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Trichinella is a zoonotic parasite, which historically has been of large concern for public health in Europe. Consequently, testing of all pigs for Trichinella has been mandatory in many European countries, even though Trichinella is almost exclusively found in outdoor or backyard production. The idea therefore emerged that auditing for biosecurity should replace testing in indoor production. In the European Union (EU), pigs raised under so-called controlled housing conditions are exempt from testing. The specific requirements for a pig production to be considered a controlled housing holding are described in Annex IV in the EU Trichinella Regulation No. 2015/1375. In Denmark, the controlled housing concept is used extensively, not to omit Trichinella testing but to allow visual-only post-mortem inspection at export-authorized abattoirs. The Danish pig industry has established a quality assurance scheme called DANISH Product Standard, which is used to assure that the pig production maintains specific standards. This paper describes how the control, including the auditing, is set up and shows how the EU requirements regarding controlled housing are controlled. Moreover, the EU requirements are compared with the recommendations issued by the International Commission on Trichinellosis and the World Organisation for Animal Health. Finally, strengths, weaknesses, opportunities and threats of the Danish way of documenting indoor finisher herds' compliance with controlled housing as specified by the EU are discussed. It is concluded that the validity of the system is high., Competing Interests: Lis Alban and Lisbeth Harm Nielsen work for an organisation which gives advice to farmers and meat- producing companies., (© 2024 The Authors. Published by Elsevier Inc. on behalf of International Association of Food and Waterborne Parasitology.)

Measuring trace element concentrations in tissue can be a valuable approach to monitor animal health status. Temporal variation in the absorption, transport, and storage of elements between different tissues can, however, complicate the assessment of element-health relationships. Here, we measured concentrations of selected essential (copper (Cu), zinc (Zn), selenium (Se)) and non-essential (arsenic (As), cadmium (Cd), lead (Pb)) trace elements within blood, liver, kidney, and hair of fallow deer (Dama dama; N=20) and red deer (Cervus elaphus; N=21). Using multivariate regression and structural equation models, we estimated direct and indirect linkages between tissue-specific trace element profiles and long- (body condition) and short-term (serum protein biomarkers for acute inflammation, infection, and malnutrition) health indicators. Trace element concentrations varied markedly and were weakly correlated among tissues, with the exception of Se. After accounting for sex- and site-differences in trace element concentrations, body condition of red deer was directly, and positively, associated to trace element status in liver and hair, but not in kidney. For both deer species, trace element status in blood was directly linked to serum protein status with an indirect positive association to deer body condition. For fallow deer, no direct association between trace element status and body condition was detected in any of the tissues, possibly because of elemental homeostasis, and because all individuals were in good clinical health. This study shows that hair can serve as an effective, non-invasive, biomarker in deer health assessments, yet, to fully uncover trace element-health relationships a variety of sample matrices is preferred., (© 2023. The Author(s).)

Urban cloudburst management may include the intentional temporary storage of flood water in green recreational areas. In cities with combined sewers, this will expose the population visiting the area to sewage and increase the risk of diarrhoeal disease. We present a unique approach to estimate the risk of diarrhoeal disease after urban flooding. The exposure scenario was: rainwater mixed with sewage flows into a park; sewage with pathogens deposit on the grass; after discharge, a baby plays on the grass and is exposed to the pathogens in the deposited sewage by hand-to-mouth transfer. The work included modelling the transport of sewage into four parks intended to be flooded during future cloudbursts. A flood simulation experiment was conducted to estimate the deposition of pathogens from sewage to grass and transfer from grass to hand. Hand-to-mouth transfer, based on literature values, was used to estimate the ingested dose of pathogens. The probability of illness was estimated by QMRA. The estimated average probability of illness varied between 0.03 and 17%. If the probability of illness is considered unacceptable, the cloudburst plans should be changed, or interventions, e.g. informing the public about the risk or restricting access to the flooded area, should be implemented.

Tralokinumab, a fully human mAb specifically targeting the IL-13 cytokine, has demonstrated clinical efficacy and safety in patients with moderate-to-severe atopic dermatitis. Tralokinumab binds IL-13 with high affinity, which prevents the interaction of IL-13 with IL-13Rα1 and subsequent signaling. Similarly, tralokinumab-bound IL-13 cannot bind to IL-13Rα2, a proposed decoy receptor that is reported to bind IL-13 with extraordinarily high affinity. It has however not been fully elucidated to what extent tralokinumab interferes with the endogenous regulation of IL-13 through IL-13Rα2. In this mechanistic study, we used biophysical, biochemical, and cellular assays to investigate the effect of tralokinumab on the interaction between IL-13 and IL-13Rα1 and IL-13Rα2, respectively, as well as the effects on IL-13Rα2-mediated IL-13 internalization. We demonstrate that IL-13Rα2 binds IL-13 with exceptionally high affinity and that tralokinumab is unable to displace IL-13 from IL-13Rα2. In contrast to this, tralokinumab is able to disrupt the IL-13/IL-13Rα1 and IL-13Rα1/IL-13/IL-4Rα complex. Furthermore, we demonstrate that whereas the IL-13/tralokinumab complex is unable to bind IL-13Rα2, any IL-13 that is not bound by tralokinumab (i.e., free IL-13) can be bound by IL-13Rα2 and subsequently internalized, regardless of the presence of tralokinumab. In summary, our study indicates that tralokinumab does not interfere with endogenous IL-13Rα2-mediated regulation of free IL-13., (© 2023 The Authors.)

Parasites can exert a substantial influence on the ecology of wildlife populations by altering host condition. Our objectives were to estimate single and multiparasite-condition relationships for fallow deer ( Dama dama ) and red deer ( Cervus elaphus ) in Denmark and to assess potential health effects along the parasite burden gradient. Fallow deer hosted on average two endoparasite taxa per individual (min = 0, max = 5) while red deer carried on average five parasite taxa per individual (min = 2, max = 9). Body condition of both deer species was negatively related to presence of Trichuris ssp. eggs while body condition of red deer was positively related to antibodies of the protozoan Toxoplasma gondii . For the remaining parasite taxa (n = 12), we either found weak or no apparent association between infection and deer body condition or low prevalence levels restricted formal testing. Importantly, we detected a strong negative relationship between body condition and the sum of endoparasite taxa carried by individual hosts, a pattern that was evident in both deer species. We did not detect systemic inflammatory reactions, yet serology revealed reduced total protein and iron concentrations with increased parasite load in both deer species, likely due to maldigestion of forage or malabsorption of nutrients. Despite moderate sample sizes, our study highlights the importance of considering multiparasitism when assessing body condition impacts in deer populations. Moreover, we show how serum chemistry assays are a valuable diagnostic tool to detect subtle and sub-clinical health impacts of parasitism, even at low-level infestation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

For the purpose of nature management and species conservation, European bison ( Bison bonasus ) are being increasingly reintroduced into nature reserves across Europe. The aim of this study was to investigate European bison's adaptability to new areas through the study of their parasite-EPG (eggs per gram feces) and dietary diversity during twelve months after translocation. We compared the parasite-EPG from introduced European bison in Lille Vildmose, Denmark, with the parasite-EPG from populations from Bornholm, Denmark, and Białowieża Forest, Poland. From March 2021 to February 2022, fecal samples were collected from three populations. Samples from Lille Vildmose were examined through flotation, sedimentation, the Baermann technique, and nanopore sequencing. Fecal samples from Bornholm and Białowieża were examined through flotation and sedimentation. Nanopore sequencing of DNA from 63 European bison's fecal samples collected during March-September in Lille Vildmose identified 8 species of nematodes within the digestive tract of the European bison, with Haemonchus contortus being the most frequently observed. In Lille Vildmose, a significantly higher excretion of nematode-EPG was observed during the summer period than in the spring, autumn, and winter. In addition, monthly differences in the excretion of nematode eggs were found, with this being significantly higher in June than in the months during autumn and winter (October-February). Significant differences in the nematode-EPG were only found between the excretion of nematode eggs in Białowieża Forest when compared to that of Lille Vildmose, with significantly higher excretion in Lille Vildmose (October-November). The results indicate that the development rates for nematodes may be affected by changes in temperature, with increasing temperatures speeding up their development time. Independent of this study design, wildlife vets together with the gamekeepers managing the herd found it necessary to treat the herd with antiparasitics for practical and animal welfare reasons in relation to translocation. Furthermore, 79 plant taxa were identified in the diet of the European bison. The broadest diet was observed in March suggesting that the European bison quickly adapted to their new habitat. The results suggest a seasonal shift in their diet, with this being most apparent from March to April.

