Inkoo and Sindbis viruses in blood sucking insects, and a serological study for Inkoo virus in semi-domesticated Eurasian tundra reindeer in Norway.

BACKGROUND: Mosquito-borne viruses pose a serious threat to humans worldwide. There has been an upsurge in the number of mosquito-borne viruses in Europe, mostly belonging to the families Togaviridae, genus Alphavirus (Sindbis, Chikungunya), Flaviviridae (West Nile, Usutu, Dengue), and Peribunyaviridae, genus Orthobunyavirus, California serogroup (Inkoo, Batai, Tahyna). The principal focus of this study was Inkoo (INKV) and Sindbis (SINV) virus circulating in Norway, Sweden, Finland, and some parts of Russia. These viruses are associated with morbidity in humans. However, there is a knowledge gap regarding reservoirs and transmission. Therefore, we aimed to determine the prevalence of INKV and SINV in blood sucking insects and seroprevalence for INKV in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) in Norway. MATERIALS AND METHODS: In total, 213 pools containing about 25 blood sucking insects (BSI) each and 480 reindeer sera were collected in eight Norwegian reindeer summer pasture districts during 2013-2015. The pools were analysed by RT-PCR to detect INKV and by RT-real-time PCR for SINV. Reindeer sera were analysed for INKV-specific IgG by an Indirect Immunofluorescence Assay (n = 480, IIFA) and a Plaque Reduction Neutralization Test (n = 60, PRNT). RESULTS: Aedes spp. were the most dominant species among the collected BSI. Two of the pools were positive for INKV-RNA by RT-PCR and were confirmed by pyrosequencing. The overall estimated pool prevalence (EPP) of INKV in Norway was 0.04%. None of the analysed pools were positive for SINV. Overall IgG seroprevalence in reindeer was 62% positive for INKV by IIFA. Of the 60 reindeer sera- analysed by PRNT for INKV, 80% were confirmed positive, and there was no cross-reactivity with the closely related Tahyna virus (TAHV) and Snowshoe hare virus (SSHV). CONCLUSION: The occurrence and prevalence of INKV in BSI and the high seroprevalence against the virus among semi-domesticated reindeer in Norway indicate that further studies are required for monitoring this virus. SINV was not detected in the BSI in this study, however, human cases of SINV infection are yearly reported from other regions such as Rjukan in south-central Norway. It is therefore essential to monitor both viruses in the human population. Our findings are important to raise awareness regarding the geographical distribution of these mosquito-borne viruses in Northern Europe.

Human myiasis caused by the reindeer warble fly, Hypoderma tarandi, case series from Norway, 2011 to 2016.

Hypoderma tarandi causes myiasis in reindeer and caribou (Rangifer tarandus spp.) in most northern hemisphere regions where these animals live. We report a series of 39 human myiasis cases caused by H. tarandi in Norway from 2011 to 2016. Thirty-two were residents of Finnmark, the northernmost county of Norway, one a visitor to Finnmark, and six lived in other counties of Norway where reindeer live. Clinical manifestations involved migratory dermal swellings of the face and head, enlargement of regional lymph nodes, and periorbital oedema, with or without eosinophilia. Most cases of human myiasis are seen in tropical and subtropical countries, and in tourists returning from such areas. Our findings demonstrate that myiasis caused by H. tarandi is more common than previously thought. Healthcare professionals in regions where there is a likelihood of human infestation with H. tarandi (regions populated by reindeer), or treating returning travellers, should be aware of the condition. All clinicians are advised to obtain a detailed travel history when assessing patients with migratory dermal swellings. On clinical suspicion, ivermectin should be given to prevent larval invasion of the eye (ophthalmomyiasis). Since H. tarandi oviposits on hair, we suggest wearing a hat as a prevention measure.

The relative plasma availabilities of ivermectin in reindeer (Rangifer tarandus tarandus) following subcutaneous and two different oral formulation applications.

