Torill Mørk

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.

Intestinal parasites of the Arctic fox in relation to the abundance and distribution of intermediate hosts.

The intestinal parasite community of Arctic foxes (Vulpes lagopus) on the Svalbard archipelago in the High Arctic was investigated in relation to the abundance and distribution of intermediate hosts. Five species of cestodes (Echinococcus multilocularis, Taenia crassiceps, Taenia polyacantha, Taenia krabbei and Diphyllobothrium sp.), ascaridoid nematodes and one unidentified acanthocephalan species were found. The cestodes E. multilocularis, T. crassiceps and T. polyacantha all showed a decreasing prevalence in the fox population with increasing distance from their spatially restricted intermediate host population of sibling voles (Microtus levis). In addition, the prevalence of E. multilocularis in a sample from the vole population was directly related to the local vole abundance. The cestode T. krabbei uses reindeer as intermediate host, and its prevalence in female foxes was positively related to the density of reindeer (Rangifer tarandus platyrhyncus). Finally, the prevalence of the ascaridoid nematodes also decreased with increasing distance from the vole population, a finding that is consistent with the idea that voles are involved in transmission, most likely as paratenic hosts. The prevalence of the remaining species (Diphyllobothrium sp. and an unidentified acanthocephalan) was very low. We conclude that the distribution and abundance of intermediate host structure the gastrointestinal parasite community of the Arctic fox on the Svalbard archipelago.

Arctic Rabies – A Review

Rabies seems to persist throughout most arctic regions, and the northern parts of Norway, Sweden and Finland, is the only part of the Arctic where rabies has not been diagnosed in recent time. The arctic fox is the main host, and the same arctic virus variant seems to infect the arctic fox throughout the range of this species. The epidemiology of rabies seems to have certain common characteristics in arctic regions, but main questions such as the maintenance and spread of the disease remains largely unknown. The virus has spread and initiated new epidemics also in other species such as the red fox and the racoon dog. Large land areas and cold climate complicate the control of the disease, but experimental oral vaccination of arctic foxes has been successful. This article summarises the current knowledge and the typical characteristics of arctic rabies including its distribution and epidemiology.SammendragArktisk rabies - en oversikt.Rabies synes å opprettholdes i de fleste arktiske regioner, og de nordlige deler av Norge, Sverige og Finland er de eneste områder av Arktis hvor rabies ikke har vært diagnostisert i nyere tid. Fjellreven er hovedvert og ser ut til å være infisert med den samme virusvarianten i hele sitt utbredelsesområde. Epidemiologien i arktiske områder synes å ha enkelte fellestrekk, men svar på essensielle spørsmål slik som opprettholdelse og spredning av sykdommen, er fortsatt ukjent. Spredning av viruset har forårsaket nye epidemier også hos andre arter som rødrev og mårhund. Store landområder og kaldt klima kompliserer kontroll av sykdommen, men eksperimentell oral vaksinasjon av fjellrev har vært vellykket. Artikkelen summerer opp typiske egenskaper ved arktisk rabies samt utbredelse og epidemiolo.

Cervid Herpesvirus 2, the Primary Agent in an Outbreak of Infectious Keratoconjunctivitis in Semidomesticated Reindeer

ABSTRACT An outbreak of infectious keratoconjunctivitis (IKC) occurred in semidomesticated reindeer (Rangifer tarandus tarandus) in Troms County, Norway, in February 2009. Twenty-eight animals with clinical symptoms and 12 apparently healthy animals were investigated. They ranged in age from calves of the year to 4-year-old animals (mean, 1.9 years; standard deviation, ±0.9). The seroprevalence of antibodies against cervid herpesvirus 2 (CvHV2) was 86% in animals with IKC and 42% in unaffected animals. For the 28 clinically affected animals, CvHV2 was detected by PCR in swabs obtained from the eye (82%), nose (64%), and vagina (24%), and CvHV2 was isolated from eye swabs from 8 animals. Virus was not isolated from clinically unaffected animals but was detected by PCR in eye swab samples from five of them. The viral activity, assessed by the ability to cause a cytopathic effect in cell culture, increased with the severity of clinical symptoms, but in severe clinical cases, virus was absent and secondary bacterial infections were dominant. Moraxella sp. isolates were obtained from seven animals, and those from two animals were identified as Moraxella bovoculi. Staphylococcus aureus, Streptococcus sp., and Arcanobacterium pyogenes were also isolated. It is concluded that CvHV2, which is endemic in reindeer, can cause IKC, probably most commonly as a primary infection of calves. This can be a very painful and devastating disease of economic importance for reindeer herders. This is the first report of CvHV2 as the primary agent of IKC in reindeer. This is also the first isolation of this virus in reindeer under natural herding conditions.