Purpose: To evaluate arteriovenous malformations (AVM) with swept-source (SS) optical coherence tomography (OCT) angiography (OCTA) in iris racemose hemangioma and compare it with traditional intravenous iris fluorescein angiography (IVFA)., Methods: A cross-sectional observational clinical study was conducted on patients with iris racemose hemangioma with the ZEISS PLEX Elite 9000 SS OCT & OCTA., Results: Three eyes of three patients were imaged. Iris racemose hemangiomas demonstrated a tortuous, well-defined, and continuous course of the AVM. The ZEISS PLEX Elite 9000 SS OCT & OCTA allowed for a detailed visualization of the ARM and was superior to IVFA in depicting small caliber, fine vessels., Conclusions: SS-OCTA may provide a dye-free, no-injection, cost-effective method comparable to spectral domain OCTA and IVFA for diagnosing and monitoring iris racemose hemangiomas for growth and vascularity.

Toxoplasma gondii is a zoonotic protozoan parasite capable of infecting possibly all warm-blooded animals including humans, and is one of the most widespread zoonotic pathogens known. Free-ranging wildlife can be valuable sentinels for oocyst contaminated environments, as well as a potential source for human foodborne infection with T. gondii. Here we aimed to determine the sero-prevalence of T. gondii in Danish wild deer populations and examine risk factors associated with increased exposure to the parasite. Blood samples were collected from 428 cervids (87 fallow deer ( Dama dama ), 272 red deer ( Cervus elaphus ), 55 roe deer ( Capreolus capreolus ) and 14 sika deer ( Cervus Nippon ) from 23 hunting sites in Denmark. The animals were shot during the hunting season 2017/2018, and screened for antibodies against T. gondii using a commercial ELISA kit. One hundred and five (24.5%) cervids were sero-positive. Sero-prevalence was significantly different between species (p < 0.05), with odds of sero-positivity being 4.5 times higher in roe deer than fallow deer, and 3.0 times higher in red deer than in fallow deer. A significant increase in sero-prevalence with age was observed, driven by a significant increase in risk in adult red deer compared to calves (OR: 13.22; 95% CI: 5.96-33.7). The only other significant risk factor associated with wild cervid T. gondii sero-positivity was fencing, with the highest exposure associated with deer from non-fenced hunting areas (OR: 2.21; 95% CI: 1.05-4.99). This study documented a widespread exposure to T. gondii in Danish cervids. Therefore the meat of the wild deer, in particular from roe deer and red deer, should be considered a significant risk of T. gondii infections to humans, if not properly cooked. Further, molecular studies to confirm the presence of infective parasitic stages in the muscles of deer used for consumption is recommended., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Background: The regulation of factor X (FX) is critical to maintain the balance between blood coagulation and fluidity., Objectives: To functionally characterize the role of the FX autolysis loop in the regulation of the zymogen and active form of FX., Methods: We introduced novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. The activity and inhibition of recombinant FX variants was quantified in pure component assays. The in vitro thrombin generation potential of the FX variants was evaluated in FX-depleted plasma., Results: The factor VIIa (FVIIa)-mediated activation and prothrombin activation was reduced, presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). K i for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, whereas ATIII inhibition in the presence of the heparin-analog Fondaparinux was modestly increased by 2- to 15-fold compared with wild-type. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FXa variants., Conclusion: The autolysis loop participates in all aspects of FX regulation. In plasma-based assays, a modest decrease in FX activation rate appeared to knock down the procoagulant response even when down regulation of FXa activity by inhibitors was reduced., (© 2021 International Society on Thrombosis and Haemostasis.)

Objective disease markers in the southern white rhinoceros ( Ceratotherium simum simum ) are in high demand. In the field, such markers are typically needed to decide whether a captured white rhinoceros is fit to cope with quarantine, transport, or both. Captive white rhinoceros have a need for unbiased biomarkers for early detection of disease. Acute phase proteins, including haptoglobin, are proteins that significantly change their plasma concentration in response to tissue perturbation or inflammation, such as that occurring during infection or neoplastic disease. Acute phase proteins are well known diagnostic tools in both human and veterinary medicine. In this study, an ELISA with commercially available anti-human haptoglobin antibodies for quantification of haptoglobin in white rhinoceros serum was developed. The validity of the haptoglobin assay and haptoglobin as a biomarker of disease was investigated with the use of serum samples from both captive and free-ranging animals with a well-described health status. The assay was precise (intra-assay and interassay reproducibility were 5.0% and 13.1%, respectively) and reliably quantified white rhinoceros haptoglobin serum concentrations consuming low volumes of sample. The assay was sensitive to the presence of free hemoglobin in the sample at levels corresponding to a visibly hemolyzed sample. Haptoglobin was readily measurable, baseline levels (in white rhinoceros with no clinical signs of disease) did not differ between genders, and a significant increase was seen in captive as well as in free-ranging white rhinoceros with inflammatory disease. Thus, haptoglobin is a positive acute phase protein in southern white rhinoceros with potential for use as an objective marker of disease.

The prevalence of Onchocerca infection in wild cervids from Denmark was studied in 119 fallow deer (Dama dama), 123 red deer (Cervus elaphus), 51 roe deer (Capreolus capreolus) and eight sika deer (Cervus Nippon) shot during the hunting season from October 2017 to January 2018 from 18 geographical locations across Denmark. The carcasses were macroscopic checked for nodules, and skin samples were examined for microfilaria. All roe deer, fallow deer and sika deer were negative for Onchocerca, while 30.9% red deer were positive for either microfilaria, nodules or both. Significantly more adult red deer (50.8%; 37.6-62.4; p < 0.0001) were infected with Onchocerca than juveniles <1 year (7.8%; 2.1-18.5), while there was an insignificant (p = 0.075) difference in prevalence observed between males (17.4%; 7.8-31.4) and females (41.7%; 30.2-53.9). Onchocerca-positive red deer were observed from 91.7% (11/12) of the sampled geographical locations. Species identification was done on adult worms from nodules taken from the lumbar region of 20 red deer of different geographical origin by sequencing the mitochondrial 12S, 16S and nad5 gene fragments. The sequences matched with previously published sequences for Onchocerca flexuosa. The high prevalence of Onchocerca infection caused by O. flexuosa in red deer in Denmark shows that Denmark has favourable vector conditions and a suitable environment for the maintenance of the parasite. To our knowledge, this is the first systematic study of Onchocerca in wild-living cervids in Denmark., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Purpose: To investigate the prevalence of Cryptosporidium spp. infection and Toxoplasma gondii antibodies in farmed and feral mink in Denmark., Methods: We examined meat juice from 235 feral mink and 306 farmed mink for T. gondii antibodies, and faecal samples from 113 feral mink and 166 farmed mink for Cryptosporidium oocyst excretion. Meat juice was analysed using a commercial indirect enzyme-linked immunosorbent assay and oocyst excretion was identified by a modified Ziehl-Neelsen method., Results: All farmed mink tested sero-negative, while 53.6% of feral mink were T. gondii sero-positive. The probability of being sero-positive for T. gondii was not associated with recent escapes from farms (p = 0.468), but was significantly higher for male feral mink (64.2%) than female feral mink (42.5%) (p = 0.0008). Only one feral mink and four farmed mink (2.4%) excreted Cryptosporidium oocysts., Conclusion: Farmed mink were all T. gondii sero-negative, whereas approximately half the feral mink were sero-positive. Cryptosporidium prevalence in farmed and feral mink were low. Overall, the public health risk of transmission of these two parasites via mink in Denmark is low., (© 2021. Witold Stefański Institute of Parasitology, Polish Academy of Sciences.)

