Humanized Transgenic Mice Are Resistant to Chronic Wasting Disease Prions From Norwegian Reindeer and Moose.

Chronic wasting disease (CWD) is the transmissible spongiform encephalopathy or prion disease affecting cervids. In 2016, the first cases of CWD were reported in Europe in Norwegian wild reindeer and moose. The origin and zoonotic potential of these new prion isolates remain unknown. In this study to investigate zoonotic potential we inoculated brain tissue from CWD-infected Norwegian reindeer and moose into transgenic mice overexpressing human prion protein. After prolonged postinoculation survival periods no evidence for prion transmission was seen, suggesting that the zoonotic potential of these isolates is low.

Chronic wasting disease in Norway-A survey of prion protein gene variation among cervids.

Susceptibility of cervids to Chronic Wasting Disease (CWD), a prion disease, can be modulated by variations in the prion protein gene (PRNP), encoding the cellular prion protein (PrPC ). In prion diseases, PrPC is conformationally converted to pathogenic conformers (PrPSc ), aggregates of which comprise infectious prions. CWD has recently been observed in its contagious form in Norwegian reindeer (Rangifer tarandus) and in novel, potentially sporadic forms, here called 'atypical CWD', in moose (Alces alces) and red deer (Cervus elaphus). To estimate relative susceptibility of different Norwegian cervid species to CWD, their non-synonymous PRNP variants were analyzed. In reindeer, seven PRNP alleles were observed and in red deer and moose two alleles were present, whereas roe deer (Capreolus capreolus) PRNP was monomorphic. One 'archetypal' PRNP allele associated with susceptibility was common to all four cervid species. The distribution of PRNP alleles differed between wild and semi-domesticated reindeer, with alleles associated with a high susceptibility occurring, on average, above 55% in wild reindeer and below 20% in semi-domesticated reindeer. This difference may reflect the diverse origins of the populations and/or selection processes during domestication and breeding. Overall, PRNP genetic data indicate considerable susceptibility to CWD among Norwegian cervids and suggest that PRNP homozygosity may be a risk factor for the atypical CWD observed in moose. The CWD isolates found in the Norwegian cervid species differ from those previously found in Canada and USA. Our study provides an overview of the PRNP genetics in populations exposed to these emerging strains that will provide a basis for understanding these strains' dynamics in relation to PRNP variability.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

High winter loads of Oestrid larvae and Elaphostrongylus rangiferi are associated with emaciation in wild reindeer calves.

The Oestrid flies Cephemyia trompe and Hypoderma tarandi and the nematode Elaphostrongylus rangiferi are important parasites of Rangifer spp. The larvae of Oestrid flies develop in the throat (C. trompe) and skin (H. tarandi) of their host during winter while E. rangiferi develop in the CNS. Oestrid pupation, and development of E. rangiferi larvae from first- (L1) to infective third- stage in the environment during summer are highly temperature dependent. We investigated the possible negative effects of these parasites on the winter body-condition of wild reindeer calves. Two year-classes (generations) of calf, born in a warm (2014) and cold (2015) summer respectively, were examined for changes in body condition between autumn and spring, in relation to the parasite load determined in the spring. The body condition in the autumn was assessed as carcass weight, while the body condition in the spring was assessed as carcass weight, supplemented by an evaluation of fat reserves in various bodily locations. Oestrids were counted directly whereas the E. rangiferi quantification was based on faecal counts of L1 larvae. The abundance of infections for Oestrids and E. rangiferi were significantly greater in the 2014 generation than in the 2015 generation. The mean carcass weight decreased between autumn and spring for the 2014 generation but increased in the 2015 generation. Emaciation in the spring was documented (fat reserve evaluation) in 42% and 7% of calves in the 2014 and 2015 cohorts, respectively. There was a significant correlation between high parasite load and the probability of emaciation. The mean summer temperature in 2014 was 2.6 °C higher than the mean for 2015, and 1.0 °C higher than the mean for the last 30-years. Our findings suggest that following a warm summer, high loads of Oestrids and E. rangiferi may cause emaciation and potentially deaths among the calves.

Distribution, prevalence and intensity of moose nose bot fly (Cephenemyia ulrichii) larvae in moose (Alces alces) from Norway.

