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Serologic evidence of Brucella spp. exposure in Atlantic walruses (Odobenus rosmarus rosmarus) and ringed seals (Phoca hispida) of Arctic Canada.

(Received 14 February 1996; accepted in revised form 17 July 1996)

ABSTRACT. The first presumptive evidence of Brucella infection in marine mammals of Arctic Canada is reported. Blood samples were collected from 248 ringed seals (Phoca hispida) and 59 Atlantic walrus (Odobenus rosmarus rosmarus) from eight locations in the Canadian Arctic between 1987 and 1994. A competitive enzyme-linked immunosorbent assay (C-ELISA), using a specific monoclonal antibody to Brucella spp. cell wall components, was used to detect anti-Brucella spp. antibodies in the samples. Sera from ten seals and seven walruses exceeded the C-ELISA threshold that indicates that cattle have been exposed to Brucella spp. Five of the positive walrus sera were suitable for the tube agglutination test. All five were confirmed positive using this test. Although the bacterium has not yet been identified, it appears that a Brucella sp. or a Brucella-like bacterium may be enzootic in these species in the Canadian Arctic. It is also possible that the very low prevalence of antibodies in ringed seals and the seemingly random distribution of seropositive animals may indicate a sporadic infection from another enzootically infected phocid or predator (e.g., Arctic fox Alopex lagopus). Or perhaps, limited epizootics may have occurred in the areas where seropositive seals were found. A similar situation could also exist in the walrus of Foxe Basin.

Key words: walrus, Odobenus rosmarus rosmarus, ringed seal, Phoca hispida, Brucella, brucellosis, competitive ELISA, tube agglutination test.

RESUME. On rapporte la premiere preuve par inference d'une infection des mammiferes marins de l'Arctique canadien par le bacille Brucella. Entre 1987 et 1994, on a effectue des prelevements sanguins sur 248 phoques anneles (Phoca hispida) et 59 morses de l'Atlantique (Odobenus rosmarus rosmarus) a huit endroits dans l'Arctique canadien. On a employe la technique immuno-enzymatique par competition ELISA, en se servant d'un anticorps monoclonal specifique aux composants de la paroi cellulaire de Brucella spp, afin de detecter les anticorps anti-Brucella sp dans les prelevements. Les serums de dix phoques et de sept morses depassaient le seuil d'ELISA qui indique que la population animale a ete exposee a Brucella spp. Cinq des serums positifs de morses se pretaient a l'epreuve d'agglutination en tube, et tous se sont reveles positifs. Bien qu'on n'ait pas encore identifie la bacterie, il semble que Brucella sp ou une bacterie semblabel a Brucella puisse etre enzootique a ces especes dans l'Arctique canadien. Il est egalement possible que la faible frequence globale d'anticorps chez le phoque annele et la distribution apparemment erratique des animaux seropositifs soient l'indice d'une infection sporadique communiquee par un autre phocide infecte par proximite dans le milieu ou encore par un predateur (p. ex., le renard arctique Alopex lagopus). Ou bien des epizooties se seraient produites dans les regions ou l'on a trouve des phoques seropositifs. Une situation semblable pourrait aussi se retrouver chez le morse du bassin de Foxe.

Mots cles: morse, Odobenus rosmarus rosmarus, phoque annele, Phoca hispida, Brucella, brucellose, ELISA par competition, epreuve d'agglutination en tube

Traduit pour la revue Arctic par Nesida Loyer.

INTRODUCTION

Brucella is a genus of gram-negative bacteria whose members can infect both domestic and wild mammals. The resulting disease is known as brucellosis. There are six recognized species of Brucella. Three species can occur as one of several biovars or strains. All but two species are pathogenic in humans (Mayfield et al., 1990). In domestic mammals, brucellosis causes abortions and other reproductive disorders. In humans, these pathogens are often associated with influenza-like symptoms, although other complications may occur.

Brucella spp. also occur in a number of wildlife species in Arctic Canada. Rangiferine brucellosis, the most common form of brucellosis in the Arctic, is caused by Brucella suis biovar 4. The disease primarily affects reindeer and caribou (Rangifer tarandus) (Neiland et al., 1968), moose (Alces alces) (Dietrich et al., 1991; Honour and Hickling, 1993), and muskox (Ovibos moschatus moschatus) (Gates et al., 1984). Brucella suis biovar 4 has also been isolated from animals that consume infected ungulate carcasses, such as wolves (Canis lupus), dogs (C. familiaris), red foxes (Lulpes vulpes), and grizzly bears (Ursus arctos) (Neiland, 1970, 1975). It has been isolated in Siberia and Alaska as well as in Canada (Neiland et al., 1968). Human brucellosis has been reported from a number of locations in Arctic Canada (Tessaro and Forbes, 1986).