The European hedgehog population is declining in Europe. It is therefore important to investigate the causes for the decline and monitor the general health of the species. We investigated the endoparasite occurrence in 299 dead European hedgehogs. Of these, endoparasites were detected in 69% of the individuals tested. We identified Crenosoma striatum , Capillaria aerophila (syn. Eucoleus aerophilus ), Capillaria spp. , coccidia, Cryptosporidium spp., Brachylaemus spp. and Capillaria hepatica . We also examined the hedgehogs for Giardia spp. and Echinococcus multilocularis but all were negative. Coccidia (n = 7, 2.5%) and Cryptosporidium spp. (n = 14, 5.2%) were only detected in individuals from Zealand, Lolland and Jutland south of the Limfjord. Single cases of Brachylaemus spp. (n = 1, 0.4%) and Capillaria hepatica (n = 1, 1.1%) were exclusively discovered in Jutland south and north of the Limfjord, respectively. These results indicate a regional difference in endoparasite species carried by European hedgehogs in Denmark. This stresses the need for hedgehogs to be cared for locally when admitted to wildlife rehabilitation centres, and to be released within their area of origin, to prevent spread of endoparasite infections among hedgehogs. Additionally, we explored the following possible determinants of parasite infection in the hedgehogs: sex, age, mortality category (in-care, natural and roadkill), infection with MRSA, genetic heterozygosity, month of death, geographical location and habitat type, and found that only age had a statistically significant effect on endoparasite prevalence, as we detected a lower occurrence of endoparasites in juvenile hedgehogs (<1 year) compared to the other age classes. However, pairwise comparisons of geographical regions did show significant differences in endoparasite occurrence: Zealand vs. Jutland south of the Limfjord and Zealand vs. Falster. We conclude that, in line with previous studies of European hedgehogs throughout their range in Western Europe, endoparasite infections are common and a natural part of their ecology., Competing Interests: The authors declare that there is no conflict of interest., (© 2021 Published by Elsevier Ltd on behalf of Australian Society for Parasitology.)

In the past few decades, the relevance of Dirofilaria immitis and Dirofilaria repens , causing cardiopulmonary and subcutaneous dirofilariosis in dogs and cats, and of Angiostrongylus vasorum , causing canine angiostrongylosis, has steadily increased in Central and Northern Europe. In this review, a summary of published articles and additional reports dealing with imported or autochthonous cases of these parasites is provided for Central (Austria, Czechia, Germany, Hungary, Luxemburg, Poland, Slovakia, Slovenia, and Switzerland) and Northern (Denmark, Finland, Iceland, Norway, and Sweden) Europe. Research efforts focusing on Dirofilaria spp. and A. vasorum have varied by country, and cross-border studies are few. The housing conditions of dogs, pet movements, the spread of competent vectors, and climate change are important factors in the spread of these nematodes. Dogs kept outside overnight are a major factor for the establishment of Dirofilaria spp. However, the establishment of invasive, diurnal, synanthropic, competent mosquito vectors such as Aedes albopictus may also influence the establishment of Dirofilaria spp. The drivers of the spread of A. vasorum remain not fully understood, but it seems to be influenced by habitats shared with wild canids, dog relocation, and possibly climatic changes; its pattern of spreading appears to be similar in different countries. Both Dirofilaria spp. and A. vasorum merit further monitoring and research focus in Europe.

Raccoon dogs have successfully invaded Europe, including Denmark. Raccoon dogs are potential vectors and reservoir hosts of several zoonotic pathogens and thus have the potential for posing a threat to both human and animal health. This study includes analysis of four zoonotic parasites, 16 tick-borne pathogens and two pathogen groups from 292 raccoon dogs collected from January 2018 to December 2018. The raccoon dogs were received as a part of the Danish national wildlife surveillance program and were hunted, found dead or road killed. The raccoon dogs were screened for Alaria alata and Echinococcus multilocularis eggs in faeces by microscopy and PCR, respectively, Trichinella spp. larvae in muscles by digestion, antibodies against Toxoplasma gondii by ELISA and screening of ticks for pathogens by fluidigm real-time PCR. All raccoon dogs tested negative for E. multilocularis and Trichinella spp., while 32.9% excreted A. alata eggs and 42.7% were T. gondii sero-positive. Five tick-borne pathogens were identified in ticks collected from 15 raccoon dogs, namely Anaplasma phagocytophilum (20.0%), Babesia venatorum (6.7%) , Borrelia miyamotoi (6.7%) , Neoehrlichia mikurensis (6.7%) and Rickettsia helvetica (60.0%). We identified raccoon dogs from Denmark as an important reservoir of T. gondii and A. alata infection to other hosts, including humans, while raccoon dogs appear as a negligible reservoir of E. multilocularis and Trichinella spp. infections. Our results suggest that raccoon dogs may be a reservoir of A. phagocytophilum ., Competing Interests: None., (© 2021 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.)

Aims: To determine inactivation of Cryptosporidium parvum oocysts and reduction of Escherichia coli and enterococci in cattle slurry added aqueous ammonia., Methods and Results: Escherichia coli, enterococci and nonviable C. parvum oocysts (DAPI+PI+) were enumerated every second day for 2 weeks in cattle slurry amended with 60 mmol l -1 aq. ammonia and compared with untreated slurry at three temperatures. Regardless of temperature, the proportion of nonviable C. parvum oocysts increased significantly faster over time in slurry with added ammonia than raw slurry (P = 0·021) corresponding to 62·0% higher inactivation (P = 0·001) at day 14. Additionally, 91·8% fewer E. coli and 27·3% fewer enterococci were observed in slurry added ammonia at day 14 compared to raw slurry., Conclusion: The addition of aqueous ammonia to raw slurry significantly reduced the viability of C. parvum oocysts and numbers of bacterial indicators. Hence, ammonia is usable at lower pathogen concentrations in slurry before application to agricultural land., Significance and Impact of the Study: Livestock waste is a valuable source of plant nutrients and organic matter, but may contain high concentrations of pathogens like E. coli and Cryptosporidium sp. that can be spread in the environment, and cause disease outbreaks. However, die-off rates of pathogens in organic waste can increase following increasing ammonia concentrations., (© 2020 The Society for Applied Microbiology.)

The feral mink population in Denmark consists of two groups of animals: mink born in the wild and mink that have recently escaped from farms. The aims of this study were to: (1) estimate the reproductive performance and mortality of the Danish mink born in the wild (wild-born) and mink escaped from farms (captive-born); (2) discuss the likelihood of a self-sustaining population of wild-born mink in Denmark; and (3) model the relationship between the pulp cavity width and the age of mink. During 2018, 247 wild caught mink were sent for necropsy at the Danish National Veterinary Institute. Based on body length, 112 were determined as captive-born and 96 as wild-born. The mean litter size ± SE of wild-born females was 7.6 ± 0.9 (range: 5-11 kits) and for captive-born females 5.9 ± 0.9 (range: 1-10 kits). The relationship between age (in months) of mink and pulp cavity width was highly significant. Individuals with a pulp cavity width of >35% were younger than one year. Based on fecundity, the turnover of the mink population was estimated to be 66%, and the yearly mortality was estimated at 69%. Hence, the population is slightly declining. In conclusion, a feral reproducing mink population in Denmark persists without a continuous influx of captive-born mink from farms.

SARS-CoV-2 infection is the cause of COVID-19 in humans. In April 2020, SARS-CoV-2 infection in farmed mink ( Neovision vision ) occurred in the Netherlands. The first outbreaks in Denmark were detected in June 2020 in three farms. A steep increase in the number of infected farms occurred from September and onwards. Here, we describe prevalence data collected from 215 infected mink farms to characterize spread and impact of disease in infected farms. In one third of the farms, no clinical signs were observed. In farms with clinical signs, decreased feed intake, increased mortality and respiratory symptoms were most frequently observed, during a limited time period (median of 11 days). In 65% and 69% of farms, virus and sero-conversion, respectively, were detected in 100% of sampled animals at the first sampling. SARS-CoV-2 was detected, at low levels, in air samples collected close to the mink, on mink fur, on flies, on the foot of a seagull, and in gutter water, but not in feed. Some dogs and cats from infected farms tested positive for the virus. Chickens, rabbits, and horses sampled on a few farms, and wildlife sampled in the vicinity of the infected farms did not test positive for SARS-CoV-2. Thus, mink are highly susceptible to infection by SARS-CoV-2, but routes of transmission between farms, other than by direct human contact, are unclear.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 μm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

The cardiopulmonary nematodes Angiostrongylus vasorum, Crenosoma vulpis, Capillaria aerophila and Aelurostrongylus abstrusus , are a cause of concern in the scientific and veterinary community, potentially causing significant disease in domestic animals. To investigate the potential of wild carnivores as reservoir hosts to these parasites, a total of 1041 animals from seven regions of Denmark were sampled: 476 raccoon dogs ( Nyctereutes procyonoides ), 367 red foxes ( Vulpes vulpes ), 123 American mink ( Neovison vison ), 31 beech martens ( Martes foina ), 30 Eurasian otters ( Lutra lutra ) and 14 polecats ( Mustela putorius ). Hearts and lungs were collected and examined for cardiopulmonary parasites. Capillaria aerophila was identified using morphology, whereas A. vasorum and C. vulpis were identified by a duplex real-time PCR, and A. abstrusus by conventional PCR. This is the first Danish report of A. vasorum and C. vulpis infections in raccoon dogs, mink and polecats, and of C. aerophila in raccoon dogs and beech martens. In addition, this is the first time A. vasorum and C. vulpis have been identified in wild animals from the island of Bornholm, just as it is the first report of C. vulpis in American mink, and C. vulpis and A. vasorum in polecats in Europe. The prevalence of A. vasorum appears to have increased in red foxes in Denmark compared to previous studies, while C. vulpis and C. aerophila prevalences are lower. Our data show that several wild carnivores can serve as reservoir hosts for A. vasorum, C. vulpis and C. aerophila in Denmark, and that A. vasorum appears more abundant than previously reported. It is speculated that the A. vasorum increase might relate to increased snail abundance, which may be due to a rise in mean yearly temperatures in Denmark., Competing Interests: None., (© 2020 The Authors.)

