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.

Infectious keratoconjunctivitis in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus): microbiological study of clinically affected and unaffected animals with special reference to cervid herpesvirus 2.

BACKGROUND: Infectious keratoconjunctivitis (IKC) is one of the most common ocular diseases in ruminants worldwide. In addition to keratitis and conjunctivitis, animals with IKC can develop uveitis, corneal ulcer, and in severe cases, blindness. The bacteria Moraxella spp. has been described as the primary causative agent of infectious bovine keratoconjunctivitis (IBK) in cattle (Bos taurus), while Chlamydia spp. and Mycoplasma conjunctivae are considered the main causative agents of IKC in sheep (Ovis aries). Previous studies indicated cervid herpesvirus 2 (CvHV2) as the primary causative agent of IKC in semi-domesticated reindeer (Rangifer tarandus tarandus). The aim of the study was to investigate the presence and prevalence of potential pathogens for IKC in reindeer, and compare the ocular microbiota of animals with IKC, with apparently healthy animals. RESULTS: Semi-domesticated reindeer (n = 341), with (n = 108) or without (n = 113) ocular clinical signs, or with no information on clinical status (n = 120), were sampled in Norway, Sweden and Finland in 2010-2014. Seroprevalence was 37.4% for alphaherpesvirus (95/254), 3.8% for gammaherpesvirus (8/211) and 7.1% for pestivirus (15/211) (ELISA). PCR analyses of conjunctival swab samples revealed a prevalence of 28.5% for CvHV2 (57/200), 11.9% for Chlamydiaceae (16/135) and 1.0% for M. conjunctivae (2/197). Bacteriological cultivation of 202 conjunctival swab samples revealed bacterial growth from 75.2% of the samples, with Moraxella spp. being isolated from 21.6% (11/51) of the animals with and 5.6% (5/84) without ocular clinical signs. A significant association (p < 0.001) existed between the presence of clinical signs of IKC and CvHV2 DNA in the affected eyes, an association that was not present for other microorganisms. CONCLUSIONS: These results support the hypothesis that CvHV2 is the primary agent of IKC in semi-domesticated reindeer in Fennoscandia, with Moraxella bovoculi being a secondary candidate, since it was isolated in two different outbreaks of IKC. Further studies should be carried out to better understand the infection biology and the pathogenesis of IKC in reindeer.

First isolation of Brucella pinnipedialis and detection of Brucella antibodies from bearded seals Erignathus barbatus.

Brucella species infecting marine mammals was first reported in 1994 and in the years since has been documented in various species of pinnipeds and cetaceans. While these reports have included species that inhabit Arctic waters, the few available studies on bearded seals Erignathus barbatus have failed to detect Brucella infection to date. We report the first isolation of Brucella pinnipedialis from a bearded seal. The isolate was recovered from the mesenteric lymph node of a bearded seal that stranded in Scotland and typed as ST24, a sequence type associated typically with pinnipeds. Furthermore, serological studies of free-ranging bearded seals in their native waters detected antibodies to Brucella in seals from the Chukchi Sea (1990-2011; 19%) and Svalbard (1995-2007; 8%), whereas no antibodies were detected in bearded seals from the Bering Sea or Bering Strait or from captive bearded seals.

Age-dependent prevalence of anti-Brucella antibodies in hooded seals Cystophora cristata.

Investigations of hooded seals Cystophora cristata have revealed high prevalences of Brucella-positive seals in the reduced Northeast Atlantic stock, compared to the increasing Northwest Atlantic stock. This study evaluated the relation between Brucella-serostatus in seals in the Northeast Atlantic stock and age, sex, body condition and reproduction. Bacteriology documented which animals and organs were B. pinnipedialis positive. No relationship was observed between Brucella-serostatus and body condition or reproductive traits. Pups (<1 mo old) had a substantially lower probability of being seropositive (4/159, 2.5%) than yearlings (6/17, 35.3%), suggesting that exposure may occur post-weaning, during the first year of life. For seals >1 yr old, the mean probability of being seropositive decreased with age, with no seropositives older than 5 yr, indicating loss of antibody titre with either chronicity or clearance of infection. The latter explanation seems to be most likely as B. pinnipedialis has never been isolated from a hooded seal >18 mo old, which is consistent with findings in this study; B. pinnipedialis was isolated from the retropharyngeal lymph node in 1 seropositive yearling (1/21, 5%). We hypothesize that this serological and bacteriological pattern is due to environmental exposure to B. pinnipedialis early in life, with a subsequent clearance of infection. This raises the question of a reservoir of B. pinnipedialis in the hooded seal food web.

