Association between Antibiotic Consumption and Resistance in Mink Production.

Antibiotic consumption is considered to be a main driver of antibiotic resistant bacteria. Mink breeding follows a distinctive seasonal reproduction cycle, and all of the mink produced in the northern hemisphere are bred, born, and pelted around the same time of year. Some of the diseases are age-related, which is reflected in the seasonal variation of antibiotic consumption. The seasonality makes mink a good model for the investigation of the association between antibiotic consumption and resistance. The objectives of this study were (1) to monitor the farm level of antibiotic resistance during one production cycle and (2) to assess the potential associations between antibiotic consumption and resistance. Twenty-four farms were included in this study (Denmark n = 20, Iceland n = 2, and The Netherlands n = 2), following a cohort of animals born in 2018. Staphylococcus delphini and Escherichia coli were isolated from samples of the carcasses and faeces and were collected randomly. The isolates were susceptibility tested and subsequently divided into the sensitive wildtype (WT) and the resistant non-wildtype (NWT) populations. The antibiotic consumption relative to the sampling periods was assessed as having a short-term or a long-term impact, i.e., in two explanatory factors. For both S. delphini and E. coli, a large between-farm variation of NWT profiles was detected. In the final multivariable, generalized linear mixed models, significant associations between NWT isolates and the consumption of specific antibiotics were found: the short-term use of tetracyclines in the growth period was associated with the occurrence of tetracycline NWT E. coli in the growth period (OR: 11.94 [1.78; 89.28]), and the long-term use of macrolide and tetracyclines was associated with the occurrence of erythromycin NWT S. delphini in the weaning period (OR: 18.2 [2.26; 321.36]) and tetracycline NWT S. delphini in the growth period (OR: 8.2 [1.27; 63.31]), respectively. Farms with zero consumption in the study years prior to sampling also had a substantial proportion of NWT isolates, indicating that NWT isolates are persistent and/or widely spread in the environment. Generally, a high occurrence of tetracycline NWTs was observed. NWT isolates with resistance against the most commonly used antibiotics were found on all the farms, stressing the need for routine surveillance and the prudent use of antibiotics. The results offer a preview of the complex relationship between consumption and resistance, demonstrating some significant associations between use and resistance. Moreover, antibiotic-resistant bacteria are present even on farms with no antibiotic consumption over extended periods, and theoretical explanations supported by the data are offered.

First finding of Streptococcus phocae infections in mink (Neovison vison).

Streptococcus phocae infection has been described in salmon, sea otters, and several families of pinnipeds. The pathology of the infected animals has mainly been located in the respiratory tract and reproductive system, and with indications of septicemia. In this study, we report the finding of S. phocae in diagnostic material from three unrelated cases of farmed mink. Since S. phocae initially has been described in pinnipeds, two isolates from wild harbor seals were included. All isolates originated from Denmark. To our knowledge, this is the first report of S. phocae infection in mink. The animals (three mink, two seals) were necropsied, and samples were collected for bacteriology, virology, and histopathology. Additionally, the S. phocae isolates were whole genome sequenced and compared to sequences of previously reported isolates from other host species. S. phocae was isolated from the lungs of one mink and one seal with bacteremia, and from one seal with pneumonia. The two remaining mink had dermal infections on the paws and S. phocae was isolated from the lesions. The analysis of the sequence data showed that the three mink isolates and one seal isolate were closely related. Further investigation is needed to conclude whether S. phocae is establishing as commensal in farmed mink and to uncover the infection related pathology in mink. Streptococcus phocae has been described as an emerging pathogen in other species, therefore future awareness and surveillance of this pathogen is crucial.

Evaluating a tylosin dosage regimen for treatment of Staphylococcus delphini infection in mink (Neovison vison): a pharmacokinetic-pharmacodynamic approach.