Background: Similar to the situation in other European countries, Danish wild boars may harbour a wide range of pathogens infectious to humans and domestic pigs. Although wild boars must be kept behind fences in Denmark, hunting and consumption of the meat may cause zoonotic transmission. Moreover, most infections of wild boars are transmissible to domestic pigs, which may have important economic consequences. The aim of this study was to investigate whether Danish wild boars were infected with bacteria and parasites transmissible to humans or domestic pigs: Brucella suis, methicillin-resistant Staphylococcus aureus (MRSA), Salmonella spp., Trichinella spp., lungworms and gastrointestinal parasites, especially Ascaris suum. This is the first study to investigate the prevalence of these important pathogens in Danish wild boars., Results: Wild boars from eight enclosures were analysed over a 5-year period. All tested wild boars were negative for B. suis (n = 240), MRSA (n = 244), Salmonella spp. (n = 115) and Trichinella spp. (n = 232), while eight parasite genera were identified in the faeces (n = 254): Ascaris suum, Capillaria sp., Cystoisospora suis, Eimeria spp., Metastrongylus sp. (lungworm), Strongyloides ransomi, Trichuris suis and strongylid eggs, i.e. strongyles not identified to the genera. Eimeria spp. and Metastrongylus sp. had the highest prevalence (92.3 and 79.5%, respectively) and were identified in wild boars from all eight enclosures, while the remaining parasite genera were present more sporadically., Conclusions: Wild boars from Denmark constitute a low risk of transmitting B. suis, MRSA, Salmonella spp. and Trichinella spp. to humans or domestic pigs, while economically important parasites transmissible to domestic pigs are highly prevalent in the wild boar population.

In Denmark, American mink (Neovison vison) have been bred for their fur since the mid-1920s. Mink escaping from farms may supply the feral population. Often, it is of biological and management interest to separate the population of feral mink (i.e. mink caught in the wild) in two groups: 1) mink born on farms i.e., escapees, and 2) mink born in the wild. In this study, two methods were used for separating feral mink into the two groups: a) Comparison of body length of farmed mink and feral mink, and b) Presence of a biomarker (tetracycline: an oral antibiotic used on mink farms). A total of 367 wild caught mink (from the mainland of Denmark and the island of Bornholm), and 147 mink from farms, collected during the period 2014-2018, were used for the analysis of body length. For the testing of tetracycline (TC) as a biomarker, 78 mink from farms where there was knowledge about TC treatment (with or without) were examined for fluorescent markings in the canine teeth. Results from both univariate analyses and Gaussian mixture model analysis demonstrated clear divisions between the mean body length (mean ± S.E., range) of farmed males (52.1 cm ± 0.4, 48-68) and farmed females (mean 44.0 ± 0.2, 40-50), and between farmed mink and wild caught mink. Mixture analysis identified two groups within each sex of the wild caught mink, one assigned to farmed mink (born in captivity) and another group of smaller mink suspected of being born in the wild. On Bornholm, the mean (±SD, range) length of males born in the wild was 43.7cm (± 0.3, 36-57) and for females 37.5cm (± 0.3, 32-45). The mean length (±SD, range) of males born in the wild in the mainland of Denmark was 42.5cm (± 2.3, 36-46) and for females 36.1cm (± 1.0, 34-37). Among the feral mink from mainland Denmark, 28.4% of males and 21.6% of females were identified as escapees, while 0% of the males and 1% of the females were identified as escapees among the wild caught mink on Bornholm. Eight percent of mink from farms using tetracycline were false negatives, while no false positives were found among mink from farms not using TC. TC fluorescence was found in five of 217 mink caught in the wild equivalent to 22% escapees in mainland Denmark. No TC markings were found in mink caught in the wild on Bornholm. In conclusion, both methods a) the body length of mink, and b) fluorescent biomarkers in canine teeth are considered as useful tools to identifing mink that have escaped from farms., Competing Interests: The authors have declared that no competing interests exist.

Aims: Patients with cancer are insufficiently represented in randomized clinical trials investigating efficacy of implantable cardioverter-defibrillators (ICDs). We aimed to describe outcomes in patients with a pre-existing diagnosis of cancer at time of ICD implantation., Methods and Results: We utilized Danish nationwide registries to identify primary and secondary prevention ICD implantations from 2007 to 2012. Multivariable Cox models were used to assess the risk of appropriate ICD therapy and mortality in patients with and without cancer at time of implantation. During a median follow-up of 2.1 years, 2935 primary prevention ICD and 2730 secondary prevention ICD implantations were identified. Out of these [289 (5.1%)] had pre-existing cancer [primary 140 (4.8%), secondary 149 (5.5%)]. No differential risk for appropriate ICD therapy was found between patients with or without cancer, [primary cancer: 19/140, no cancer: 380/2795, hazard ratio (HR) = 1.07 (0.67-1.69)] and [secondary cancer: 42/149, no cancer: 699/2581, HR = 1.28 (0.93-1.75)]. In primary patients, cancer was not associated with higher risk of 1-year [cancer: 10/140, no cancer: 133/2795, HR = 1.20 (0.84-2.28)] or all-time mortality [cancer: 22/140, no cancer: 339/2795, HR = 1.13 (0.74-1.75)]. In secondary patients, cancer was associated with a higher 1-year [cancer: 19/149, no cancer: 108/2581, HR = 2.62 (1.60-4.29)] and all-time mortality [cancer: 44/149, no cancer: 315/2581, HR = 2.36 (1.71-3.24)]., Conclusion: Implantable cardioverter-defibrillators were implanted in a minority of cancer patients. No difference in risk of appropriate therapy was observed between cancer and non-cancer patients, regardless of implant indication. Cancer was associated with increased mortality in secondary prevention ICD patients, but not in primary prevention ICD patients. In secondary prevention ICD patients, the majority of deaths were attributable to cancer., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths., (© 2018 Novo Nordisk A/S. Journal of Thrombosis and Haemostasis © 2018 International Society on Thrombosis and Haemostasis.)

Aims: Electrical storm (ES) is a serious arrhythmic syndrome that is characterized by recurrent episodes of ventricular arrhythmias. Electrical storm is associated with increased mortality and morbidity despite the use of implantable cardioverter-defibrillators (ICDs). Predicting ES could be essential; however, models for predicting this event have never been developed. The goal of this study was to construct and validate machine learning models to predict ES based on daily ICD remote monitoring summaries., Methods and Results: Daily ICD summaries from 19 935 patients were used to construct and evaluate two models [logistic regression (LR) and random forest (RF)] for predicting the short-term risk of ES. The models were evaluated on the parts of the data not used for model development. Random forest performed significantly better than LR (P < 0.01), achieving a test accuracy of 0.96 and an area under the curve (AUC) of 0.80 (vs. an accuracy of 0.96 and an AUC of 0.75). The percentage of ventricular pacing and the daytime activity were the most relevant variables in the RF model., Conclusion: The use of large-scale machine learning showed that daily summaries of ICD measurements in the absence of clinical information can predict the short-term risk of ES.