BACKGROUND: Overwintering (breeding) reindeer (Rangifer tarandus tarandus) are commonly treated with ivermectin against parasitic infestations once yearly in autumn-winter roundups. The only preparations registered to reindeer are those for subcutaneous injection. However, also oral extra-label ivermectin administration is used. Twenty-six, 8-month-old reindeer calves were randomly allocated into three groups. Group 1 (n = 9) received oral ivermectin mixture (Ivomec® vet mixt. 0.8 mg/ml, oral ovine liquid drench formulation), Group 2 (n = 9) oral ivermectin paste (Ivomec® vet 18.7 mg/g equine paste), and Group 3 (n = 8) subcutaneous injection of ivermectin (Ivomec® 10 mg/ml vet inj.), each group at a dose of 200 µg/kg body weight. Blood samples were collected at treatment and at days 1, 2, 3, 6, 9 and 16 post treatment. Plasma concentrations of ivermectin were determined by high-pressure liquid chromatography (HPLC) with fluorescence detection. RESULTS: The peak plasma concentration (Cmax) was reached by 2 days after each treatment. The Cmax and Area Under Curve (AUC) differed significantly between the groups: Cmax was 30.2 ± 3.9, 14.9 ± 5.7 and 63.1 ± 13.1 ng/ml, and AUC∞ was 2881 ± 462, 1299 ± 342 and 6718 ± 1620 ng*h/ml for groups 1, 2 and 3, respectively (mean ± standard deviation). CONCLUSIONS: The differences in plasma concentrations of ivermectin are concomitant with earlier observed differences in antiparasitic efficacy, which discounts the use of the equine paste in reindeer in favour of the oral ovine liquid drench formulation, or preferably, the reindeer-registered subcutaneous injection formulation.

West Greenland harbour porpoises assayed for antibodies against Toxoplasma gondii: false positives with the direct agglutination method.

We assayed blood/tissue fluid samples from 20 harbour porpoises Phocoena phocoena from western Greenland coastal waters for antibodies against the protozoan parasite Toxoplasma gondii by the direct agglutination test (DAT). Nine individuals (45%) were interpreted to be seropositive at 1:40 dilution and 4 (20%) were seropositive up to 1:160. Samples from these individuals were assayed by an enzyme-linked immunosorbent assay (ELISA), and tissue samples of the DAT-positive animals were tested by a nested polymerase chain reaction (nPCR). Results from both methods were negative, suggesting the absence of infection in the tested animals. After chloroform clean-up, all were negative when re-assayed by DAT. We concluded that infection with T. gondii was absent in all 20 animals, despite the initially positive DAT results, and that the false positives resulted from non-specific adherence to tachyzoites in the DAT assay which could be removed by the chloroform clean-up method. Our results suggest that detecting antibodies against T. gondii using the DAT or the modified agglutination technique, particularly on samples from Arctic marine animals which often are rich in lipids, may lead to false positive results. For such samples, the use of ELISA or PCR on available tissue samples may be advocated as confirmatory tests in order to avoid false positives and overestimating seroprevalence.

Relationships between persistent organic pollutants and circulating immunoglobulin-Y in black-legged kittiwakes and Atlantic puffins.

Have Although persistent organic pollutants (POPs) may affect the immune system, few field studies actually examined this effect. There are indications that POP exert effects on the immune system; however, in the Arctic ecosystem data are scarce. The aim of this study was to examine immune functions in two medium trophic-positioned seabirds, the black-legged kittiwakes (Rissa tridactyla) and Atlantic puffins (Fratercula arctica). Overall POP concentrations were higher in kittiwakes than puffins and males had significantly higher concentrations than females. Mean concentrations of total polychlorinated biphenyls (ΣPCB9) were 4700 ± 200 and 9600 ± 1400 ng/g lipid weight and 2800 ± 180 and 3900 ± 200 ng/g lipid weight in female and male kittiwake and puffin blood, respectively. Levels of immunoglobulin-Y (IgY) in blood of kittiwakes were not markedly affected by concentrations of POP. Similarly, the primary IgY response to tetanus toxoid was not affected by POP concentrations in a subsample of immunized kittiwakes. In puffins, there were significant correlations between the IgY-response and some of the POPs, but with low explanatory values. These results suggest that POPs concentrations were lower than, or just at the threshold level for effects of the proposed IgY biomarker. It is also conceivable that the IgY levels are not a suitable endpoint for evaluating perturbation of the immune system in free-living seabirds.

