Influenza A(H10N7) virus in dead harbor seals, Denmark.

Since April 2014, an outbreak of influenza in harbor seals has been ongoing in northern Europe. In Denmark during June-August, 152 harbor seals on the island of Anholt were found dead from severe pneumonia. We detected influenza A(H10N7) virus in 2 of 4 seals examined.

Application of Machine Learning for Automating Behavioral Tracking of Captive Bornean Orangutans (Pongo Pygmaeus).

This article applies object detection to CCTV video material to investigate the potential of using machine learning to automate behavior tracking. This study includes video tapings of two captive Bornean orangutans and their behavior. From a 2 min training video containing the selected behaviors, 334 images were extracted and labeled using Rectlabel. The labeled training material was used to construct an object detection model using Create ML. The use of object detection was shown to have potential for automating tracking, especially of locomotion, whilst filtering out false positives. Potential improvements regarding this tool are addressed, and future implementation should take these into consideration. These improvements include using adequately diverse training material and limiting iterations to avoid overfitting the model.

Hepatitis E virus variant in farmed mink, Denmark.

Hepatitis E virus (HEV) is a zoonotic virus for which pigs are the primary animal reservoir. To investigate whether HEV occurs in mink in Denmark, we screened feces and tissues from domestic and wild mink. Our finding of a novel HEV variant supports previous findings of HEV variants in a variety of species.

Climate-associated drivers of plasma cytokines and contaminant concentrations in Beaufort Sea polar bears (Ursus maritimus).

Assessing polar bear (Ursus maritimus) immune function in relation to environmental stressors, including habitat change, nutritional stress, pathogen prevalence, and pollution, has been identified as critical for improved understanding of the species' health. The objectives of this study were two-fold: 1) to assess the role of climate-associated factors (habitat use, body condition) in explaining the plasma concentrations of contaminants in southern Beaufort Sea (SB) polar bears, and 2) to investigate how climate-associated factors, contaminant concentrations, and pathogen sero-prevalence influence the plasma concentrations of immune-signaling proteins called cytokines. A commercially available multiplex canine cytokine panel was validated for the quantification of five pro- and anti-inflammatory cytokines in polar bear plasma: tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), IL-8, IL-10, and interferon gamma-induced protein 10 (IP-10). This panel was then used to measure cytokine concentrations in 49 SB polar bears sampled in the springs of 2013 and 2014. Mean ∑PCBs (plasma), ∑OCs (plasma), and THg (hair) were 13.01 ± 1.52 ng g-1 w.w. (range: 0.17-52.63), 19.46 ± 1.17 ng g-1 w.w. (range: 6.63-45.82), and 0.49 µg g-1 d.w. (range: 0.99-15.18), respectively. Top models explaining variation in concentrations of plasma PCBs, plasma OC pesticides, and hair THg in SB polar bears included body mass index and/or habitat use (onshore versus offshore), with higher contaminant concentrations in leaner and/or offshore bears. Plasma cytokine concentrations were influenced most strongly by plasma PCBs and age, with little to no influence found for plasma OCs or hair THg concentrations, habitat use, or pathogen sero-prevalence. The lack of association between cytokines and these latter variables is likely due to a temporal disconnect between measured endpoints. The change of polar bear habitat use, feeding ecology, and body condition with ongoing climate warming is affecting exposure to contaminants and pathogens, with potential adverse consequences on a well-balanced immune system.

Aleutian mink disease virus in free-ranging mink from Sweden.

Aleutian mink disease (AMD) is a chronic viral disease in farmed mink and the virus (AMDV) has been found in many free-ranging mink (Neovison vison) populations in Europe and North America. In this study, AMDV DNA and AMDV antibodies were analysed in 144 free-ranging mink hunted in Sweden. Associations between being AMDV infected (defined as positive for both viral DNA and antibodies) and the weight of the spleen, liver, kidneys, adrenal glands and body condition were calculated and the sequences of ten AMDV isolates were analysed in order to characterize the genetic relationships. In total, 46.1% of the mink were positive for AMDV antibodies and 57.6% were positive for AMDV DNA. Twenty-two percent of the mink tested on both tests (n = 133) had dissimilar results. The risk of having AMDV antibodies or being positive for AMDV DNA clearly increased with age and the majority of the mink that were two years or older were infected. Few macroscopic changes were found upon necropsy. However, the relative weight of the spleen was sexually dimorphic and was found to be slightly, but significantly (p = 0.006), heavier in AMDV infected male mink than uninfected. No association between AMDV infection and body condition, weight of the kidneys, liver or adrenal glands were found. Several different strains of AMDV were found across the country. Two of the AMDV sequences from the very north of Sweden did not group with any of the previously described groups of strains. In summary, AMDV seems to be prevalent in wild mink in Sweden and may subtly influence the weight of the spleen.

Comparison of histological lesions in mink with acute hemorrhagic pneumonia associated with Pseudomonas aeruginosa or Escherichia coli.

