Application of hydraulic modelling and quantitative microbial risk assessment (QMRA) for cloudburst management in cities with combined sewer systems.

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.

Effect of Translocation on Host Diet and Parasite Egg Burden: A Study of the European Bison (Bison bonasus).

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 Bialowieza 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 Bialowieza 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 Bialowieza 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.

The raccoon dog (Nyctereutes procyonoides) as a reservoir of zoonotic diseases in Denmark.

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.

Dirofilaria spp. and Angiostrongylus vasorum: Current Risk of Spreading in Central and Northern Europe.

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.

An investigation of endoparasites and the determinants of parasite infection in European hedgehogs (Erinaceus europaeus) from Denmark.

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.

Prevalence of Toxoplasma gondii and Cryptosporidium in Feral and Farmed American Mink (Neovison vison) in Denmark.

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.

Estimation of the Age and Reproductive Performance of Wild-Born and Escaped Mink (Neovison vison) Caught in the Wild in Denmark.

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 in Danish Mink Farms: Course of the Epidemic and a Descriptive Analysis of the Outbreaks in 2020.

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.

Cardiopulmonary nematodes of wild carnivores from Denmark: Do they serve as reservoir hosts for infections in domestic animals?

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.

Surveillance of important bacterial and parasitic infections in Danish wild boars (Sus scrofa).

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.

Methods for the identification of farm escapees in feral mink (Neovison vison) populations.

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.

Toxoplasma gondii seroprevalence in extensively farmed wild boars (Sus scrofa) in Denmark.

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.

Patterns of Fasciola hepatica infection in Danish dairy cattle: implications for on-farm control of the parasite based on different diagnostic methods.

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.

Serum antibody responses in pigs trickle-infected with Ascaris and Trichuris: Heritabilities and associations with parasitological findings.

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.

Parasite population dynamics in pigs infected with Trichuris suis and Oesophagostomum dentatum.

The aim of the present study was to investigate the population dynamics and potential interactions between Trichuris suis and Oesophagostomum dentatum in experimentally co-infected pigs, by quantification of parasite parameters such as egg excretion, worm recovery and worm location. Forty-eight helminth naïve pigs were allocated into four groups. Group O was inoculated with 20 O. dentatum L3/kg/day and Group T with 10 T. suis eggs/kg/day. Group OT was inoculated with both 20 O. dentatum L3/kg/day and 10 T. suis eggs/kg/day, while Group C was kept as an uninfected control group. All inoculations were trickle infections administered twice weekly and were continued until slaughter. Faecal samples were collected from the rectum of all pigs at day 0, and twice weekly from 2 to 9 weeks post first infection (wpi). Six pigs from each group were necropsied 5 wpi and the remaining 6 pigs from each group were necropsied 10 wpi. The faecal egg counts (FEC) and total worm burdens of O. dentatum were dramatically influenced by the presence of T. suis, with significantly lower mean FECs and worm burdens at 5 and 10 wpi compared to single infected pigs. Furthermore, in the presence of T. suis we found that O. dentatum was located more posteriorly in the gut. The changes in the Trichuris population were less prominent, but faecal egg counts, worm counts 5 wpi (57% recovered vs. 39%) and the proportion of infected animals at 10 wpi were higher in Group OT compared to Group T. The location of T. suis was unaffected by the presence of O. dentatum. These results indicate an antagonistic interaction between T. suis and O. dentatum which is dominated by T. suis.