Infestation by Hypoderma tarandi in reindeer calves from northern Finland--prevalence and risk factors.

Serum samples from 953 reindeer (Rangifer t. tarandus) calves-of-the-year from 21 reindeer herding co-operatives in Finland in slaughter season 2006/2007 were assayed for antibodies against Hypodermin C by an ELISA detecting IgG. Data on presence of Hypoderma tarandi larvae on 12,327 reindeer hides from 17 of the 21 herding co-operatives in slaughter season 2005/2006 were included for support. ELISA showed a seroprevalence of 60.9%, with no significant difference between females and males, and increase with latitude (southernmost and northernmost co-operatives examined, Pudasjärvi and Kaldoaivi, 11.8 and 100% of seropositives, respectively). The proportion of larva positive hides (range 0.5-60% per co-operative) was low compared to the proportion of seropositives. Also the proportion of larva positive hides increased with latitude. Our findings indicated that high latitude combined with open landscape, presence of low vegetation and high reindeer density provided more favorable conditions for sustaining of high degree of warble fly infestation, and furthermore, that any possible effect of ivermectin treatment on infestation rate was ruled out by the higher effect by the above factors.

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 (r s = 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 parapoxvirus infection (contagious ecthyma) in semi-domesticated reindeer (Rangifer tarandus tarandus).

Contagious ecthyma (contagious pustular dermatitis, orf) occurs world-wide in sheep and goats and is caused by orf virus (genus Parapoxvirus, family Poxviridae). Contagious ecthyma outbreaks have been described in semi-domesticated reindeer (Rangifer tarandus tarandus) in Sweden, Finland and Norway, occasionally with high mortality. Fourteen one-year-old reindeer were corralled in mid-April. One week after arrival, two animals received a commercial live orf virus vaccine for sheep (Scabivax(®)) on scarified skin of the medial thigh. Four weeks later, the two vaccinated and six additional animals were inoculated in scarified oral mucosa with parapoxvirus obtained from reindeer with clinical contagious ecthyma. The remaining six reindeer were kept as sentinels, sharing feed and water with the inoculated animals. A small whitish lesion appeared on the inoculation site and the labial skin-mucosa junction of three animals five days post inoculation (p.i.). Twelve days p.i., typical ecthyma lesions were visible on the inoculation site in six of eight animals, including both vaccinees. Four inoculated animals (including both vaccinees) and one sentinel seroconverted 12 days p.i., and five animals (including one sentinel) seroconverted 20 days p.i. No contagious ecthyma-like lesions were detected in the sentinels. All animals were euthanized at 26-29 days p.i. Histological examination of lesions showed proliferative dermatitis with epidermal hyperplasia, hyperkeratosis, intra-epithelial pustules and ulcers. Orf virus DNA was detected in mandibular lymph nodes, tonsils and mucosal lesions of four animals, including one sentinel, which showed that virus transmission took place. The commercial orf virus vaccine may be difficult to administer due to the need for close-cropping and its zoonotic nature, and did not indicate significant protection, although the latter has to be verified with a larger number of animals.

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 64-99 g/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.

Moritella viscosa bypasses Atlantic salmon epidermal keratocyte clearing activity and might use skin surfaces as a port of infection.