High host density combined with climate change may lead to invasion of harmful parasites in cervid (host) populations. Bot flies (Diptera: Oestridae) are a group of ectoparasites that may have strong impact on their hosts, but data on the current distribution, prevalence and intensity of the moose nose bot fly (Cephenemyia ulrichii) in Scandinavia are lacking. We estimated prevalence and intensity of nose bot fly larvae in 30 moose from southern and 79 moose from central Norway. All larvae detected were identified as the moose nose bot fly. We found surprisingly high prevalence in these areas, which are up to 1300 km south-southwest of the first published location in Norway and west of the distribution in Sweden. Prevalence (0.44-1.00) was higher in areas with higher moose density. Parasite intensity in hunter killed moose was higher in central Norway (mean 5.7) than southern Norway (mean 2.9), and in both regions higher in calves and yearlings than adults. Fallen moose had higher parasite intensity (mean 9.8) compared to hunter killed moose in the subsample from central Norway, suggesting a link to host condition or behavior. Our study provides evidence of parasite range expansion, and establishing monitoring appears urgent to better understand impact on host populations.

Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America.

Chronic wasting disease (CWD) is a relentless epidemic disorder caused by infectious prions that threatens the survival of cervid populations and raises increasing public health concerns in North America. In Europe, CWD was detected for the first time in wild Norwegian reindeer (Rangifer tarandus) and moose (Alces alces) in 2016. In this study, we aimed at comparing the strain properties of CWD prions derived from different cervid species in Norway and North America. Using a classical strain typing approach involving transmission and adaptation to bank voles (Myodes glareolus), we found that prions causing CWD in Norway induced incubation times, neuropathology, regional deposition of misfolded prion protein aggregates in the brain, and size of their protease-resistant core, different from those that characterize North American CWD. These findings show that CWD prion strains affecting Norwegian cervids are distinct from those found in North America, implying that the highly contagious North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases. In addition, Norwegian CWD isolates showed an unexpected strain variability, with reindeer and moose being caused by different CWD strains. Our findings shed light on the origin of emergent European CWD, have significant implications for understanding the nature and the ecology of CWD in Europe, and highlight the need to assess the zoonotic potential of the new CWD strains detected in Europe.

Chronic wasting disease associated with prion protein gene (PRNP) variation in Norwegian wild reindeer (Rangifer tarandus).

The emergence of CWD in Europe in 2016 and the first natural infection in wild reindeer warranted disease management. This led to the testing of 2424 hunted or culled reindeer during 2016-2018, from the infected subpopulation in the Nordfjella mountain range in Southern Norway. To identify any association between PRNP variation and CWD susceptibility, we characterized the open reading frame of the PRNP gene in 19 CWD positive reindeer and in 101 age category- and sex-matched CWD negative controls. Seven variant positions were identified: 6 single nucleotide variants (SNVs) and a 24 base pair (bp) deletion located between nucleotide position 238 and 272, encoding four instead of five octapeptide repeats. With a single exception, all variant positions but one were predicted to be non-synonymous. The synonymous SNV and the deletion are novel in reindeer. Various combinations of the non-synonymous variant positions resulted in the identification of five PRNP alleles (A-E) that structured into 14 genotypes. We identified an increased CWD risk in reindeer carrying two copies of the most common allele, A, coding for serine in position 225 (Ser225) and in those carrying allele A together with the 24 bp deletion.

First Detection of Chronic Wasting Disease in a Wild Red Deer (Cervus elaphus) in Europe.

Chronic wasting disease (CWD) is a fatal contagious prion disease naturally occurring in cervids in North America. In 2016, CWD was detected in wild reindeer (Rangifer tarandus) and moose (Alces alces) in Norway. Here, we report the first known naturally infected wild Norwegian red deer (Cervus elaphus).

Novel Type of Chronic Wasting Disease Detected in Moose (Alces alces), Norway.

