG, A. W. SMITH, J. C. SWEENEY,B.W.DE LAPPE,R.W.RISEBROUGH, L.

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