Moritella viscosa is considered the main causative agent of winter ulcer disease in salmonid fish. In order to obtain more details on route of infection, we challenged Atlantic salmon (Salmo salar) epidermal keratocytes with M. viscosa and performed an Atlantic salmon immersion challenge. Although keratocytes were able to remove M. viscosa from surfaces, their engulfment capability appeared inefficient with reduced ability to reepithelialise superficial wounds (scale less skin surfaces) challenged with the bacterium. The immersion challenge revealed a significant connection between exposure area and mortality. Enhanced invasion ability and mortality was observed by M. viscosa exposure of the head and gill region compared to exposure of: the right side of the body; the left side of the body; or the body from pectoral to caudal fin (p=0.04). Ulcer development corresponded to area exposed (p=0.002), suggesting skin ulcer formation to result primarily from direct skin surface colonization. Ulceration of surfaces exposed to M. viscosa in parallel with occurrence of septicaemia suggests that both skin and gills may act as possible initiation sites for M. viscosa infections.

Rabies in the arctic fox population, Svalbard, Norway.

Arctic foxes, 620 that were trapped and 22 found dead on Svalbard, Norway (1996-2004), as well as 10 foxes trapped in Nenets, North-West Russia (1999), were tested for rabies virus antigen in brain tissue by standard direct fluorescent antibody test. Rabies antigen was found in two foxes from Svalbard and in three from Russia. Blood samples from 515 of the fox carcasses were screened for rabies antibodies with negative result. Our results, together with a previous screening (1980-1989, n=817) indicate that the prevalence of rabies in Svalbard has remained low or that the virus has not been enzootic in the arctic fox population since the first reported outbreak in 1980. Brain tissues from four arctic foxes (one from Svalbard, three from Russia) in which rabies virus antigen was detected were further analyzed by reverse-transcriptase polymerase chain reaction direct amplicon sequencing and phylogenetic analysis. Sequences were compared to corresponding sequences from rabies virus isolates from other arctic regions. The Svalbard isolate and two of the Russian isolates were identical (310 nucleotides), whereas the third Russian isolate differed in six nucleotide positions. However, when translated into amino acid sequences, none of these substitutions produced changes in the amino acid sequence. These findings suggest that the spread of rabies virus to Svalbard was likely due to migration of arctic foxes over sea ice from Russia to Svalbard. Furthermore, when compared to other Arctic rabies virus isolates, a high degree of homology was found, suggesting a high contact rate between arctic fox populations from different arctic regions. The high degree of homology also indicates that other, and more variable, regions of the genome than this part of the nucleoprotein gene should be used to distinguish Arctic rabies virus isolates for epidemiologic purposes.

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.

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.

First isolate of Toxoplasma gondii from arctic fox (Vulpes lagopus) from Svalbard.

Cats are considered essential for the maintenance of Toxoplasma gondii in nature. However, T. gondii infection has been reported in arctic fox (Vulpes lagopus) from the Svalbard high arctic archipelago where felids are virtually absent. To identify the potential source of T. gondii, we attempted to isolate and genetically characterize the parasite from arctic foxes in Svalbard. Eleven foxes were trapped live in Grumant (78 degrees 11'N, 15 degrees 09'E), Svalbard, in September 2005 and 2006. One of the foxes was found to be seropositive to T. gondii by the modified agglutination test (MAT). The fox was euthanized and its heart and brain were bioassayed in mice for the isolation of T. gondii. All 10 mice inoculated with brain tissue and one of the five inoculated with heart developed MAT antibodies, and tissue cysts were found in the brains of seropositive mice. Two cats fed tissues from infected mice shed T. gondii oocysts. Genotyping using 10 PCR-RFLP markers and DNA sequencing of gene loci BSR4, GRA6, UPRT1 and UPRT2 determined the isolate to be Type II strain, the predominant T. gondii lineage in the world.

Serosurvey for Toxoplasma gondii in arctic foxes and possible sources of infection in the high Arctic of Svalbard.