Chronic wasting disease (CWD) persists in cervid populations of North America and in 2016 was detected for the first time in Europe in a wild reindeer in Norway. We report the detection of CWD in 3 moose (Alces alces) in Norway, identified through a large scale surveillance program. The cases occurred in 13-14-year-old female moose, and we detected an abnormal form of prion protein (PrPSc) in the brain but not in lymphoid tissues. Immunohistochemistry revealed that the moose shared the same neuropathologic phenotype, characterized by mostly intraneuronal deposition of PrPSc. This pattern differed from that observed in reindeer and has not been previously reported in CWD-infected cervids. Moreover, Western blot revealed a PrPSc type distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. These findings suggest that these cases in moose represent a novel type of CWD.

Molecular identification of Sarcocystis halieti n. sp., Sarcocystis lari and Sarcocystis truncata in the intestine of a white-tailed sea eagle (Haliaeetus albicilla) in Norway.

An emaciated white-tailed sea eagle (Haliaeetus albicilla) from Western Norway was found and nursed briefly before it died. The necropsy revealed that the principal cause of death was an inflammation and occlusion of the bile ducts. A secondary finding was the presence in the intestinal mucosa of numerous sporulated Sarcocystis oocysts measuring 21.8-22.8 × 16.0-17.0 µm. The aim of this study was to identify these oocysts to species level using molecular methods. Genomic DNA was extracted from 10 mucosal scrapings containing oocysts and subjected to PCR amplification and sequencing of four DNA regions: the 18S and 28S rRNA genes, the ITS1 region and the cox1 gene. DNA of three previously known Sarcocystis spp. was identified, but only two of these, Sarcocystis halieti n. sp. and Sarcocystis lari, both employing sea birds as intermediate hosts, were considered to have used the sea eagle as a definitive host and to have formed oocysts in its intestine. The third species found, Sarcocystis truncata, employs red deer as intermediate hosts and seems to use felids as definitive hosts based on its phylogenetic position and prevalence. The sea eagle had probably recently ingested portions of one of the latter hosts (red deer or cat/lynx) containing stages (sarcocysts/oocysts) and thus DNA of S. truncata. The species S. halieti and S. lari could only be unambiguously separated from their most closely related congeners on the basis of their ITS1 sequences. This is the first report of Sarcocystis oocysts in sea eagles and the first identification to species level of Sarcocystis oocysts in any type of eagle. The sea eagle also acted as intermediate host of an unidentified Sarcocystis spp. as evidenced by the finding of six thin-walled sarcocysts in a histological section of cardiac muscle.

Yew (Taxus) intoxication in free-ranging cervids.

Wild ruminants, including deer species (cervids) have incorrectly been regarded as refractory to yew (Taxus) intoxication. This assumption has been based upon anecdotal observations of individual deer browsing on yew over time without apparent adverse effect. A single case of yew intoxication was reported in a free-ranging Norwegian moose (Alces alces) in 2008. The current report describes five additional cases of yew toxicosis in moose, seven in roe deer (Capreolus capreolus) and two in reindeer (Rangifer tarandus tarandus), all in Norway. The animals were found dead during the winter, close to or within gardens containing yew plants showing signs of browsing. Gross findings included lung congestion and edema, thoracic and pericardial effusion, bilateral heart dilatation, epi- and endocardial hemorrhage, and enlarged (congested) spleen. Yew plant remnants were detected in the rumen of all animals with the exception of a single moose. Histology revealed multifocal acute myocardial degeneration and necrosis with hemorrhage in roe deer, but not in the two other species. A qualitative high performance liquid chromatography-ion trap mass spectrometry analysis was used to tentatively identify five major Taxus alkaloids (taxines) in crude yew extracts and in heart and liver samples from the moose cases. All five major taxines were detected with good signal/noise ratio in tissue samples from the four moose with visible ruminal yew content, whereas lower levels of taxines were detected in the moose without visible ruminal yew content. Possible differences in interspecies tolerance to taxines and role of individual protective adaptation are discussed.

First detection of European bat lyssavirus type 2 (EBLV-2) in Norway.