There have been few studies of Brucella spp. in marine mammals. There is preliminary evidence of Brucella spp. in a few stranded harbour seals (Phoca vitulina), harbour porpoises (Phocoena phocoena), and a common dolphin (Delphinus delphis) from the Scottish coast, although the bacterium was not identified (Ross et al., 1994). A culture of Brucella sp. isolated from an aborted bottlenose dolphin (Tursiops truncatus) fetus from California did not belong to any known species or biovars (Ewalt et al., 1994). An initial survey in Alaska found no evidence of Brucella spp. in 12 Pacific walrus (Odobenus rosmarus divergens) (Seagers et al., 1995).

There have been no reported surveys for Brucella spp. in marine mammals in the Canadian Arctic. However, these mammals are an important source of food in most communities. Their health and the impact of consuming arctic marine mammals on human health are, therefore, of utmost importance to Northerners. Here we report the first indirect evidence of Brucella spp. in Atlantic walrus (Odobenus rosmarus rosmarus) and ringed seals (Phoca hispida) from the Canadian Arctic.

METHODS

As part of ongoing marine mammal sampling programs (e.g., Garlich-Miller, 1994), blood, reproductive tracts and other tissues were collected and held frozen during Inuit subsistence hunts throughout the Canadian Arctic. Ringed seals were sampled between 1992 and 1994 at seven locations (Fig. 1). Walrus were sampled between 1987 and 1993 at two locations in Foxe Basin (Fig. 1). Usually, blood was collected within hours of death, frozen whole, and maintained at -20[degrees]C before testing. For some samples, the serum was separated before initial freezing. Prior to testing, both blood and sera were centrifuged at 1000 x g for ten minutes.

The sera were tested for anti-Brucella spp. antibodies by two competitive enzyme-linked immunosorbent assays (C-ELISA) (Nielsen et al., 1992, 1995). The C-ELISAs are robust in this application. The tests allow the detection of Brucella-specific antibodies in a number of animal species and also distinguish between Brucella spp. and other related gram-negative bacteria, even when whole blood samples are used (Nielsen et al., 1992). Briefly, the C-ELISA assays involve the adsorption of Brucella spp. smooth lipopolysaccharide (s-LPS) antigen or polysaccharide antigen conjugated with poly-L-lysine to a solid plastic matrix. Diluted (1:50) test serum or blood and a mouse monoclonal antibody (MAB) specific for antigenic determinants of the polysaccharide are then added. The serum dilutions and antibody solutions were mixed simultaneously in a microtiter plate containing the bound antigen. The mixtures were incubated for one to two hours. In the first assay, the MAB was labelled with horseradish peroxidase. The subsequent chromagen mixture was added after the incubation step. In the second assay, goat antimouse IgG antibody conjugated to horseradish peroxidase was added. After incubation, enzyme substrate and a chromagen were added, and the subsequent coloured product was measured photometrically. If the test serum contained Brucella-specific antibodies, they competed with the MAB for antigenic sites, thereby inhibiting the binding of the MAB and the subsequent colour development. A threshold at 30% or greater inhibition of MAB binding was selected on the basis of results obtained with bovine sera (Nielsen et al., 1995). Sera which met or exceeded this threshold are believed to be indicative of previous exposure to Brucella spp. These sera will be referred to as positive. The C-ELISA positive sera were tested by the standard tube agglutination test (MacMillan, 1992). Only unhaemolyzed sera were suitable for this test, and a tube agglutination titer of 20 or greater was considered positive.

Ages of the seals and walrus were estimated by counting dental annuli in the canine teeth (McLaren, 1958; Garlich-Miller et al., 1993). Reproductive tracts of adult females that had serologic evidence of exposure were thawed, dissected, and examined for gross evidence of reproductive failure or abnormalities.

RESULTS AND DISCUSSION

Approximately 4% (10 of 248) of the seals and 12% (7 of 59) of the walrus were positive in the C-ELISA tests (Table 1). Five of the seven positive walrus sera were suitable for confirmation using the tube agglutination test. All were positive at dilutions of 1:20, 1:40, or 1:80. None of the seal samples were suitable for tube agglutination analysis since they were badly haemolyzed. Broughton et al. (1970) found Brucella spp. agglutinating antibody titers of 1:25 or greater in 4.37% (14 of 320) of the caribou from Kaminuriak, Northwest Territories and in 8.74% (148 of 1692) of sera from reindeer in the Mackenzie Delta. These data seem consistent with the prevalences we report from seals and walrus.


[Part 1 of 2]

TABLE 1. Serum antibody prevalence of Brucella spp. in two species of
marine mammals from Arctic Canada.

