Kjetil Åsbakk

The prevalence of Toxoplasma gondii in polar bears and their marine mammal prey: evidence for a marine transmission pathway?

Little is known about the prevalence of the parasite Toxoplasma gondii in the arctic marine food chain of Svalbard, Norway. In this study, plasma samples were analyzed for T. gondii antibodies using a direct agglutination test. Antibody prevalence was 45.6% among polar bears (Ursus maritimus), 18.7% among ringed seals (Pusa hispida) and 66.7% among adult bearded seals (Erignathus barbatus) from Svalbard, but no sign of antibodies were found in bearded seal pups, harbour seals (Phoca vitulina), white whales (Delphinapterus leucas) or narwhals (Monodon monoceros) from the same area. Prevalence was significantly higher in male polar bears (52.3%) compared with females (39.3%), likely due to dietary differences between the sexes. Compared to an earlier study, T. gondii prevalence in polar bears has doubled in the past decade. Consistently, an earlier study on ringed seals did not detect T. gondii. The high recent prevalence in polar bears, ringed seals and bearded seals could be caused by an increase in the number or survivorship of oocysts being transported via the North Atlantic Current to Svalbard from southern latitudes. Warmer water temperatures have led to influxes of temperate marine invertebrate filter-feeders that could be vectors for oocysts and warmer water is also likely to favour higher survivorship of oocycts. However, a more diverse than normal array of migratory birds in the Archipelago recently, as well as a marked increase in cruise-ship and other human traffic are also potential sources of T. gondii.

Direct high-resolution genotyping of Toxoplasma gondii in arctic foxes (Vulpes lagopus) in the remote arctic Svalbard archipelago reveals widespread clonal Type II lineage

Characterization of Toxoplasma gondii genotypes in hosts living in remote, isolated regions is important for elucidating the population structure and transmission mode of this parasite. Herein, we report the results of direct genotyping of T. gondii in brain tissue of arctic foxes (Vulpes lagopus) from the remote, virtually cat-free, high arctic islands of Svalbard. DNA extracts from brains of 167 seropositive arctic foxes (including four cases of fatal toxoplasmosis) and 11 seronegative arctic foxes were genotyped at 10 loci (SAG1, SAG2, SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, and Apico) using the polymerase chain reaction-restriction fragment length polymorphism method. Of the 167 samples from seropositive foxes (including toxoplasmosis cases), 31 were genotyped at all 10 loci and 24 were genotyped at four to nine loci. To ensure confidence in T. gondii strain genotyping, samples for which less than four loci were genotyped were not considered positive. None of the 11 samples from seronegative foxes was positive for the 10 markers. Of the 55 samples that genotyped positively, 46 were of the Type II strain, 7 were of the Type III strain, and 2 were of atypical T. gondii strains. Five representative samples of the three genotypes were sequenced at loci SAG2, SAG3, GRA6, PK1, and UPRT-1. The DNA sequences confirmed the genotyping results. This study shows that the archetype Type II T. gondii strain, which is most widely distributed in North America and Europe, also predominates in arctic foxes on the Svalbard archipelago. This suggests that the T. gondii at this location originate from continental Europe and that transmission may be mediated by migrating birds. This study highlights the significance of long-distance transport of T. gondii and demonstrates that high-resolution genotyping protocols are useful for direct genetic studies of T. gondii when isolation of live parasites is infeasible.

Antibodies against Toxoplasma gondii in Fennoscandian reindeer — association with the degree of domestication

To measure the prevalence of antibodies against Toxoplasma gondii in Fennoscandian reindeer, we examined 2577 serum samples collected between 1993 and 1996 from slaughtered reindeer from Finnmark county, Norway, and from several locations in Finland. The overall prevalence in this sample was 0.9%, and the titres of the seropositives in the Direct Agglutination Test (DAT) were between 1:40 and 1:162000. Logistic regression associated seropositivity with the age of the animal, the odds ratio (OR) of adults was four when compared with calves. Seropositivity was also positively associated with corral feeding, which was used in the analysis as an indicator of domestication. No significant association was found with sex, or the frost sum of the pasture area.

Prevalence of antibodies against Toxoplasma gondii in polar bears (Ursus maritimus) from Svalbard and East Greenland.

Abstract Serum samples from 419 polar bears (Ursus maritimus) from Svalbard and the Barents Sea (collected 1990–2000) and 108 polar bears from East Greenland (collected 1999–2004) were assayed for antibodies against Toxoplasma gondii using the modified agglutination test. Antibody prevalences were 3.6% among cubs dependent on their mothers and 21.4% among subadults and adults. Among subadults and adults there was an interaction between population and sex, with similar prevalences among females (Svalbard = 19.5%, Greenland = 18.0%), but a high frequency among Svalbard males (28.7%) as compared to Greenland males (5.8%). The pattern was also significant after correcting for differences in age distribution. The sex-population interaction term is believed to be connected to area- and sex-specific feeding ecology. The prevalences of antibodies against T. gondii in Svalbard and Greenland were high compared to previously reported findings in polar bears from Russian and Alaskan areas.