BACKGROUND: In Europe, bat rabies is primarily attributed to European bat lyssavirus type 1 (EBLV-1) and European bat lyssavirus type 2 (EBLV-2) which are both strongly host-specific. Approximately thirty cases of infection with EBLV-2 in Daubenton's bats (Myotis daubentonii) and pond bats (M. dasycneme) have been reported. Two human cases of rabies caused by EBLV-2 have also been confirmed during the last thirty years, while natural spill-over to other non-flying mammals has never been reported. Rabies has never been diagnosed in mainland Norway previously. CASE PRESENTATION: In late September 2015, a subadult male Daubenton's bat was found in a poor condition 800 m above sea level in the southern part of Norway. The bat was brought to the national Bat Care Centre where it eventually displayed signs of neurological disease and died after two days. EBLV-2 was detected in brain tissues by polymerase chain reaction (PCR) followed by sequencing of a part of the nucleoprotein gene, and lyssavirus was isolated in neuroblastoma cells. CONCLUSIONS: The detection of EBLV-2 in a bat in Norway broadens the knowledge on the occurrence of this zoonotic agent. Since Norway is considered free of rabies, adequate information to the general public regarding the possibility of human cases of bat-associated rabies should be given. No extensive surveillance of lyssavirus infections in bats has been conducted in the country, and a passive surveillance network to assess rabies prevalence and bat epidemiology is highly desired.

Low Copper Levels Associated with Low Carcass Weight in Wild Red Deer ( Cervus elaphus ) in Norway.

Copper (Cu) deficiency is associated with several disease syndromes, including poor growth, in farmed red deer ( Cervus elaphus ), but little is known of the consequences of low Cu levels in free-ranging populations. Low hepatic Cu levels have been documented in several wild red deer populations along the west coast of Norway, with the lowest values found on the island of Hitra. We studied the relationship between liver Cu concentration and slaughter weight in 63 red deer calves and 69 yearlings shot on Hitra during the autumn hunting season of 2009. Less than half of each age class had adequate Cu levels (>20 µg/g dry weight) and 14% of the calves and 28% of the yearlings had levels indicating deficiency (<13 µg/g). For yearlings, but not calves, there was a significant increase in slaughter weight with increasing hepatic Cu level. The differences between yearlings and calves could be linked to differences in physiologic Cu status of the two age classes, in that red deer calves are born with much higher levels of hepatic Cu than are their mothers. Our data demonstrate an association between low levels of Cu and reduced growth rate in a free-ranging cervid population.

First case of chronic wasting disease in Europe in a Norwegian free-ranging reindeer.

Chronic wasting disease (CWD) is a fatal contagious prion disease in cervids that is enzootic in some areas in North America. The disease has been found in deer, elk and moose in the USA and Canada, and in South Korea following the importation of infected animals. Here we report the first case of CWD in Europe, in a Norwegian free-ranging reindeer in Southern Norway. The origin of the disease is unknown. Until now a low number of cervids, and among them a few reindeer, have been tested for CWD in Norway. Therefore the prevalence of CWD is unknown.

A geographic cluster of malignant catarrhal fever in moose (Alces alces) in Norway.

Three cases of lethal sheep-associated malignant catarrhal fever (SA-MCF) in free-ranging moose (Alces alces) were diagnosed in Lesja, Norway, December 2008-February 2010. The diagnosis was based on PCR identification of ovine herpesvirus 2 (OvHV-2) DNA (n = 3) and typical histopathologic lesions (n = 1). To study the possibility of subclinical or latent MCF virus (MCFV) infection in this moose population and in red deer (Cervus elaphus), we examined clinically normal animals sampled during hunting in Lesja 2010 by serology and PCR. Sera from 63 moose and 33 red deer were tested for antibodies against MCFV by competitive-inhibition enzyme-linked immunosorbent assay. To test for MCFVs, a consensus PCR for herpesviral DNA was run on spleen samples from 23 moose and 17 red deer. All samples were antibody and PCR negative. Thus, there is no evidence of previous exposure, subclinical infection, or latent infection in this sample. This seasonal cluster of SA-MCF cases (2008-10) may be attributable to exposure of moose to lambs when OvHV-2 shedding is presumed to be maximal, compounded by an unusual extended grazing period by sheep in the autumn.

Resurrection and redescription of Varestrongylus alces (Nematoda: Protostrongylidae), a lungworm of the Eurasian moose (Alces alces), with report on associated pathology.