Serological screening for tick-borne encephalitis virus in eight Norwegian herds of semi-domesticated reindeer (Rangifer tarandus tarandus).

Tick-borne encephalitis virus (TBEV) is found in Ixodes ricinus ticks throughout the area where viable tick populations exist. In Norway, TBEV is found in I. ricinus from the south coast until Brønnøy municipality in Nordland County and the range of the vector is expanding due to changes in climate, vegetation, host animals and environmental conditions. TBEV might thus have the potential to establish in new areas when I. ricinus expand its geographical distribution. At present, there is little knowledge on the status of the virus in high-altitude areas of inland regions in Norway. It has previously been indicated that reindeer may be an important sentinel species and indicator of the spread of ticks and TBEV in high-altitude regions. In this study, 408 semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus) from eight herds, from Tana in Troms and Finnmark County in northern Norway to Filefjell in Innlandet and Viken Counties in southern Norway, were screened for TBEV antibodies using a commercial enzyme-linked immunosorbent assay (ELISA). We found 16 TBEV reactive reindeer samples by ELISA; however, these results could not be confirmed by the serum neutralization test (SNT). This could indicate that a flavivirusand not necessarily TBEV, may be circulating among Norwegian semi-domesticated reindeer. The results also indicate that TBEV was not enzootic in Norwegian semi-domesticated reindeer in 2013-2015. This knowledge is important as an information base for future TBEV and flavivirus surveillance in Norway.

Evidence of alphaherpesvirus infections in Alaskan caribou and reindeer.

BACKGROUND: The reindeer (Rangifer tarandus tarandus) industry in Alaska began with animals imported from Siberia (Russia) in the 1890's. Cervid herpes virus 2 (CvHV2) is endemic in reindeer in Scandinavia. We sought to determine if the same virus, or similar herpesviruses, were circulating in Alaskan reindeer and caribou (Rangifer tarandus granti). Serum samples from 292 reindeer were collected during annual reindeer handlings (1988-2005) near Nome, Alaska. In 2005, swab samples were collected from 40 calves from this herd, near Nome, Alaska. In 2007, ocular and nasal swab samples were collected from 30 apparently healthy reindeer calves near Wales, Alaska. Samples of plasma and white blood cells were collected from three Alaskan caribou herds, Mulchatna (n = 24), Teshekpuk (n = 34) and the Western Arctic (n = 87) in 2009. RESULTS: Of 292 reindeer samples tested by ELISA for antibodies against alphaherpesvirus (bovine herpesvirus 1 as antigen), seroprevalence was 47% (136/292) and adult reindeer had higher seroprevalence than yearlings. The overall seroprevalence for caribou was 60% (87/145), with no significant differences among caribou herds. A virus neutralization test of 20 samples from both reindeer and caribou showed that ELISA positive samples always neutralized CvHV2 to a greater extent than BoHV1 or elk herpesvirus (ElkHV), indicating that CvHv2 is the most likely virus circulating. PCR of nasal and ocular swabs sampled from 30 reindeer calves in Wales, Alaska (2007) yielded four CvHV2 positive samples. PCR amplicons of the expected size (294 bp) were obtained from 2 of the 36 buffy coats samples from caribou, and the amplicon sequences were consistent with CvHV2. CONCLUSIONS: This study shows that Alaskan reindeer and Caribou are infected with an alphaherpesvirus. Based on sequence similarity, CvHV-2 is the most likely virus. Further studies should be conducted to determine the impact of this infection on the health of these animals.