Toxoplasma gondii is a zoonotic parasite of worldwide importance. In this study, we estimated T. gondii seroprevalence in extensively farmed wild boars in Denmark, where little is known about T. gondii in animal hosts. Our study focused on wild boars because they are considered good indicator species for the presence of T. gondii, and wild boar meat is used for human consumption. Serum samples from 101 wild boars collected in 2016-2018 from five different locations from the continental part of Denmark, Jutland, were screened for anti-T. gondii antibodies. The samples were analysed using a commercial indirect enzyme-linked immunosorbent assay (ELISA). Samples from 28 (27.7%) of the 101 wild boars tested positive with the ELISA. The odds for a wild boar to test seropositive were higher if it was sampled during the hunting season 2017-2018 than during 2016-2017 and if it was reported to be at least 1 year old than if it was younger (logistic regression model with the two variables: odds ratios 17.5 and 3.9, respectively). A substantial proportion of the investigated extensively farmed wild boars had been exposed to T. gondii. Moreover, the parasite appeared widespread, at least in the continental part of Denmark, Jutland, as seropositive wild boars were found from all five sampled locations. Assuming seropositivity indicates hosting viable parasites, consumption of undercooked wild boar meat from Denmark is a potential source of T. gondii infections to other hosts, including humans.

Background: Bovine fasciolosis is an economically important livestock disease in Europe, and represents a particular challenge for organic farms, where cattle are grazed extensively and the use of anthelmintic is limited. A two-year longitudinal study was conducted on two conventional and two organic Danish dairy farms to examine the current temporal trend of F. hepatica infection on-farm, and to gather data of practical relevance for parasite control. Data were collected both at the herd and individual level using currently available diagnostic methods: a commercial serum antibody ELISA, a commercial copro-antigen ELISA, faecal egg counts, and monthly bulk tank milk (BTM) ELISA. The temporal patterns (animal age, farm-level temporal trends and seasonality) in the animal-level test results were analysed by generalised additive mixed models (GAMM)., Results: Patterns of infection differed substantially between the farms, due to different grazing management and anthelmintic use. However, animals were first infected at the age of 1.5-2 years (heifers), and most at-risk animals sero-converted in autumn, suggesting that summer infections in snails prevail in Denmark. Our results also suggest that the lifespan of the parasite could be over 2 years, as several cows showed signs of low grade infection even after several years of continuous indoor housing without access to freshly-cut grass. The serum antibody ELISA was able to detect infection first, whereas both copro-antigen ELISA and faecal egg counts tended to increase in the same animals at a later point. Decreasing BTM antibody levels were seen on the two farms that started anthelmintic treatment during the study., Conclusions: While important differences between farms and over time were seen due to varying grazing management, anthelmintic treatment and climatic conditions, the young stock was consistently seen as the main high-risk group and at least one farm also had suspected transmission (re-infection) within the lactating herd. Careful interpretation of test results is necessary for older cows as they can show persistent infections several years after exposure has stopped. Rigorous treatment regimens can reduce BTM ELISA values, but further research is needed to develop a non-medicinal approach for sustainable management of bovine fasciolosis.

Capillaria plica is a parasitic nematode belonging to the family Capillariidae. The adult parasites reside in the urinary tract of wild and domestic canines. The infection is most often asymptomatic, but can cause a wide range of symptoms including urinary bladder inflammation, pollacisuria, dysuria and hematuria. Canines acquire the infection by ingesting the intermediate host, the earthworm (Lumbricidae). Epidemiological studies on C. plica infection in wildlife are few and only one previous Danish study examined the prevalence in red foxes, while studies on prevalence in other animals are limited. We examined the urine sediment or urinary bladder from 375 Raccoon dogs ( Nyctereutes procyonoides ), 247 red foxes ( Vulpes vulpes ), 20 beech martens ( Martes foina ), 16 wild mink ( Neovison vison ), 14 otters ( Lutra lutra ), nine European polecats ( Mustela putorius ), three European badgers ( Meles meles ) and one golden jackal ( Canis aureus ) received as a part of Danish wildlife surveillance. Capillaria plica was detected in 73.7% of red foxes, 20.0% of beech martens, 0.5% of raccoon dogs, and in the Golden jackal. Red foxes originating from all 5 regions of Denmark were infected, although with a significantly higher prevalence in the three regions in Jutland compared to Region Zealand.

A survey was conducted on 30 Danish mink farms from April to October 2016 to determine the prevalence and species of Eimeria in Danish farmed mink. In total, 2.6% of mink faecal samples (108/4140) were positive for Eimeria vison-like oocysts by microscopy, with 24.8% (78/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts (n = 20) identified Eimeria vison-like oocysts measuring 21.0 × 13.8 μm with a length/width (L/W) ratio of 1.5. Phylogenetic analysis of 18S rRNA sequences (1221 bp) from three positive mink indicated that Eimeria vison-like shared the highest genetic similarity to Eimeria sp. ex Apodemus agrarius from a Striped field mouse (A. agrarius) from the Czech Republic (99.6%). Analysis of a shorter region of 18S (531 bp) revealed that the E. vison-like genotype sequences grouped in the same clade and shared 97.7% similarity with E. furonis. At the cytochrome c oxidase subunit I (COI) locus, mink-derived sequences were not available from GenBank and phylogenetic analysis placed the novel E. vison-like in a clade with E. cf. ictidea (99.4% similarity) from a black footed ferret (Mustela nigripes) from Canada.

In Western Europe, the Echinococcus multilocularis lifecycle is predominantly sylvatic, typically involving red foxes (Vulpes vulpes) as the main definitive hosts with Microtus spp. and Arvicola spp. as intermediate hosts. During a 4-year surveillance study (2012-2015), Danish red foxes and raccoon dogs (n = 1345) were examined for E. multilocularis. Moreover, 134 insectivores and rodents collected in South Jutland during spring and summer 2016 were examined for the presence of metacestodes. The sedimentation and counting technique and molecular typing were used to identify E. multilocularis infections in the carnivores, while the rodent livers were examined macro- and microscopically for parasite lesions. Following morphological identification of E. multilocularis adult worms, the identity was verified by sequence analysis of the 12S rRNA gene in most cases (n = 13). Echinococcus multilocularis infection was demonstrated in 19 red foxes (Vulpes vulpes) originating from only two specific areas of South Jutland, namely Højer and Grindsted, and in two raccoon dogs (Nyctereutes procyonoides), originating from Højer. In Højer, 28.5% (CI 95% 11.7-45.3) of the examined red foxes were E. multilocularis positive per year. Moreover, positive red foxes were identified each year from 2012 to 2015, while E. multilocularis positive red foxes were only identified in Grindsted in 2013 (4.0%) and 2014 (6.4%). In contrast, all collected rodents were negative for E. multilocularis. We conclude that E. multilocularis is locally endemic in South Jutland with a high local prevalence in Højer.

Background: Choice of left ventricular pacing vector (LVPV) affects the QRS-duration (QRSd) in patients with Cardiac Resynchronization Therapy (CRT). It is not known whether testing all LVPVs reduces QRSd compared to device-based “standard-programming”., Methods: In patients implanted with CRT several ECGs were recorded for each usable LVPV (no phrenic nerve stimulation and threshold <3.5 V) and during “standard-programming” after device-based optimization of AV/VV delays., Results: 22 consecutive patients were included. Average QRSd reduction after CRT + “standard-programming” was 27.3 ± 22 ms. Additional QRSd-reduction was possible in 4 patients by changing the LVPV, and in 5 other patients after optimization of AV- and VV delays without changing LVPV., Conclusions: Shortening of QRSd compared to “standard-programming” was possible approximately 40% of these patients treated with CRT by testing all LVPVs and re-optimizing AV/VV delays during follow-up. Studies of clinical effects are needed.

Purpose: Shortening of the QRS duration (QRSd) is often used to guide device optimization and reprogramming in patients with cardiac resynchronization therapy (CRT). Detecting the small changes expected during reprogramming requires that the QRSd can be measured with high precision, but this has never been studied in patients with CRT. In this study, we wanted to assess the precision of automated QRSd measurement in patients treated with CRT using two commonly available electrocardiographs., Methods: Patients treated with CRT were recruited during routine follow-up in our pacemaker clinic. In all participants, a number of immediate successive ECGs were recorded with the GE MAC 5500 (Mac55) and the GE MAC 1600 (Mac16). Data were analyzed with a linear mixed model., Results: A total of 785 12-lead ECGs were recorded in 36 patients with an average of 11.2 and 10.6 ECGs per patient with the Mac55 and Mac16, respectively. The Mac55 measured the QRSd longer by 10.3 milliseconds (ms) (95% CI 7.1-13.5 ms, p < 0.001) and with significantly smaller standard deviation for repeated measurements (6.3 vs. 10.4 ms, p < 0.001). Limits of agreement were ± 17.5 and ± 28.8 ms for the Mac55 and Mac16, respectively., Conclusions: Automated measurement of QRSd in patients with CRT shows low precision with limits of agreement of ± 17.5 and ± 28.8 ms for repeated measurements in two commercially available electrocardiographs. Device programming solely by QRSd changes should be done with caution, and clinical effects should be demonstrated in future trials. Device programming based on QRSd changes should be done with caution until the ability of this measure to predict clinical outcome can be demonstrated in prospective study.