BACKGROUND: Varestrongylus alces, a lungworm in Eurasian moose from Europe has been considered a junior synonym of Varestrongylus capreoli, in European roe deer, due to a poorly detailed morphological description and the absence of a type-series. METHODS: Specimens used in the redescription were collected from lesions in the lungs of Eurasian moose, from Vestby, Norway. Specimens were described based on comparative morphology and integrated approaches. Molecular identification was based on PCR, cloning and sequencing of the ITS-2 region of the nuclear ribosomal DNA. Phylogenetic analysis compared V. alces ITS-2 sequences to these of other Varestrongylus species and other protostrongylids. RESULTS: Varestrongylus alces is resurrected for protostrongylid nematodes of Eurasian moose from Europe. Varestrongylus alces causes firm nodular lesions that are clearly differentiated from the adjacent lung tissue. Histologically, lesions are restricted to the parenchyma with adult, egg and larval parasites surrounded by multinucleated giant cells, macrophages, eosinophilic granulocytes, lymphocytes. The species is valid and distinct from others referred to Varestrongylus, and should be separated from V. capreoli. Morphologically, V. alces can be distinguished from other species by characters in the males that include a distally bifurcated gubernaculum, arched denticulate crura, spicules that are equal in length and relatively short, and a dorsal ray that is elongate and bifurcated. Females have a well-developed provagina, and are very similar to those of V. capreoli. Morphometrics of first-stage larvae largely overlap with those of other Varestrongylus. Sequences of the ITS-2 region strongly support mutual independence of V. alces, V. cf. capreoli, and the yet undescribed species of Varestrongylus from North American ungulates. These three taxa form a well-supported crown-clade as the putative sister of V. alpenae. The association of V. alces and Alces or its ancestors is discussed in light of host and parasite phylogeny and host historical biogeography. CONCLUSIONS: Varestrongylus alces is a valid species, and should be considered distinct from V. capreoli. Phylogenetic relationships among Varestrongylus spp. from Eurasia and North America are complex and consistent with faunal assembly involving recurrent events of geographic expansion, host switching and subsequent speciation.

Mycoplasma ovipneumoniae--a primary cause of severe pneumonia epizootics in the Norwegian Muskox (Ovibos moschatus) population.

The Norwegian muskox (Ovibos moschatus) population lives on the high mountain plateau of Dovre and originates from animals introduced from Greenland. In the late summers of 2006 and 2012, severe outbreaks of pneumonia with mortality rates of 25-30% occurred. During the 2012 epidemic high quality samples from culled sick animals were obtained for microbiological and pathological examinations. High throughput sequencing (pyrosequencing) of pneumonic lung tissue revealed high concentrations of Mycoplasma ovipneumoniae in all six animals examined by this method and Pasteurella multocida subsp. multocida in four animals, whereas no virus sequences could be identified. Mycoplasma ovipneumoniae and P. multocida multocida were also isolated by culture. Using real time PCR on lung swabs, M. ovipneumoniae was detected in all of the 19 pneumonic lungs examined. Gross pathological examination revealed heavy consolidations primarily in the cranial parts of the lungs and it also identified one case of otitis media. Histologically, lung lesions were characterized as acute to subacute mixed exudative and moderately proliferative bronchoalveolar pneumonia. Immunohistochemical (IHC) examination revealed high load of M. ovipneumoniae antigens within lung lesions, with particularly intensive staining in the neutrophils. Similar IHC finding were observed in archived lung tissue blocks from animals examined during the 2006 epidemic. An M. ovipneumoniae specific ELISA was applied on bio-banked muskox sera from stray muskoxen killed in the period 2004-2013 and sick muskoxen culled, as well as sera from wild reindeer (Rangifer tarandus tarandus) on Dovre and muskoxen from Greenland. Serology and mycoplasma culturing was also carried out on sheep that had been on pasture in the muskox area during the outbreak in 2012. Our findings indicated separate introductions of M. ovipneumoniae infection in 2006 and 2012 from infected co-grazing sheep. Salt licks shared by the two species were a possible route of transmitting infection.

Acute fatal toxoplasmosis in a Great Spotted Woodpecker (Dendrocopos major).