Pathogen Exposure in White Whales (Delphinapterus leucas) in Svalbard, Norway.

The Svalbard white whale (Delphinapterus leucas) population is one of the smallest in the world, making it particularly vulnerable to challenges such as climate change and pathogens. In this study, serum samples from live captured (2001−2016) white whales from this region were investigated for influenza A virus (IAV) antibodies (Abs) (n = 27) and RNA (n = 25); morbillivirus (MV) Abs (n = 3) and RNA (n = 25); Brucella spp. Abs; and Toxoplasma gondii Abs (n = 27). IAV Abs were found in a single adult male that was captured in Van Mijenfjorden in 2001, although no IAV RNA was detected. Brucella spp. Abs were found in 59% of the sample group (16/27). All MV and T. gondii results were negative. The results show that Svalbard white whales have been exposed to IAV and Brucella spp., although evidence of disease is lacking. However, dramatic changes in climate and marine ecosystems are taking place in the Arctic, so surveillance of health parameters, including pathogens, is critical for tracking changes in the status of this vulnerable population.

A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata).

Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population.

Infectious Disease Outbreak Associated With Supplementary Feeding of Semi-domesticated Reindeer.

Supplementary winter feeding of semi-domesticated reindeer (Rangifer tarandus tarandus) has become more common in Sweden and Norway due to reindeer pasture fragmentation and climatic conditions. With increased corralling and feeding, often associated with animal stress, increased animal-to-animal contact, and poor hygienic conditions, an altered range of health challenges and diseases may emerge. An outbreak of three different infectious diseases appeared simultaneously in a reindeer herd in Norrbotten County, Sweden. The animals were corralled and fed silage. Several animals in poor body condition stopped eating, with drool and discoloration of the hair coat around the mouth. There were large, black, necrotic lesions on the tongue and gingiva, with holes perforating the chin, indicative of oral necrobacillosis and Fusobacterium spp. infection. Simultaneously, animals were seen with proliferative lesions in the oral mucosa and on the lips, characteristic of contagious ecthyma and Orf virus infection. Furthermore, three animals had keratoconjunctivitis suggesting exposure to cervid herpesvirus 2 (CvHV2) and possibly secondary bacterial infections. DNA specific for Fusobacterium necrophorum and ORFV was detected in relevant tissue samples. Antibodies against CvHV2 were detected in 10 of 13 diseased and in four of 11 apparently healthy reindeer. Nine animals were found dead or were euthanized during the outbreak. Health risk factors associated with feeding and corralling may severely impact animal welfare and the herder's economy, and may represent an underestimated cost when replacing natural grazing with feeding.

HEALTH SURVEY OF BOREAL CARIBOU (RANGIFER TARANDUS CARIBOU) IN NORTHEASTERN BRITISH COLUMBIA, CANADA.

Boreal woodland caribou (Rangifer tarandus caribou) are listed as threatened across Canada, and a basic understanding of their health status is lacking. From December 2012 to April 2013, we investigated multiple health indices for adult female boreal caribou (n=163) captured from seven herds in NE British Columbia, Canada. Health indices included physical characteristics, physiologic and trace mineral status, exposure to or infection with selected pathogens, and measures of chronic stress and inflammation, including serum amyloid A, haptoglobin, and hair cortisol concentration. Key findings were exposure to the bacterium Erysipelothrix rhusiopathiae in 14% of individuals, mild to severe hair loss associated with winter tick (Dermacentor albipictus) infestations in 76% of caribou from December to early February and 81% from late February to early April, and evidence of trace mineral deficiencies with 99% and 34% of individuals deficient in copper and selenium, respectively. Seroprevalence for exposure to selected pathogens was: alphaherpesvirus (63%), pestivirus (1%), Besnoitia spp. (60%), and Neospora caninum (2%). All animals were seronegative to Brucella spp. and Toxoplasma gondii. Mycobacterium avium ssp. paratuberculosis was not detected in any fecal samples. Parasite eggs or larvae, including Parelaphostrongylus andersoni (36%), Skrjabinema spp. (1%), Strongyle-type eggs (11%), Moniezia-type eggs (8%), and nematodirines (3%), were detected on fecal examination, but at low intensity. Blood biochemistry values and hair cortisol concentrations were within ranges previously reported in Rangifer tarandus sspp. Some significant differences among herds were noted, including antler morphology, exposure to Besnoitia spp., and concentrations of serum amyloid A, copper, cobalt, manganese, and iron.