Serosurvey for Trichinella in polar bears (Ursus maritimus) from Svalbard and the Barents Sea

Blood samples of live-caught polar bears (Ursus maritimus) from Svalbard collected 1991-2000 (Period 1) and 2006-2008 (Period 2) and from the pack ice of the Barents Sea collected in Period 1, were assayed for antibodies against Trichinella spp. by ELISA. Of 54 cubs-of-the-year included in the Period 1 sample, 53 were seronegative, indicating that exposure to Trichinella infected meat is uncommon during the first months of life for polar bears in the Svalbard region. Of 30 mother-offspring pairs, 18 mothers were seropositive with seronegative offspring (n=27), suggesting (1) that maternal antibodies had dropped to levels below detection limit by the time of capture in April (offspring approximately 4 months old), and (2) supporting experimental studies in other animal models showing that vertical transmission of Trichinella spp. is uncommon. Bear 1 year and older had higher prevalence in Svalbard (78%) than in the Barents Sea (51%). There was no temporal change in prevalence for bears from Svalbard during the time between the two periods. The prevalence increased with age in both sexes. A positive correlation was found between anti-Toxoplasma gondii and anti-Trichinella spp. antibodies.

A protein A/G indirect enzyme-linked immunosorbent assay for the detection of anti-Brucella antibodies in Arctic wildlife

A species-independent indirect enzyme-linked immunosorbent assay (iELISA) based on chimeric protein A/G was established for the detection of anti-Brucella antibodies in Arctic wildlife species and compared to previously established brucellosis serological tests for hooded seals (Cystophora cristata), minke whales (Balaenoptera acutorostrata), sei whales (Balaenoptera borealis), fin whales (Balaenoptera physalus), and polar bears (Ursus maritimus), as well as bacteriology results for reindeer and caribou (Rangifer tarandus sp.). The protein A/G iELISA results were consistent with the other serological tests with Cohen kappa values between 0.47 and 0.92, and the protein A/G iELISA can thus offer a technically simple method for these species yielding results consistent with established brucellosis serological tests. Receiver operator characteristics analysis proved that the reindeer and caribou protein A/G iELISA results were consistent with the bacteriological gold standard with an area under the curve of 0.99, and the protein A/G iELISA was thus validated as a sensitive and specific serological method for the detection of anti-Brucella antibodies in reindeer and caribou. The binding of the antibodies from the respective species to protein A and G were also evaluated in the iELISA. The antibodies from hooded seals and polar bears reacted stronger to protein A than to G. The sei whale, fin whale, reindeer, and caribou antibodies reacted stronger to protein G than to A. The minke whale antibodies reacted to both protein A and G. There was a strong correlation (rs = 0.88–0.98) between the optical density results obtained with the iELISA with protein A/G and protein A or G, showing that protein A/G is as well suited as protein A or G for the detection of anti-Brucella antibodies in these species with the iELISA.

Experimental trichinellosis in reindeer.

Six female reindeer calves were inoculated intraruminally with various doses of Trichinella muscle larvae. Four calves were inoculated with T. nativa, receiving 15,000 (n = 1), 5,000 (1), and 2,500 (2) larvae each. Two calves were inoculated with 5,000 T. spiralis larvae each. Blood samples were collected twice per week for total white blood cell (WBC) and differential counts and for serology using enzyme-linked immunosorbent assay (ELISA) based on T. spiralis excretory–secretory antigen. On day 56, the calves were slaughtered and muscle samples were examined according to the standard digestion method for Trichinella larvae. Blood samples were also collected twice a week from 4 uninoculated, but otherwise similar, reindeer calves corralled separately. Both the total WBC and eosinophil counts of the inoculated animals were, on average, higher during the experimental period. All the inoculated calves seroconverted, showing an increase in the optical density (OD) in the ELISA starting between day 23 and day 27 postinoculation. Very few muscle larvae (<0.08 larvae/g [lpg]) were to be found from the animals inoculated with T. nativa, but about 4 and 6 lpg were recovered from the masseter muscles of those inoculated with T. spiralis.

Participation of antigens related to the psoriasis associated antigen, pso p27, in immune complex formation in patients with ankylosing spondylitis.