A juvenile male Great Spotted Woodpecker (Dendrocopos major), found dead at a bird feeding station in central Norway in September 2011, was examined postmortem. Its lungs were consolidated and edematous, and its spleen was enlarged. The main histopathologic diagnoses included acute protozoal necrotizing interstitial pneumonia, splenitis, and hepatitis. Toxoplasma gondii parasites were identified with immunohistochemistry in all examined organs: lung, heart, liver, kidney, and spleen. Direct multilocus genotyping of the parasites revealed that the woodpecker was killed by a T. gondii strain belonging to genotype II. This is the first report of naturally acquired fatal generalized toxoplasmosis in a Dendrocopos species.

Malignant catarrhal fever virus identified in free-ranging musk ox (Ovibos moschatus) in Norway.

To study the epizootiology of malignant catarrhal fever viruses (MCFV), serum and spleen samples collected in 2004-2011 from a free-ranging musk ox (Ovibos moschatus) population in Dovrefjell, Norway, were examined. Sera were tested for antibodies against MCFV by competitive enzyme-linked immunosorbent assay and spleen samples were examined by a consensus polymerase chain reaction (PCR) for herpesviral DNA and sequencing identification. The study included 101 musk oxen, of which 61 were examined with both tests. Antibodies against MCFV were found in 65 of 72 musk oxen (90%). Antibody prevalence increased with age from 67% in calves to 96% in adults. We detected MCFV DNA by consensus PCR in 67 of 90 spleen samples tested (74%). The prevalence of PCR-positive musk oxen increased with age from 60% in calves to 81% in adults. Fifty (82%) of the 61 animals subjected to both PCR and serology were positive in both tests. Sequencing analysis showed that all PCR-positive animals were infected with a MCFV previously identified in musk oxen in North America. The results suggest that MCFV-Muskox is enzootic in the Dovrefjell musk ox population and that many calves seem to be infected early in life. This is the first report of MCFV-Muskox infection in free-ranging musk ox outside North America.

Ixodes ricinus infestation in free-ranging cervids in Norway--a study based upon ear examinations of hunted animals.

Prevalence, abundance and instar composition of Ixodes ricinus as found on one ear collected from 1019 moose (Alces alces), red deer (Cervus elaphus) and roe deer (Capreolus capreolus), shot during hunting (August-December) 2001-2003, are reported. The animals originated from 15 coastal municipalities (CM), seven municipalities bordering to coastal municipalities (BCM) and four inland municipalities (IM), in Norway, between latitudes 58-66° N. I. ricinus occurred endemically in all CM and BCM up to 63°30' N, whereas it was non-endemic further north and in the IM. This geographical distribution of the tick along the coast of southern Norway was largely in accordance with that reported as far back as the 1940s. Our results therefore did not indicate any large scale northwards expansion of I. ricinus in Norway during the 60 year-period between the two studies. However, the prevalence of infestation and tick abundance were significantly higher in CM as compared to BCM. The prevalence and abundance by month were highest during August and September, gradually decreasing towards December. The considerable prevalence of ticks in November, as well as findings in December, would seem to indicate a prolonged tick season as compared with the studies carried out 60 years ago. A total of 8920 ticks were isolated from 439 of the 603 animals examined in endemic municipalities, and the maximum number of ticks found on one single ear was 204. Attached adult ticks were primarily found among the long hairs at base of the ear, whereas nymphs and larvae were seen all over the outer surface of the pinna, for larvae especially at the edge and tip of the ear. Nymphs were the dominant instar, constituting 74% of the total tick count. The proportion of larvae and adult ticks was 13% and 12%. A significantly higher proportion of adult ticks and lower proportion of immature stages were found in moose, as compared to red deer and roe deer. The same apparently size-associated preference of adult ticks was also found for adult animals (all species) as compared to calves. Other grossly detected ectoparasites included the lice Solenopotes burmeisteri in red deer and Damalinia meyeri in roe deer, and the deer ked fly, Lipoptena cervi, in moose and roe deer. This is believed to be the first systematic study on the instar composition by I. ricinus infestation in free-ranging cervids. The examination of ears from hunted cervids should be recognized as a rational way of obtaining data on the geographical distribution and abundance of this tick in nature.