A Multi-Pathogen Screening of Captive Reindeer (Rangifer tarandus) in Germany Based on Serological and Molecular Assays.

Captive reindeer in German zoos and wildlife parks live outside their natural geographic range and are exposed to a variety of viral, bacterial and protozoan pathogens, some host-specific and some which they are not exposed to in their native habitat. Reindeer blood samples and ticks collected in 2013 from 123 reindeer at 16 different zoological facilities were available from a previous study. The aims of this study were to assess the serological status of these animals with regards to various microorganisms as well as to test ticks (Ixodes ricinus) and blood samples for the presence of Anaplasma spp. DNA in order to evaluate the exposure of captive reindeer in Germany to a variety of pathogens. A total of 119 or 118 serum samples were screened (ELISA) and antibodies were detected (seropositive/tested, prevalence, confidence interval) against alphaherpesvirus (24/119, 20.3%, CI: 13.9-28.3), bluetongue virus (BTV; 4/119, 3.4%, CI: 1.0-8.7), malignant catarrhal fever related gammaherpesvirus (MCFV-related gammaherpesvirus; 7/119, 5.9%, CI: 2.7-11.9), pestivirus (5/118, 4.2%, CI: 1.6-9.8), Schmallenberg virus (SBV; 70/118, 59.3%, CI: 50.3-67.8), smooth Brucella spp. (1/118; 0.9%, CI: 0-5.1), Neospora caninum (5/118, 4.2%, CI: 1.6-9.8), and Toxoplasma gondii (62/119, 52.1%, CI: 43.2-60.9). These results suggested the exposure of reindeer to all tested pathogens. Moreover, real-time PCR for Anaplasma phagocytophilum targeting the partial msp2 gene was performed on DNA extracted from whole blood samples from reindeer (n = 123) and from ticks (n = 49) collected from 22 reindeer in seven different facilities. In addition to the real-time PCR, a semi-nested PCR for the partial groEL gene, and a nested PCR targeting the partial 16S rRNA gene were performed. DNA of A. phagocytophilum was detected in 17 reindeer (13.8%) and 15 ticks (30.6%). Three of the five reindeer with ticks having A. phagocytophilum DNA also had such DNA in blood. These results indicate that captive reindeer can be exposed to several ruminant pathogens that they hitherto had no known exposure to through their natural geographical distribution and habitats as shown for Culicoides-borne BTV and SBV. Further, captive reindeer may serve as reservoir hosts for pathogens circulating in local domestic, captive, and wild ruminant species and populations and arthropod vectors.

Contaminants in Atlantic walruses in Svalbard part 1: Relationships between exposure, diet and pathogen prevalence.

This study investigated relationships between organohalogen compound (OHC) exposure, feeding habits, and pathogen exposure in a recovering population of Atlantic walruses (Odobenus rosmarus rosmarus) from the Svalbard Archipelago, Norway. Various samples were collected from 39 free-living, apparently healthy, adult male walruses immobilised at three sampling locations during the summers of 2014 and 2015. Concentrations of lipophilic compounds (polychlorinated biphenyls, organochlorine pesticides and polybrominated diphenyl ethers) were analysed in blubber samples, and concentrations of perfluoroalkylated substances (PFASs) were determined in plasma samples. Stable isotopes of carbon and nitrogen were measured in seven tissue types and surveys for three infectious pathogens were conducted. Despite an overall decline in lipophilic compound concentrations since this population was last studied (2006), the contaminant pattern was similar, including extremely large inter-individual variation. Stable isotope ratios of carbon and nitrogen showed that the variation in OHC concentrations could not be explained by some walruses consuming higher trophic level diets, since all animals were found to feed at a similar trophic level. Antibodies against the bacteria Brucella spp. and the parasite Toxoplasma gondii were detected in 26% and 15% of the walruses, respectively. Given the absence of seal-predation, T. gondii exposure likely took place via the consumption of contaminated bivalves. The source of exposure to Brucella spp. in walruses is still unknown. Parapoxvirus DNA was detected in a single individual, representing the first documented evidence of parapoxvirus in wild walruses. Antibody prevalence was not related to contaminant exposure. Despite this, dynamic relationships between diet composition, contaminant bioaccumulation and pathogen exposure warrant continuing attention given the likelihood of climate change induced habitat and food web changes, and consequently OHC exposure, for Svalbard walruses in the coming decades.