Fatal inanition in reindeer (Rangifer tarandus tarandus): Pathological findings in completely emaciated carcasses

BackgroundIn a project to determine the causes of winter mortality in reindeer in Finnmark County, northern Norway, the most frequent diagnosis turned out to be complete emaciation, despite several of the reindeer having been given silage for up to 4 weeks before they died. The present paper describes autopsy results and other findings in these animals.MethodsAutopsies were made of 32 reindeer carcasses, and 28 of these were diagnosed as completely emaciated based on lack of visible fat and serous atrophy of subepicardial and bone marrow fat. Other investigations of the carcasses included histology, bacteriology, parasitology (counting of macro parasites and faecal egg counting), analysis of vitamin E and selenium in liver, chemical and botanical analysis of rumen content, analysis of lipid content in femur bone marrow and estimation of muscle atrophy by use of a muscle index.ResultsMain findings were: Low carcass weight, severe muscle atrophy, hemosiderosis in liver and spleen, subcutaneous oedema (18%) and effusions to body cavities (18%). Two types of lipofuscin granula were identified in the liver: One type occurred in liver endothelial cells of all carcasses, while the other type occurred in hepatocytes, and prevailed in adult animals. Abomasal haemorrhages, consistent with previously described stress lesions, was present in 68% of the carcasses. Diarrhoea occurred in 2 cases, and loose faecal consistency was associated with silage feeding. Rumen content was low in crude protein. Grass dominated rumen content in silage-fed carcasses, while reindeer on natural pastures had mainly woody plants, mosses and litter in rumen. Stem dominated the grass fraction in rumens with high grass content, indicating ruminal indigestion as a cause of emaciation in silage fed animals. Some cases had heavy infestation of parasites such as warble fly larvae (Hypoderma tarandi), throat bot larvae (Cephenemyiae trompe) and lung nematodes.ConclusionLack of appropriate amounts and/or appropriate quality of feed has been the main cause of emaciation, though heavy infestation of parasites may have contributed to the emaciation in some cases.

Evidence of parapox-, alphaherpes- and pestivirus infections in carcasses of semi-domesticated reindeer (Rangifer tarandus tarandus) from Finnmark, Norway

During March to May 2000, 48 carcasses of semi-domesticated reindeer (Rangifer tarandus tarandus) were collected on winter pastures and calving grounds from two herds in western Finnmark and two herds in eastern Finnmark, northern Norway. The animals were autopsied and blood and tissue samples were collected for serology (alphaherpes- and pestivirus; virus neutralization test) and polymerase chain reaction (PCR; parapoxvirus; B2L gene) investigations. Autopsy revealed that 39 of 48 animals (81%) had died of emaciation. Parapoxvirus-specific DNA was detected in samples from 6 of 48 animals (12.5%; liver, parotid salivary gland and/or pulmonary lymph nodes). A DNA sequence of 376 base pairs from a PCR amplicon obtained from a liver sample from one animal showed 98-99% identity with orf virus strain Orf-11 and reindeer parapoxvirus isolates from Norway and Finland (1992 and 1994), 92-93% similarity with pseudocowpoxvirus and 87% similarity with bovine papular stomatitis virus. Alphaherpes- and pestivirus antibodies were detected in 10% and 33% of the animals, respectively. These results indicates that parapoxvirus, presumably orf-virus, is present among reindeer also in Finnmark, although contagious ecthyma has never been reported in reindeer in this important reindeer herding area. Furthermore, they show that herpes- and pestiviruses are still endemic in reindeer herds in Finnmark. The nature of these viruses and their impact on reindeer health and reproduction and reindeer herding economy should be further addressed, as well as the possibility that these viruses may be transferred between reindeer and domestic animals in this region. Abstract in Norwegian / Sammendrag: I lopet av perioden mars-mai 2000 ble 48 reinsdyrkadavre (Rangifer tarandus tarandus) samlet inn fra vinterbeiter og kalvingsomrader fra to flokker i Vest-Finnmark og to i Ost-Finnmark, Norge. Dyrene ble obdusert, og blod og vevsprover ble samlet for pavisning av antistoffer mot alfaherpes- og pestivirus i blod(serologi) og tilstedevaerelse av parapoxvirus-DNA i vev (Polymerase kjedereaksjon, PCR; parapoxvirus B2L genet). Obduksjonen viste at 39 av de 48 dyrene (81%) hadde dodd av avmagring. Parapoxvirus-spesifikt DNA ble funnet i prover av lever, spyttkjertel (Gl. parotis) og/eller lungelymfeknuter fra 6 av de 48 dyrene (12,5%). En DNA sekvens pa 376 basepar fra PCR-oppformeringsproduktet fra en leverprove hadde 98-99% likhet med orf-virus (Orf-11) og parapoxvirus isolert fra reinsdyr i Norge og Finland (1992 og 1994), 92-93% likhet med pseudocowpoxvirus og 87% likhet med bovint papulaer stomatittvirus, hvorav de to siste parapoxvirusartene er assosiert med storfe. Disse resultatene viser at ogsa reinsdyr i Finnmark er infisert av parapoxvirus, til tross for at sykdommen munnskurv ikke enna er rapportert hos rein i dette fylket. Alfaherpes- og pestivirus antistoffer ble funnet hos henholdsvis 10% og 33% av dyrene. Dette er i samsvar med tidligere funn pa slaktedyr, og viser at disse virusinfeksjonene er endemiske hos rein i Finnmark. Det er viktig a avklare hvilken rolle disse virusinfeksjonene spiller for reinsdyrenes helse og naeringens okonomi. Videre bor det avklares i hvilken grad disse virustypene er i stand til a smitte mellom rein og husdyr.