Samples (blood or tissue fluid) from 594 arctic foxes (Alopex lagopus), 390 Svalbard reindeer (Rangifer tarandus platyrhynchus), 361 sibling voles (Microtus rossiaemeridionalis), 17 walruses (Odobenus rosmarus), 149 barnacle geese (Branta leucopsis), 58 kittiwakes (Rissa tridactyla), and 27 glaucous gulls (Larus hyperboreus) from Svalbard and nearby waters were assayed for antibodies against Toxoplasma gondii using a direct agglutination test. The proportion of seropositive animals was 43% in arctic foxes, 7% in barnacle geese, and 6% (1 of 17) in walruses. There were no seropositive Svalbard reindeer, sibling voles, glaucous gulls, or kittiwakes. The prevalence in the arctic fox was relatively high compared to previous reports from canid populations. There are no wild felids in Svalbard and domestic cats are prohibited, and the absence of antibodies against T. gondii among the herbivorous Svalbard reindeer and voles indicates that transmission of the parasite by oocysts is not likely to be an important mechanism in the Svalbard ecosystem. Our results suggest that migratory birds, such as the barnacle goose, may be the most important vectors bringing the parasite to Svalbard. In addition to transmission through infected prey and carrion, the age-seroprevalence profile in the fox population suggests that their infection levels are enhanced by vertical transmission.

Prolonged persistence of fecally excreted ivermectin from reindeer in a sub-arctic environment.

In December 2001 and 2002, feces from reindeer calves treated with ivermectin were distributed on plots established on two types of forested reindeer pasture in northern Finland. The ungrazed plots were on an enclosure that had been fenced to prevent reindeer access for the last 6 years. The grazed plots were on an area that had been heavily stocked by reindeer during the last 5 years. After enclosures had been established, reindeer and large wildlife were prevented from entering by a fence. Topsoil samples (feces, vegetation, and soil) were collected monthly during the summers of the following 2 years, over a period of from 25 to 95 weeks after deposition. The samples were analyzed for ivermectin using HPLC. Although ivermectin degradation rapidly took place during the first spring, considerable residual ivermectin could be measured throughout the sampling time, showing that ivermectin in feces on pasture may not be photodegraded as rapidly as previously believed. The results support the need for further environmental evaluation studies on the use of ivermectin to control reindeer parasites.

Dynamics of antibodies against hypodermin C in reindeer infested with the reindeer warble fly, Hypoderma tarandi.

Serum samples from 25 reindeer (Rangifer tarandus tarandus) were assayed for antibody against hypodermin C (HyC) using an ELISA. Nineteen animals were calves (born in 1998, 1999 or 2001) and six were adults (3-10 years old at first blood collection). The samples were collected over periods of 4 months (calves born in 2001) or 27 months (adults and calves born in 1998 and 1999), the latter encompassing three Hypoderma tarandi infestation seasons. The calves received antibodies against HyC from their mothers, either by placental transfer or through the ingestion of colostrum. The low level at 3 h postpartum compared to the high level 3 days after birth in one calf suggests that the antibodies are transferred through colostrum. The levels of antibody of maternal origin decreased rapidly and reached low levels by mid-July, which coincides with the onset of the major Hypoderma ovipositioning season in this region. The calves thus did not appear to be protected by antibody against HyC when they were exposed to H. tarandi infestation for the first time. Antibody levels increased following infestation and reached a maximum during November or December, which coincides with when the H. tarandi larva stops migrating after it has reached the site under the skin of the back of the host and develops further. Levels declined thereafter and reached a nadir during the following summer. After the subsequent re-infestation, the increase in levels occurred at least 1 month earlier than with the first infestation. Levels remained elevated throughout the year after repeated infestations. This implies that the antibodies persist after the annual exit of mature larvae from the animal, and after larvae have been killed by application of ivermectin. Levels in adults, however, declined significantly with age, and levels were significantly lower in animals that were 4-11 years old than in 1-year-old animals during the same 1-year period. This supports the contention that the functional capacity of the immune system declines gradually with age. The study demonstrated that HyC is potentially useful for serological diagnosis of hypodermosis in reindeer, but the persistence of antibodies complicates interpretation of antibody-based surveillance programme data in all cases other than first-time exposure.