Brucella Antibodies in Alaskan True Seals and Eared Seals-Two Different Stories.

Brucella pinnipedialis was first isolated from true seals in 1994 and from eared seals in 2008. Although few pathological findings have been associated with infection in true seals, reproductive pathology including abortions, and the isolation of the zoonotic strain type 27 have been documented in eared seals. In this study, a Brucella enzyme-linked immunosorbent assay (ELISA) and the Rose Bengal test (RBT) were initially compared for 206 serum samples and a discrepancy between the tests was found. Following removal of lipids from the serum samples, ELISA results were unaltered while the agreement between the tests was improved, indicating that serum lipids affected the initial RBT outcome. For the remaining screening, we used ELISA to investigate the presence of Brucella antibodies in sera of 231 eared and 1,412 true seals from Alaskan waters sampled between 1975 and 2011. In eared seals, Brucella antibodies were found in two Steller sea lions (Eumetopias jubatus) (2%) and none of the 107 Northern fur seals (Callorhinus ursinus). The low seroprevalence in eared seals indicate a low level of exposure or lack of susceptibility to infection. Alternatively, mortality due to the Brucella infection may remove seropositive animals from the population. Brucella antibodies were detected in all true seal species investigated; harbor seals (Phoca vitulina) (25%), spotted seals (Phoca largha) (19%), ribbon seals (Histriophoca fasciata) (16%), and ringed seals (Pusa hispida hispida) (14%). There was a low seroprevalence among pups, a higher seroprevalence among juveniles, and a subsequent decreasing probability of seropositivity with age in harbor seals. Similar patterns were present for the other true seal species; however, solid conclusions could not be made due to sample size. This pattern is in accordance with previous reports on B. pinnipedialis infections in true seals and may suggest environmental exposure to B. pinnipedialis at the juvenile stage, with a following clearance of infection. Furthermore, analyses by region showed minor differences in the probability of being seropositive for harbor seals from different regions regardless of the local seal population trend, signifying that the Brucella infection may not cause significant mortality in these populations. In conclusion, the Brucella infection pattern is very different for eared and true seals.

Concomitant Temperature Stress and Immune Activation may Increase Mortality Despite Efficient Clearance of an Intracellular Bacterial Infection in Atlantic Cod.

The environmental temperature has profound effects on biological systems of marine aquatic organisms and plays a critical role in species distribution and abundance. Particularly during the warmer seasons, variations in habitat temperature may introduce episodes of stressful temperatures which the organisms must adapt to and compensate for to maintain physiological homeostasis. The marine environment is changing and predicted raises in water temperatures will affect numerous marine species. Translocation of pathogens follow migration of species and alternations in physical environmental parameters may have influence upon the virulence of pathogens, as well as the hosts immune responses. While pathogenicity of many true pathogens is expected to increase following climate induced temperature stress, the impact from environmental stressors on the occurrence and severity of opportunistic infections is unknown. Here we describe how thermal stress in the cold-water species Atlantic cod influenced the fish immune responses against an opportunistic intracellular bacterium. Following experimental infection with Brucella pinnipedialis at normal water temperature (6°C) and sub-optimal temperature (15°C), cod cleared the intracellular bacteria more rapidly at the highest temperature. The overall immune response was faster and of higher amplitude at 15°C, however, a significant number of cod died at this temperature despite efficient clearance of infection. An increased growth rate not affected by infection was observed at 15°C, confirming multiple energy demanding processes taking place. Serum chemistry suggested that general homeostasis was influenced by both infection and increased water temperature, highlighting the cumulative stress responses (allostatic load) generated by simultaneous stressors. Our results suggest a trade-off between resistance and tolerance to survive infection at sub-optimal temperatures and raise questions concerning the impact of increased water temperatures on the energetic costs of immune system activation in aquatic ectotherms.