Hemorrhagic pneumonia can be a major cause of mortality in farmed mink in the fall. In its classic form, hemorrhagic pneumonia is caused by the bacterium Pseudomonas aeruginosa. In recent years, however, outbreaks of this type of pneumonia that are associated with hemolytic Escherichia coli have also occurred in farmed mink. The purpose of this study was to compare histological lesions of acute hemorrhagic pneumonia associated with both P. aeruginosa and E. coli in mink, including a description of tissue distribution of pathogens, in an attempt to differentiate between the 2 disease entities based on histopathology. The study included material submitted for diagnostic investigation to the National Veterinary Institute in Denmark from 2006 to 2009. Altogether, 19 cases of hemorrhagic pneumonia with a pure lung culture of P. aeruginosa and 18 cases of hemorrhagic pneumonia with a pure lung culture of E. coli were examined. Formalin-fixed paraffin-embedded lung tissue obtained from the mink was examined by histology and fluorescence in-situ hybridization (FISH). It was possible to detect a slight histological difference between hemorrhagic pneumonia caused by P. aeruginosa and by E. coli, as P. aeruginosa was most often found surrounding blood vessels and lining the alveoli, while E. coli showed a more diffuse distribution in the lung tissue. Furthermore, P. aeruginosa often elicited a very hemorrhagic response in the lung, while infection with E. coli was associated with a higher frequency of alveolar edema and mild lymphoid cuffing in the lungs.

À l'automne, la pneumonie hémorragique peut être une cause majeure de mortalité chez les visons d'élevage. Dans sa forme classique, la pneumonie hémorragique est causée par la bactérie Pseudomonas aeruginosa. Au cours des dernières années toutefois, des poussées de cas de ce type de pneumonie associées à des isolats hémolytiques d'Escherichia coli se sont produits chez des visons d'élevage. Le but de cette étude était de comparer les lésions histologiques de pneumonie hémorragique aiguë associée à P. aeruginosa et E. coli, incluant une description de la distribution tissulaire des agents pathogènes, dans une tentative de différencier les deux maladies en se basant sur l'histopathologie. L'étude incluait du matériel soumis pour diagnostic à l'Institut National Vétérinaire du Danemark entre 2006 et 2009. Au total, 19 cas de pneumonie hémorragique avec culture pure de P. aeruginosa et 18 cas de pneumonie hémorragique avec une culture pure d'E. coli ont été examinés. Du tissu pulmonaire provenant des visons fixé à la formaline et enrobé de paraffine a été soumis à un examen histologique et par hybridation avec fluorescence in-situ (FISH). Il a été possible de détecter une légère différence histologique entre la pneumonie hémorragique causée par P. aeruginosa et E. coli, P. aeruginosa étant retrouvé plus fréquemment entourant les vaisseaux sanguins et tapissant les alvéoles, alors qu'E. coli montrait une distribution plus diffuse dans le tissu pulmonaire. De plus, P. aeruginosa causait souvent une réponse très hémorragique dans le poumon, alors que l'infection par E. coli était associée avec une fréquence plus élevée d'oedème alvéolaire et de présence de manchons lymphoïdes dans les poumons.(Traduit par Docteur Serge Messier).

Effect of infectious dose and season on development of hemorrhagic pneumonia in mink caused by Pseudomonas aeruginosa.

Hemorrhagic pneumonia is an acute and fatal disease of farmed mink caused by Pseudomonas aeruginosa. The pathogenesis of this disease has not yet been resolved. Mink are the only animals known to be susceptible to acute, contagious, and fatal lung infections caused by P. aeruginosa. The purpose of this study was to investigate the correlation between dose-response and season of infection and to clarify whether Danish mink are carriers of P. aeruginosa on their nasal mucosa during the season for hemorrhagic pneumonia. To elucidate the pathogenesis of the disease, an infectious dose-response trial was carried out on adult mink and mink kits, both in the season for hemorrhagic pneumonia (November) as well as out of season (July). It proved difficult to infect mink via the intra-nasal route. Only 4 out of 60 infected mink developed clinical disease and were euthanized, all of them in November, illustrating that predisposing factors in the mink itself and not infectious dose might be crucial for disease development. We were able to culture P. aeruginosa from the nasal cavity of the clinically healthy experimental mink 8 d after inoculation. This indicated that the mink can carry P. aeruginosa on their nasal mucosa without developing the disease. It was not possible, however, to culture P. aeruginosa from the nasal cavity of clinically healthy mink obtained from farms in November, which indicates that the organism is not a normal part of the nasal mucosal flora of mink.