Cervid herpesvirus 2 experimentally reactivated in reindeer can produce generalized viremia and abortion.

Cervid herpesvirus 2 (CvHV2) has never been isolated from reindeer in Norway, but serological data and investigations by PCR indicate that the virus is endemic in the country, with horizontal and vertical transmission, systemic spread, and latency in the trigeminal ganglion. In this study two seropositive reindeer, one of which was pregnant, were administered dexamethasone, to reactivate CvHV2 latent infection. One control animal received sterile water. All animals including the control reactivated, as shown by amplification of CvHV2 DNA from nasal swabs. The pregnant animal showed lesions in the lip mucosa 10 days after the first dexamethasone injection and CvHV2 was visualized by electron microscopy and isolated from those lesions, as well as from nasal and vaginal swabs. On day 13 she aborted and CvHV2 was isolated from both the aborted calf and the mother. CvHV2 was isolated from the other animal administered dexamethasone. Despite amplification of viral DNA in the control animal, it was never possible to isolate the virus. Molecular characterization of the new isolates confirmed these to be CvHV2, and similar to the previous known strain Salla82. Present results represent the first isolation of CvHV2 in Norway and reconfirm that this virus can cause systemic infections in reindeer even after reactivation episodes, and infect the fetus in utero despite a prompt immune response. While it is not possible to atribute the abortion to CvHV2 alone, present data together with previous reports of vertical transmission of CvHV2 and neonatal death, point to an abortogenic potential, which should be further investigated.

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.

Experimental Infection of Reindeer with Cervid Herpesvirus 2

ABSTRACT Cervid herpesvirus 2 (CvHV2) has been isolated from reindeer (Rangifer tarandus tarandus), and serological data indicate that in reindeer this virus is endemic in Fennoscandia, Alaska, Canada, and Greenland. CvHV2 has been described as a cause of subclinical genital infections in reindeer, but little information on primary infections exists. In this study, six seronegative and presumably pregnant reindeer were allocated to one of two groups. Two animals were inoculated with CvHV2 intratracheally, and two animals intravaginally, with one control animal in each group receiving sterile water. Mild hyperthermia and serous discharges from the vagina and nose were observed. No abortions were recorded, but one calf died shortly after birth. Inoculated animals seroconverted and had neutralizing antibodies after days 7 to 10 postinfection. CvHV2 was detected by PCR in nasal and vaginal swabs from animals in both groups but could be isolated only from nasal swabs in the respiratory group and from vaginal swabs in the genital group. CvHV2 was detected by PCR in various organs and tissues postmortem. In control animals, the virus could not be isolated in spite of PCR-positive nasal and vaginal swab samples and some degree of positive immunostaining. One of the animals that were inoculated intratracheally developed a hemorrhagic, necrotizing bronchopneumonia, which was CvHV2 positive by PCR and immunohistochemistry. We conclude that CvHV2 can cause systemic infection, that both genital and respiratory inoculations can lead to virus shedding, and that the virus can infect the fetus in utero.

A screening for canine distemper virus, canine adenovirus and carnivore protoparvoviruses in Arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) from Arctic and sub-Arctic regions of Norway

ABSTRACT Canine distemper virus (CDV), canine adenovirus (CAdV) and canine parvovirus type 2 (CPV-2) cause disease in dogs (Canis familiaris). These, or closely related viruses, may also infect wild carnivores. The aim of this study was to investigate exposure to CDV, CAdV and CPV-2 among fox populations in Norway. Arctic foxes (n = 178) from High-Arctic Svalbard were investigated for antibodies against CDV. Arctic foxes (n = 301) from Svalbard and red foxes from Low-Arctic (n = 326) and sub-Arctic (n = 74) regions in Finnmark County, Norway, were investigated for antibodies against CAdV and for the presence of carnivore protoparvovirus DNA in spleen and mesenteric lymph nodes using polymerase chain reaction. Seroprevalence against CDV in Arctic foxes decreased from 25% (1995/96) to 6% (2001/02), whereas the seroprevalence against CAdV increased from 25–40% during the seasons 1995/96 to 2001/02 to 68% for the last study year (2002/03). In red foxes, the seroprevalence against CAdV varied between 31% and 67% for the seasons 2004/05 to 2007/08, increasing to 80% for the last study year. Carnivore protoparvovirus DNA was not detected in any of the 301 Arctic foxes and the 265 red foxes investigated. These results show that CDV and CAdV are enzootic in the Arctic fox population (Svalbard), and that CAdV is enzootic in both the Low-Arctic and sub-Arctic red fox populations (Finnmark). Further studies are needed to better understand the infection biology and the impact of CDV and CAdV in these fox populations, and if viruses may be shared between foxes and other carnivores, including dogs.