Application of real-time quantitative PCR assays for detecting marine Brucella spp. in fish.

Brucella ceti and Brucella pinnipedialis have been documented as occurring in marine mammals, and B. ceti has been identified in 3 naturally acquired human cases. Seroconversion and infection patterns in Pacific Northwest harbor seals ( Phoca vitulina richardii) and North Atlantic hooded seals ( Cystophora cristata) indicate post-weaning exposure through prey consumption or lungworm infection, suggesting fish and possibly invertebrates play an epizootiologic role in marine Brucella transmission and possible foodborne risk to humans. We determined if real-time quantitative PCR (qPCR) assays can detect marine Brucella DNA in fish DNA. Insertion sequence (IS) 711 gene and sequence type (ST)27 primer-probe sets were used to detect Brucella associated with marine mammals and human zoonotic infections, respectively. First, DNA extracts from paired-species fish (containing 2 species) samples were tested and determined to be Brucella DNA negative using both IS 711 and ST27 primer-probe sets. A representative paired-species fish DNA sample was spiked with decreasing concentrations of B. pinnipedialis DNA to verify Brucella detection by the IS 711 primer-probe within fish DNA. A standard curve, developed using isolated DNA from B. pinnipedialis, determined the limit of detection. Finally, the IS 711 primer-probe was used to test Atlantic cod ( Gadus morhua) DNA extracts experimentally infected with the B. pinnipedialis hooded seal strain. In culture-positive cod tissue, the IS 711 limit of detection was ~1 genome copy of Brucella. Agreement between culture and PCR results for the 9 positive and 9 negative cod tissues was 100%. Although a larger sample set is required for validation, our study shows that qPCR can detect marine Brucella in fish.

Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model.

Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea.

Brucella pinnipedialis hooded seal (Cystophora cristata) strain in the mouse model with concurrent exposure to PCB 153.

Brucellosis, a worldwide zoonosis, is linked to reproductive problems in primary hosts. A high proportion of Brucella-positive hooded seals (Cystophora cristata) have been detected in the declined Northeast Atlantic stock. High concentrations of polychlorinated biphenyls (PCBs) have also been discovered in top predators in the Arctic, including the hooded seal, PCB 153 being most abundant. The aim of this study was to assess the pathogenicity of Brucella pinnipedialis hooded seal strain in the mouse model and to evaluate the outcome of Brucella spp. infection after exposure of mice to PCB 153. BALB/c mice were infected with B. pinnipedialis hooded seal strain or Brucella suis 1330, and half from each group was exposed to PCB 153 through the diet. B. pinnipedialis showed a reduced pathogenicity in the mouse model as compared to B. suis 1330. Exposure to PCB 153 affected neither the immunological parameters, nor the outcome of the infection. Altogether this indicates that it is unlikely that B. pinnipedialis contribute to the decline of hooded seals in the Northeast Atlantic.

Entrance and survival of Brucella pinnipedialis hooded seal strain in human macrophages and epithelial cells.

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72-96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary.

Entry and elimination of marine mammal Brucella spp. by hooded seal (Cystophora cristata) alveolar macrophages in vitro.

A high prevalence of Brucellapinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal (Cystophoracristata); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B. pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B. pinnipedialis reference strain (NCTC 12890) from harbor seal (Phocavitulina), B. ceti reference strain (NCTC 12891) from harbor porpoise (Phocoenaphocoena) and a B. ceti Atlantic white-sided dolphin (Lagenorhynchusacutus) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B. pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains tested were able to establish a persistent infection in primary alveolar macrophages from hooded seal.

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.