Species      Location                          Dates


Ringed seal
             Pangnirtung    65[degrees]22'N, 66[degrees]30'W   November '92 - February '93
             Arctic Bay     73[degrees]01'N, 85[degrees]07'W   April - June '93
             Resolute Bay   74[degrees]40'N, 95[degrees]00'W   June '93
             Eureka         80[degrees]00'N, 86[degrees]00'W   May - June '94
             Paulatuk       69[degrees]35'N, 123[degrees]40'W  August - September '93
                                               August - September '94
             Sachs Harbour  71[degrees]59'N, 125[degrees]15'W  July '93
             Holman Island  70[degrees]39'N, 101[degrees]31'W  May - July '93
                                               May - July '94

             All locations

Walrus
             lgloolik       69[degrees]23'N, 81[degrees]40'W   July - August '87
                                               July - August '88
                                               July - August '93

             Hall Beach     68[degrees]47'N, 81[degrees]13'W   July - August '93

             All locations

[Part 2 of 2]

TABLE 1. Serum antibody prevalence of Brucella spp. in two species of
marine mammals from Arctic Canada.

Species      Number  Positive
             tested  No.   %

Ringed seal
                 75     7   9
                 38     0   0
                  8     0   0
                 17     2  12
                  9     0   0
                 38     0   0
                  3     0   0
                 31     0   0
                 29     1   3

                248    10   4

Walrus
                 16     1   6
                 36     6  17
                  3     0   0

                  4     0   0



59 7 12

The seropositive ringed seals came from three widely distant sites (Pangnirtung, Eureka, and Holman Island). These areas are separated by 1250 to 2100 km, and no seropositive seals have been detected at locations between them. A chi-square goodness-of-fit test was performed on ringed seal data for locations with more than 15 samples. Results indicated the observed frequency of positive seals was significantly different among sites (p < 0.05, 1 d.f.). This difference was apparently due to the lack of positive sera from Arctic Bay and Paulatuk. Among the sites with positive animals, there were no significant differences from the expected frequency (p > 0.05, 1 d.f.). However, as the expected prevalence values were less than five for all cells, these statistical results should be viewed with caution. All the walrus samples originated from one population in the Foxe Basin (Richard and Campbell, 1988).

Seropositive walrus ranged in age from 10 to 13 years and five of the seven were female, while seropositive ringed seals ranged from 0 to 18 years and seven of the ten were female. However, there were too few data to examine age or sex differences statistically. The highest frequency of positive reactions for walrus was in 1988. However, there were too few data to test for temporal trends.

Reproductive status was available for three seropositive female walrus and one of the positive female ringed seals. All appeared normal. Another seropositive adult ringed seal was nursing an apparently healthy pup when collected. One researcher ate uncooked parts of one of the seropositive seals from Eureka and tested negative for Brucella spp. exposure approximately one year later. Presumably the Inuit who subsist on seals and walrus are exposed to this bacterium too. However, there is no evidence suggesting that prevalence of brucellosis in humans who live where we found serologic evidence of Brucella spp. in seals and walruses is different from its prevalence in other areas in the Northwest Territories (A. Corriveau, Territorial Epidemiologist, Government of the Northwest Territories, pers. comm. 1996).

There are several possible explanations for the serologic test results obtained in this survey: (1) The agent which elicited the antibody response may be a known strain of Brucella that may or may not be pathogenic in marine mammals. (2) The agent may be a new strain of Brucella. (3) Positive serologic test results may be due to some other bacterium that mimics Brucella in the ELISA and agglutination tests. However, these tests are quite specific for Brucella in terrestrial mammals (Nielsen et al., 1992), and we think that this explanation is unlikely.

The very low prevalence of anti-Brucella spp. antibodies in ringed seals and the random distribution of seropositive animals may also indicate sporadic infection, perhaps from another enzootically infected phocid or from a predator such as the Arctic fox (Alopex lagopus). Alternatively, limited epizootics may have occurred in the areas where seropositive seals were found. Walruses could also have been infected in a similar manner. Research is presently underway to isolate the responsible bacterium. In addition, we hope to determine the number and geographic range of other marine mammal species that have been exposed.

ACKNOWLEDGEMENTS

We thank J. Alikamik, K. Fisher, J. Memogama, J. Garlich-Miller, J. Ruben, and S. Innes and the Hunters and Trappers Associations of the communities involved for the collection of the samples. Seals were aged by T.G. Smith and H. Wright; walrus, by J. Garlich-Miller. The manuscript benefited from the thoughtful comments of S. Cosens, A. Corriveau, B. Souter, and B. Stewart. The study was funded by the Fisheries Joint Management Committee of the Inuvialuit Settlement Region, the Department of Fisheries and Oceans, Canada, and Agriculture and Agri-Food Canada. The authors wish to acknowledge the contributions of D. Gall and J. M. Lapointe, Animal Disease Research Institute, Agriculture and Agri-Food Canada, Nepean, Ontario.
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Author:Ole Nielsen; Klaus Nielsen; Robert E.A. Stewart
Publication:Arctic
Date:Dec 1, 1996
Words:2313
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