Analysis of five serum samples and three synovial fluids from patients with ankylosing spondylitis (AS) and five serum samples from healthy blood donors for the presence of antibodies cross reacting with the Fc part of rabbit IgG (rheumatoid factors (RFs] using an isotype specific, enzyme linked immunosorbent assay (ELISA) showed only insignificant amounts of free RFs, while IgG RFs were observed in alkaline dissociated circulating immune complexes (CICs). Only insignificant amounts of free antibodies reacting with the psoriasis associated antigen pso p27 could be detected in the samples, while extensive amounts of IgG antibodies and moderate amounts of IgM antibodies reacting with pso p27 were detected in alkaline dissociated CICs from the patients. Pso p27 has been reported to share a common determinant with the Fc part of human IgG. Removal of the RF activity from the CICs of patients with AS by absorption with IgG resulted in a decrease of the anti-pso p27 activity. Monoclonal anti-pso p27 antibodies in a sandwich ELISA were used to detect antigens cross reacting with pso p27. A positive reaction was observed in all serum CICs and in one of the synovial fluid CICs. The data indicate that antigens related to pso p27 participate in CIC formation in AS and may also be responsible for the elicitation of rheumatoid factors in patients with AS.

Treatment of reindeer with ivermectin - effect on dung insect fauna

Ivermectin is an antiparasitic drug widely used in reindeer (Rangifer tarandus (L.)) in Fennoscandia and North America. Most of the ivermectin injected in the animal is excreted unchanged in the faeces. Several reports show that ivermectin in cattle dung disrupts colonisation and survival of beneficial dung breeding insects. The present study investigated the effect of ivermectin on the reindeer dung fauna. Four reindeer calves (males, 6 months of age) were injected subcutaneously with standard doses of ivermectin (0.2 mg/kg body weight) in early December. The daily produced faeces was collected until day 30 after treatment, and the concentration of ivermectin was determined by high pressure liquid chromatography (HPLC) with fluorescence detection. The highest concentration measured (mean 1632 ng/g faeces (dry weight), range 907 to 2261 ng/g among the animals) was on day 4 after treatment. The concentration decreased gradually to 28 ng/g (range 6 to 58 ng/g) on day 30. Faeces portions from day 4 and from untreated reindeer were placed in the field on 2-4 July and recollected on 13-22 September in order to detect possible differences in decomposition fauna between the samples. The most important coprophilous beetles (Apbodius spp.) and flies (Scatbophaga spp.) were not detected in this winter dung whether it contained ivermectin or not, probably because of the dry consistency and small size of the pellets. On the other hand, these insects (larvae and imagines) were common in summer dung, which had been deposited naturally in the field and later placed together with the ivermectin-containing winter dung for comparison. The summer dung has a more soft and lumpy consistency. Treatment in autumn or early winter implies that the bulk of the ivermectin from the animal will be present in faeces with winter consistency, since this bulk portion is excreted during the first 30 days after treatment. This dry and pelleted faeces is not utilized by the important coprophilous insecr species, and the current practice of treatment of reindeer with ivermectin in autumn or early winter is therefore the regime representing the least danger of harmful influence on the coprophilous fauna and their contribution to the dung decomposition process.

SERUM CHEMISTRY AND ANTIBODIES AGAINST PATHOGENS IN ANTARCTIC FUR SEALS, WEDDELL SEALS, CRABEATER SEALS, AND ROSS SEALS

Information on health parameters, such as antibody prevalences and serum chemistry that can reveal exposure to pathogens, disease, and abnormal physiologic conditions, is scarce for Antarctic seal species. Serum samples from Antarctic fur seals (Arctocephalus gazella, n=88) from Bouvetøya (2000–2001 and 2001–2002), and from Weddell seals (Leptonychotes weddellii, n=20), Ross seals (Ommatophoca rossii, n=20), and crabeater seals (Lobodon carcinophagus, n=9) from the pack-ice off Queen Maud Land, Antarctica (2001) were analyzed for enzyme activity, and concentrations of protein, metabolites, minerals, and cortisol. Adult Antarctic fur seal males had elevated levels of total protein (range 64–99g/l) compared to adult females and pups (range 52–79 g/l). Antarctic fur seals had higher enzyme activities of creatine kinase, lactate dehydrogenase, and amylase, compared to Weddell, Ross, and crabeater seals. Antibodies against Brucella spp. were detected in Weddell seals (37%), Ross seals (5%), and crabeater seals (11%), but not in Antarctic fur seals. Antibodies against phocine herpesvirus 1 were detected in all species examined (Antarctic fur seals, 58%; Weddell seals, 100%; Ross seals, 15%; and crabeater seals, 44%). No antibodies against Trichinella spp., Toxoplasma, or phocine distemper virus (PDV) were detected (Antarctic fur seals were not tested for PDV antibodies). Antarctic seals are challenged by reduced sea ice and increasing temperatures due to climate change, and increased anthropogenic activity can introduce new pathogens to these vulnerable ecosystems and represent a threat for these animals. Our data provide a baseline for future monitoring of health parameters of these Antarctic seal species, for tracking the impact of environmental, climatic, and anthropogenic changes in Antarctica over time.