La pneumonie hémorragique est une maladie aiguë et fatale du vison d'élevage causée par Pseudomonas aeruginosa. La pathogénie de cette maladie n'a pas encore été résolue. Les visons sont les seuls animaux connus à être susceptible à l'infection pulmonaire aiguë, contagieuse et fatale causée par P. aeruginosa. Le but de ce travail était d'étudier la corrélation entre la dose-réponse et la saison d'infection et de clarifier si les visons danois sont porteurs de P. aeruginosa sur leur muqueuse nasale durant la saison pour la pneumonie hémorragique. Afin d'élucider la pathogénie de la maladie, un essai de dose infectieuse-réponse a été réalisé chez des visons adultes et des jeunes visons, durant la saison pour la pneumonie hémorragique (novembre) aussi bien qu'hors-saison (juillet). Il s'est avéré difficile d'infecter les visons via la voie intra-nasale. Seulement 4 des 60 visons infectés ont développé la maladie clinique et ont été euthanasiés, tous en novembre, illustrant ainsi que des facteurs prédisposants chez le vison lui-même et non la dose infectante pourraient être cruciaux pour le développement de la maladie. Nous avons été en mesure de cultiver P. aeruginosa à partir de la cavité nasale des visons expérimentaux cliniquement sains 8 j après l'inoculation. Ceci indique que le vison peut être porteur de P. aeruginosa au niveau de la muqueuse nasale sans que la maladie ne se développe. Il n'a pas été possible toutefois de cultiver P. aeruginosa de la cavité nasale de visons cliniquement sains obtenus des fermes en novembre, ce qui indique que le microorganisme ne fait pas partie de la flore normale de la muqueuse nasale du vison.(Traduit par Docteur Serge Messier).

An enzyme-linked immunosorbent assay for detection of avian influenza virus subtypes H5 and H7 antibodies.

BACKGROUND: Avian influenza virus (AIV) subtypes H5 and H7 attracts particular attention because of the risk of their potential pathogenicity in poultry. The haemagglutination inhibition (HI) test is widely used as subtype specific test for serological diagnostics despite the laborious nature of this method. However, enzyme-linked immunosorbent assays (ELISAs) are being explored as an alternative test method.H5 and H7 specific monoclonal antibodies were experimentally raised and used in the development of inhibition ELISAs for detection of serological response specifically directed against AIV subtypes H5 and H7. The ELISAs were evaluated with polyclonal chicken anti-AIV antibodies against AIV subtypes: H1N2, H5N2, H5N7, H7N1, H7N7, H9N9, H10N4 and H16N3. RESULTS: Both the H5 and H7 ELISA proved to have a high sensitivity and specificity and the ELISAs detected H5 and H7 antibodies earlier during experimental infection than the HI test did. The reproducibility of the ELISA's performed at different times was high with Pearson correlation coefficients of 0.96-0.98. CONCLUSIONS: The ELISAs are a potential alternative to the HI test for screening of large amounts of avian sera, although only experimental sera were tested in this study.

Establishment of stably transfected cells constitutively expressing the full-length and truncated antigenic proteins of two genetically distinct mink astroviruses.

Astroviruses are becoming a growing concern in veterinary and public health. To date there are no registered vaccines against astrovirus-induced disease, mostly due to the difficulty to cultivate astroviruses to high titer for vaccine development using conventional techniques. As means to circumvent this drawback, we have developed stably transfected mink fetal cells and BHK21 cells constitutively expressing the full-length and truncated capsid proteins of two distinct genotypes of mink astrovirus. Protein expression in these stably transfected cells was demonstrated by strong signals as evaluated by in-situ PLA and IFA, and confirmed by Western blotting. The recombinant full-length and truncated proteins induced a high level of antibodies in mink, evaluated by ELISA, demonstrating their immunogenicity. In a challenge experiment in mink, a reduction in presentation clinical signs and virus shedding was observed in mink kits born from immunized females. The gene integration and protein expression were sustained through cell passage, showing that the used approach is robust and reliable for expression of functional capsid proteins for vaccine and diagnostic applications.

High prevalence of Aleutian mink disease virus in free-ranging mink on a remote Danish island.

Aleutian mink disease virus (AMDV) causes severe disease in farmed mink (Neovison vison) worldwide. In Denmark, AMDV in farmed mink has been confined to the northern part of the mainland since 2002. From 1998 to 2009, samples from 396 free-ranging mink were collected from mainland Denmark, and a low AMDV antibody prevalence (3% of 296) was found using countercurrent immune electrophoresis. However, on the island of Bornholm in the Baltic Sea, a high prevalence (45% of 142 mink) was detected in the free-ranging mink. Aleutian mink disease virus was detected by polymerase chain reaction in 32 of 49 antibody-positive free-ranging mink on Bornholm, but not in mink collected from other parts of Denmark. Sequence analysis of 370 base pairs of the nonstructural gene of the AMDV of 17 samples revealed two clusters with closest similarity to Swedish AMDV strains.

Implementation and validation of a sensitive PCR detection method in the eradication campaign against Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs (n=299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.

Genetic diversity and phylogenetic analysis of the attachment glycoprotein of phocine distemper viruses of the 2002 and 1988 epizootics.

To investigate the possible origin and spread of the dramatic re-emergent 2002 distemper epizootic observed among seals in Danish Waters, we have sequenced wild-type genes of the attachment (H) glycoproteins of viruses from both the 2002 and 1988 epizootics. Phylogenetic analysis of the H genes of phocine distemper virus (PDV) together with other morbilliviruses, suggests that the re-emergent 2002 PDV is more closely related to a putative recent ancestral PDV than the 1988 PDV isolates. Moreover, upsurges of distemper disease in land-living carnivores linked in time and locality to the 2002 seal epizootic in Danish Waters was investigated and determined to be caused by canine distemper virus, the closest relative of PDV, revealing no direct epidemiological link to the seal epizootics.