Background: The nematophagous fungus Pochonia chlamydosporia can degrade ascarid (e.g. Ascaridia galli) eggs in agar and soil in vitro. However, it has not been investigated how this translates to reduced infection levels in naturally exposed chickens. We thus tested the infectivity of soil artificially contaminated with A. galli (and a few Heterakis gallinarum) eggs and treated with P. chlamydosporia. Sterilised and non-sterilised soils were used to examine any influence of natural soil biota., Methods: Unembryonated eggs were mixed with sterilised (S)/non-sterilised (N) soil, either treated with the fungus (F) or left as untreated controls (C) and incubated (22 °C, 35 days) to allow eggs to embryonate and fungus to grow. Egg number in soil was estimated on days 0 and 35 post-incubation. Hens were exposed to the soil (SC/SF/NC/NF) four times over 12 days by mixing soil into the feed. On day 42 post-first-exposure (p.f.e.), the hens were euthanized and parasites were recovered. Serum A. galli IgY level and ascarid eggs per gram of faeces (EPG) were examined on days -1 and 36 (IgY) or 40 p.f.e. (EPG)., Results: Egg recovery in SF soil was substantially lower than in SC soil, but recovery was not significantly different between NF and NC soils. SF hens had a mean worm count of 76 whereas the other groups had means of 355-453. Early mature/mature A. galli were recovered from SF hens whereas hens in the other groups harboured mainly immature A. galli. Heterakis gallinarum counts were low overall, especially in SF. The SF post-exposure IgY response was significantly lower while EPG was significantly higher compared to the other groups., Conclusions: Pochonia chlamydosporia was very effective in reducing ascarid egg numbers in sterilised soil and thus worm burdens in the exposed hens. However, reduced exposure of hens shifted A. galli populations toward a higher proportion of mature worms and resulted in a higher faecal egg excretion within the study period. This highlights a fundamental problem in ascarid control: if not all eggs in the farm environment are inactivated, the resulting low level infections may result in higher contamination levels with associated negative long-term consequences.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (K D ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation., Summary: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (K D ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood., (© 2018 International Society on Thrombosis and Haemostasis.)

Aims: To evaluate the association between an apical vs. non-apical right ventricular lead position (RV-LP) and clinical outcome in a large nationwide cohort of patients treated with cardiac resynchronization therapy (CRT)., Methods and Results: We included consecutive Danish patients receiving a CRT device from 2008 to 2012, identified from the Danish Pacemaker and ICD Register. Endpoints were mortality, mortality or hospitalization for heart failure (HF), and clinical response (improvement ≥1 New York Heart Association class at follow-up). Subgroup analysis was made for ischaemic heart disease (IHD) and non-IHD. Cox and logistic regression analyses were used to calculate adjusted HR (aHR) and adjusted odds ratios with 95% confidence intervals (CI). A total of 2883 patients received a CRT device during the period. We excluded 301 patients that did not meet standard CRT indication. In 2391 (93%) of the remaining 2582 patients the RV-LP was defined as apical in 647 (27%) and non-apical in 1744 (73%). After mean 3.2 ± 2 years, 660 patients had died, 1275 patients were hospitalized for HF, and 1021 were responders. With a non-apical RV-LP the aHR for mortality was 0.93 (95% CI 0.0.79-1.10, P = 0.40) and the aHR for the combined endpoint of mortality or HF-hospitalization was 0.89 (95% CI 0.79-0.99, P = 0.03). These findings were significant only in patients with non-IHD. There was no association between clinical response and RV-LP., Conclusion: A non-apical RV-LP is associated with lower risk of meeting the combined endpoint of mortality or hospitalization for HF in patients with CRT. In subgroup analysis, this association was present only in patients with non-IHD.

Viability assessment of Cryptosporidium parvum oocysts is crucial for evaluation of the public health significance of this important zoonotic protozoon. Viability is commonly assessed in wet mounts after acid pretreatment and staining with fluorogenic vital dyes. However, in some studies, oocyst viability is evaluated in dry mounts after staining in suspension. Here, we evaluate the effect of acid pretreatment in nine replicate samples and compare the assessment of oocyst viability after evaluation in wet and dry mounts, respectively. Although acid pretreatment had no significant effect on the viability scores, data obtained by scoring oocysts in dry mounts resulted in ∼25% underestimation of the proportion of viable oocyst (82.5% ± 0.9% [wet mount +acid], 57.7% ± 2.3% [dry mount, ÷ acid], 76.0% ± 1.7% [wet mount, ÷ acid]), while the proportions of nonviable oocysts (DAPI+/PI+) were comparable for wet and dry mounts (9.7% ± 0.4% [wet mount +acid], 12.1 ± 1.5% [dry mount, ÷ acid], 15.5% ± 1.1% [wet mount, ÷ acid]).

We report Taenia ovis infection in Danish sheep for the first time. In spring 2016, the metacestode stage of T. ovis was at slaughter observed in heart muscles, diaphragm and skeletal muscles from approx. a third of all sheep from one specific farm localised in South Jutland. The diagnosis was confirmed by molecular typing of the mitochondrial cytochrome c oxidase I (cox1) gene. Three newly imported dogs were suspected but the definitive host was unidentifiable. The finding is not regulated in the meat control procedures. However, infected meat is usually condemned due to aesthetic reasons causing economic losses. Thus, finding of T. ovis is of concern to sheep meat producers in the area, as the infection could have spread further on to other farms., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aims: We aimed to examine the use of guideline recommended beta-blocker therapy prior to and after primary prevention implantable cardioverter defibrillator (ICD) implantation in a 'real-life' setting., Methods and Results: From the Danish Pacemaker and ICD Registry we identified all 1st-time primary prevention ICD and cardiac resynchronization therapy defibrillator (CRT-D) implantations in Denmark from 2007-12 (n = 2935). Use of beta-blocker, type and dose was acquired through the Danish Prescription Registry. According to guideline recommendations, we defined target daily doses as ≥50 mg carvedilol and ≥200 mg metoprolol. Prior to implantation 2427 of 2935 (83%) patients received beta-blocker therapy, with 2166 patients (89%) having initiated treatment 3 months or more prior to implantation. The majority of patients was prescribed carvedilol (52%) or metoprolol (41%). Patients on carvedilol reached target dosages more frequently than patients on metoprolol, with 39% of patients on carvedilol and 26% of patients on metoprolol at the time of implantation (P < 0.001 for all time-points). Increase in proportion of patients reaching target daily doses was observed for both carvedilol and metoprolol after ICD implantation. Carvedilol treatment was a strong predictor for being on target dose of BB at time of implant, as was treatment with angiotensin-converting enzyme inhibitors and/or spironolactone, no history of myocardial infarction, younger age and less pronounced heart failure symptoms., Conclusion: In a real-life setting of primary prevention ICD patients, 39% and 26% of patients were titrated to optimal target dose of carvedilol or metoprolol prior to implantation. A higher proportion of patients on carvedilol reached target dose, as compared with metoprolol., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Decysin-1 (ADAMDEC1) is an orphan ADAM-like metalloprotease with unknown biological function and a short domain structure. ADAMDEC1 mRNA has previously been demonstrated primarily in macrophages and mature dendritic cells. Here, we generated monoclonal antibodies (mAbs) against the mature ADAMDEC1 protein, as well as mAbs specific for the ADAMDEC1 pro-form, enabling further investigations of the metalloprotease. The generated mAbs bind ADAMDEC1 with varying affinity and represent at least six different epitope bins. Binding of mAbs to one epitope bin in the C-terminal disintegrin-like domain efficiently reduces the proteolytic activity of ADAMDEC1. A unique mAb, also recognizing the disintegrin-like domain, stimulates the caseinolytic activity of ADAMDEC1 while having no significant effect on the proteolysis of carboxymethylated transferrin. Using two different mAbs binding the disintegrin-like domain, we developed a robust, quantitative sandwich ELISA and demonstrate secretion of mature ADAMDEC1 protein by primary human macrophages. Surprisingly, we also found ADAMDEC1 present in human plasma with an approximate concentration of 0.5 nM. The presence of ADAMDEC1 both in human plasma and in macrophage cell culture supernatant were biochemically validated using immunoprecipitation and Western blot analysis demonstrating that ADAMDEC1 is secreted in a mature form.

Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase, the thrombin-generating complex of factor Xa (FXa) and factor Va (FVa), during the initiation of coagulation. This inhibition requires binding of a conserved basic region within TFPIα to a conserved acidic region in FXa-activated and platelet-released FVa. In this study, the contribution of interactions between TFPIα and the FXa active site and FVa heavy chain to prothrombinase inhibition were examined to further define the inhibitory biochemistry. Removal of FXa active site binding by mutation or by deletion of the second Kunitz domain (K2) of TFPIα produced 17- or 34-fold weaker prothrombinase inhibition, respectively, establishing that K2 binding to the FXa active site is required for efficient inhibition. Substitution of the TFPIα basic region uncharged residues (Leu252, Ile253, Thr255) with Ala (TFPI-AAKA) produced 5.8-fold decreased inhibition. This finding was confirmed using a basic region peptide (Leu252-Lys261) and Ala substitution peptides, which established that the uncharged residues are required for prothrombinase inhibitory activity but not for binding the FVa acidic region. This suggests that the uncharged residues mediate a secondary interaction with FVa subsequent to acidic region binding. This secondary interaction seems to be with the FVa heavy chain, because the FV Leiden mutation weakened prothrombinase inhibition by TFPIα but did not alter TFPI-AAKA inhibitory activity. Thus, efficient inhibition of prothrombinase by TFPIα requires at least 3 intermolecular interactions: (1) the TFPIα basic region binds the FVa acidic region, (2) K2 binds the FXa active site, and (3) Leu252-Thr255 binds the FVa heavy chain.

Purpose: To evaluate the effect of lymphadenectomy (LND) in conjunction with nephroureterectomy on cancer-specific mortality (CSM) and overall survival (OS) for patients with muscle-invasive UTUC., Methods: A retrospective, multicenter study of patients with UTUC, clinical stage N0M0, who underwent nephroureterectomy between January 2008 and December 2014 was conducted. Outcome measures were OS and CSM., Results: In total, 298 patients underwent robot-assisted or laparoscopic radical nephroureterectomy with a final histological diagnosis of UTUC. LND was performed in 46 (15.4%). One hundred and seventy-two patients (62%) had non-muscle-invasive disease (NMID); 105 patients (38%) had muscle-invasive disease (MID). Median time of follow-up was 43.5 months (95% CI 36.0-47.2). For patients with MID, the 5-year cumulative incidence of all-cause mortality and CSM was 73.5% (95% CI 60.4-86.6) and 52.4% (95% CI 38.9-65.9), respectively (p < 0.0001). There was no significant difference in OS between patients with N1 and patients with N0 disease (p = 0.53). The 5-year OS rates were 30.5% (95% CI 6.6-54.4) and 25.7% (95% CI 10.9-40.5), respectively. This study is limited by its retrospective nature. There may also have been bias in the selection of patients undergoing LND., Conclusions: Five-year OS and CSM are comparable between patients with N1 and N0 MID. This evidence may support the use of the LND procedure in patients with muscle-invasive UTUC.

Aims: The validity of registry data on defibrillator lead performance is described only sparsely, despite its clinical importance. This study investigated the validity of defibrillator lead performance registry data in a nationwide and population-based registry., Methods and Results: We identified all reported surgical interventions due to defibrillator lead events in the Danish Pacemaker and ICD Register (DPIR) from 2000 to 2013. Medical records of all patients (n = 753) were examined blinded for 5 predefined intervention types and 18 reasons for lead intervention. The overall level of agreement for the types of lead intervention had a positive predictive value (PPV) of 89.4% [95% confidence interval (CI): 87.0-91.5%] and an adjusted agreement (κ value) of 0.81 (95% CI: 0.77-0.85) representing an almost perfect match. Regarding the reasons for lead intervention, the overall PPV was 63.0% (95% CI: 54.8-61.7%) with a κ value of 0.60 (95% CI: 0.55-0.64) representing a moderate match with only few subcategories having low PPV. By redistribution of the specific reasons for lead interventions into three categories commonly used to report lead performance, the overall PPV improved to 87.9% (95% CI: 85.2-90.2%) with a κ value of 0.82 (95% CI:0.78-0.86) representing an almost perfect match., Conclusion: The validity of data on defibrillator lead performance recorded in the DPIR is excellent for the specific types of lead intervention and good for the specific reasons for defibrillator lead intervention. The validity of the less detailed overall reasons for lead interventions commonly used to report lead performance is also excellent. These findings indicate high registry data quality appropriate for scientific analysis and industry-independent post-marketing surveillance., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Aims: The objective of this study was to investigate whether it is safe to perform 1.5-Tesla magnetic resonance imaging (MRI) scans in pacemaker (PM) patients without pulse oximetry or electrocardiogram monitoring and with no special specific absorption rate (SAR) or time limits, provided that the PMs are interrogated and programmed to asynchronous mode prior to the scan., Methods and Results: This study reports the outcome of 207 MRI scans on PM patients at Rigshospitalet, Copenhagen University Hospital from June 2010 to September 2013. All MRIs were performed with the PMs in asynchronous mode and without additional monitoring. There were no adverse events registered among the PM patients during the study period. The only statistically significant change after MRI scans was a small, but clinically insignificant increase in atrial sense. No occurrences of reprogramming to power-on-reset were registered., Conclusion: It is possible to perform MRI scans relatively safely in PM patients without additional monitoring or change in the normal MRI protocol, given that the PM has been assessed and reprogrammed prior to MRI. This is especially important to remember in the acute setting where MRI scans may be delayed when monitoring facilities are unavailable., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Inappropriate implantable cardioverter-defibrillator (ICD) shock therapy is painful, stressful, and typically occurs unexpected in conscious patients and may be related to a less favourable prognosis. In our institution, we have observed four cases of multiple inappropriate ICD shocks during reset to VVI backup mode. All four patients were implanted with a St Jude Medical ICD since 2010. The reset to VVI backup mode happens as a 'safety' response when the ICD encounters errors in the software or hardware often due to electromagnetic interference. The ICD then operates in a simple mode, with only a ventricular fibrillation (VF) zone starting at 146 b.p.m., with shock therapy only and changes in sensitivity settings making the ICD more sensitive. In all cases, the reason for the multiple inappropriate shocks was that the VF zone was reached due to exercise-induced sinus tachycardia or due to oversensing during sinus rhythm. The VVI backup mode has to balance between protection from failure of ICD therapy during life-threatening ventricular arrhythmias and from inappropriate shocks. It seems the non-programmable parameters in VVI backup mode of St Jude Medical ICDs carry an unacceptable high risk of inappropriate shocks during normal rhythm as illustrated by our four cases. A higher VF zone comparable with the zones chosen by the other manufacturer would give a better balance, since it is very unlikely that a patient will need shock therapy urgently for slow ventricular tachycardia., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.)

Aims: In a nationwide cohort of primary (PP-ICD) and secondary prevention (SP-ICD) implantable cardioverter defibrillator (ICD) patients, we aimed to investigate the association between co-morbidity burden and risk of appropriate ICD therapy and mortality., Methods and Results: We identified all patients >18 years, implanted with first-time PP-ICD (n = 1873) or SP-ICD (n = 2461) in Denmark from 2007 to 2012. Co-morbidity was identified in administrative registers of hospitalization and drug prescription from pharmacies. Co-morbidity burden was defined as the number of pre-existing non-ICD indication-related co-morbidities including atrial fibrillation, diabetes, chronic obstructive pulmonary disease, chronic renal disease, liver disease, cancer, chronic psychiatric disease, and peripheral and/or cerebrovascular disease, and divided into four groups (co-morbidity burden 0, 1, 2, and ≥3). Through Cox models, we assessed the impact of co-morbidity burden on appropriate ICD therapy and mortality. Increasing co-morbidity burden was not associated with increased risk of appropriate therapy, irrespective of implant indication [all hazard ratios (HRs) 1.0-1.4, P = NS]. Using no co-morbidities as reference, increasing co-morbidity burden was associated with increased mortality risk in PP-ICD patients (co-morbidity burden 1, HR 2.1; comorbidity burden 2, HR 3.7; co-morbidity burden ≥3, HR 6.6) (all P < 0.001) and SP-ICD patients (co-morbidity burden 1, HR 2.2; co-morbidity burden 2, HR 3.8; co-morbidity burden ≥3, HR 5.8). With increasing co-morbidity burden, an increasing frequency of patients died without having utilized their device, with 72% PP-ICD and 45% SP-ICD patients with co-morbidity burden ≥3 dying without prior appropriate ICD therapy., Conclusion: Increasing co-morbidity burden was not associated with increased risk of appropriate ICD therapy. With increasing co-morbidity burden, mortality increased, and a higher proportion of patients died, without ever having utilized their device., (© 2016 The Authors. European Journal of Heart Failure © 2016 European Society of Cardiology.)

Background: Non-response to cardiac resynchronization therapy (CRT) might be due to insufficient resynchronization as a result of a sub-optimal left ventricular lead positon (LV-LP)., Objective: To evaluate the impact of different LV-LPs on mortality and symptomatic improvement in a large cohort of patients treated with CRT., Methods: We performed a nationwide cohort study on consecutive patients receiving a CRT device from 1997 to 2012 registered in the Danish pacemaker and ICD register. The LV-LP was defined clockwise in a left anterior oblique (LAO) view and categorized as anterior (≤2 o'clock), lateral (2 to 4 o'clock) or posterior (>4 o'clock), and as basal, mid-ventricular, or apical in a right anterior oblique (RAO) view. Outcomes were all cause mortality and clinical response (improvement in NYHA class). Adjusted hazard ratio (aHR) and odds ratio (aOR) with 95% confidence intervals (CI) were calculated using Cox and logistic regression analysis., Results: A total of 2594 patients were included. A lateral LV-LP, (aHR 0.77, 95% CI 0.64-0.92, p=0.004), and a posterior LV-LP, (aHR 0.71 95% CI 0.53-0.97, p=0.029) were associated with lower mortality as compared to an anterior LV-LP. A lateral LV-PV was associated with higher clinical response rate as compared to an anterior LV-LP (aOR 1.37, 1.03-1.83, p=0.032). No statistically significant associations were observed between LV-LP in the RAO view and mortality or clinical response., Conclusion: An anterior left ventricular lead position is associated with increased all-cause mortality and lower clinical response rate in patients treated with CRT and should be avoided., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: The best choice of defibrillator lead in patients with routine implantable cardioverter-defibrillator (ICD) is not settled. Traditionally, most physicians prefer dual-coil leads but the use of single-coil leads is increasing., Objective: The purpose of this study was to compare clinical outcomes in patients with single- and dual-coil leads., Methods: All 4769 Danish patients 18 years or older with first-time ICD implants from 2007 to 2011 were included from the Danish Pacemaker and ICD Register. Defibrillator leads were 38.9% single-coil leads and 61.1% dual-coil leads. The primary end point was all-cause mortality. Secondary end points were lowest successful energy at implant defibrillation testing, first shock failure in spontaneous arrhythmias, structural lead failure, and lead extraction outcomes., Results: Single-coil leads were associated with lower all-cause mortality with an adjusted hazard ratio of 0.85 (95% confidence interval 0.73-0.99; P = .04). This finding was robust in a supplementary propensity score-matched analysis. However, dual-coil leads were used in patients with slightly higher preimplant morbidity, making residual confounding by indication the most likely explanation for the observed association between lead type and mortality. The lowest successful defibrillation energy was higher using single-coil leads (23.2 ± 4.3 J vs 22.1 ± 3.9 J; P < .001). No significant differences were observed for other secondary end points showing high shock efficacies and low rates of lead failures and extraction complications., Conclusion: Shock efficacy is high for modern ICD systems. The choice between single-coil and dual-coil defibrillator leads is unlikely to have a clinically significant impact on patient outcomes in routine ICD implants., (Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Although pigs are commonly infected with Cryptosporidium spp. and Giardia duodenalis, including potentially zoonotic species or genotypes, little is known about age-related infection levels, seasonal differences and genetic variation in naturally infected pigs raised in organic management systems. Therefore, the current study was conducted to assess seasonal and age-related variations in prevalence and infection intensity of Cryptosporidium and Giardia, evaluate zoonotic potential and uncover correlations between species/genotypes, infection intensity and faecal consistency. Shedding of oocysts and cysts ((oo-)cysts) was monitored at quarterly intervals (September 2011-June 2012) in piglets (n = 152), starter pigs (n = 234), fatteners (n = 230) and sows (n = 240) from three organic farms in Denmark. (oo-)Cysts were quantified by immunofluorescence microscopy; and 56/75 subsamples from Cryptosporidium infected pigs were successfully analysed by PCR amplification and partial sequencing of the small subunit (SSU) 18S rRNA and hsp70genes, while 13/67 Giardia subsamples were successfully analysed by amplification and partial sequencing of the 18S rRNA and the gdh genes. Altogether, Cryptosporidium or Giardia infections were observed in 40.9% (350/856) and 14.0% (120/856) of the pigs, respectively, including 8.2% (70/856) infected with both parasites. Prevalence, intensity of infections and presence of Cryptosporidium species varied significantly between age-groups; 53.3% piglets, 72.2% starter pigs, 40.4% fatteners and 2.9% sows were infected with Cryptosporidium, whereas 2.0% piglets, 27.4% starter pigs, 17.8% fatteners and 5.0% sows were infected with Giardia. The overall prevalence was stable throughout the year, except for dual-infections that were more prevalent in September and December (p < 0.05). The infection intensity was age-related for both parasites, and dual-infected pigs tended to excrete lower levels of oocysts compared to pigs harbouring only Cryptosporidium. Likewise, pigs infected with Cryptosporidium scrofarum excreted fewer oocysts (mean CPG: 54,848 ± 194,508CI: 9085-118,781) compared to pigs infected with Cryptosporidium suis (mean OPG: 351,035 ± 351,035CI: 67,953-634,117). No correlation between faecal consistency and (oo-)cyst excretion levels was observed. Of the successfully genotyped isolates, 38/56 (67.9%) were C. scrofarum and 18/56 (32.1%) were C. suis, while the livestock specific G. duodenalis Assemblage E was detected in 11/13 (84.6%) isolates and the potentially zoonotic Assemblage A was identified in 2/13 (15.4%) isolates. Piglets exclusively hosted C. suis, with one exception, while starter pigs and fatteners predominantly hosted C. scrofarum. As organic pigs are partly reared outdoors, environmental contamination with Cryptosporidium and Giardia is inevitable. Nevertheless, the present data indicate that the potential public health risk associated with both of these parasites in Danish organic pig production seems to be negligible., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A humoral immune response following helminth infection in pigs is well documented. However, it has been difficult to confirm the existence of antibody mediated resistance against the large roundworm, Ascaris suum, and whipworm, Trichuris suis, in experimental settings by correlating worm burdens or egg excretion with specific antibody levels. We set out to investigate the association between worm load and T. suis and A. suum specific serum antibody levels (IgG1, IgG2 and IgA) against excretory-secretory products of adults and third stage larvae, respectively, measured at 0, 7 and 14 weeks p.i. in a trickle-infected F1-resource-population of crossbred pigs (n=195). Furthermore, we wanted to determine the heritability of these antibody isotypes during the course of infection. Most pigs remained infected with A. suum throughout the experiment while they expelled T. suis between 7 and 14 weeks post infection (p.i.). Parasite specific IgG1 and IgA were significantly (P<0.001) elevated after 7 and 14 weeks of infection, whereas parasite specific IgG2 levels only changed slightly at 14 weeks p.i.. However, the observed association between specific antibody isotype levels and faecal egg counts and macroscopic worm load was weak. The relative heritabilities of the different parasite specific isotypes were assessed and resulted in significant heritability estimates for parasite specific IgG1 and IgA. The highest heritabilities were found for A. suum specific IgG1 (h(2)=0.41 and 0.46 at 7 and 14 weeks p.i., respectively). Thus, the present study demonstrates that host genetic factors influence the IgG1 and IgA antibody isotype responses specific to two of the most common gastrointestinal nematodes of swine whereas specific antibody levels were poorly associated with egg excretion and the presence of macroscopic worms., (Copyright © 2015. Published by Elsevier B.V.)