Staphylococcus delphini is one of the most common pathogens isolated from mink infections, especially dermatitis. Tylosin (TYL) is used frequently against these infections, although no evidence-based treatment regimen exists. This study aimed to explore the dosage of TYL for infections caused by S. delphini in mink. Two animal experiments with a total of 12 minks were conducted to study the serum pharmacokinetic (PK) characteristics of TYL in mink after 10 mg/kg IV and oral dosing, respectively. The concentration of TYL in serum samples collected before and eight times during 24 h after TYL administration was quantitated with liquid chromatography quadrupole time-of-flight mass spectrometry, and the TYL disposition was analyzed using non-linear mixed effect analysis. The pharmacodynamics (PD) of TYL against S. delphini were studied using semi-mechanistic modeling of in vitro time-kill experiments. PKPD modeling and simulation were done to establish the PKPD index and dosage regimen. The disposition of TYL was described by a two-compartmental model. The area under the free concentration-time curve of TYL over the minimum inhibitory concentration of S. delphini (fAUC/MIC) was determined as PKPD index with breakpoints of 48.9 and 98.7 h for bacteriostatic and bactericidal effect, respectively. The calculated daily oral dose of TYL was 2378 mg/kg, which is 238-fold higher than the currently used TYL oral dosage regimen in mink (10 mg/kg). Accordingly, sufficient TYL concentrations are impossible to achieve in mink plasma, and use of this drug for extra-intestinal infections in this animal species must be discouraged.

Validating an empiric sulfadiazine-trimethoprim dosage regimen for treatment of Escherichia coli and Staphylococcus delphini infections in mink (Neovison vison).

Antimicrobial agents are used extensively off-label in mink, as almost no agents are registered for this animal species. Pharmacokinetic (PK) and pharmacodynamic (PD) data are required to determine antimicrobial dosages specifically targeting mink bacterial pathogens. The aims of this study were to assess, in a PKPD framework, the empirical dosage regimen for a combination of trimethoprim (TMP) and sulfadiazine (SDZ) in mink, and secondarily to produce data for future setting of clinical breakpoints. TMP and SDZ PK parameters were obtained experimentally in 22 minks following IV or oral administration of TMP/SDZ (30 mg/kg, i.e. 5 mg/kg TMP and 25 mg/kg SDZ). fAUC/MIC with a target value of 24 hr was selected as the PKPD index predictive of TMP/SDZ efficacy. Using a modeling approach, PKPD cutoffs for TMP and SDZ were determined as 0.062 and 16 mg/L, respectively. By incorporating an anticipated potentiation effect of SDZ on TMP against Escherichia coli and Staphylococcus delphini, the PKPD cutoff of TMP was revised to 0.312 mg/L, which is above the tentative epidemiological cutoffs (TECOFF) for these species. The current empirical TMP/SDZ dosage regimen (30 mg/kg, PO, once daily) therefore appears adequate for treatment of wild-type E. coli and S. delphini infections in mink.

Visualization of intestinal infections with astro- and sapovirus in mink (Neovison vison) kits by in situ hybridization.

Clarification of the infection microbiology remains a challenge in the pre-weaning diarrhea (PWD) syndrome in farmed mink (Neovison vison). Duodenal, jejunal and colon sections from 36 mink kits with PWD were systematically examined by chromogen in situ hybridization targeting two incriminated viruses: mink astrovirus and mink sapovirus. Using the RNAscope® 2.5 HD Duplex Assay, astrovirus and sapovirus were visualized and simultaneously demonstrated in the gut tissue. Both viruses infect enterocytes in the small intestine with a specific localization pattern; astrovirus affects the two apical thirds of the villi, whereas sapovirus generally affects the basal parts of the villi. Furthermore, we demonstrated that astrovirus in mink does not target the goblet cells. This is the first time astro- and calicivirus have been visualized in mink kit gut tissue, and these findings might be important in clarification of the impact of these viruses in the PWD syndrome.

Employing MIC Data for Mink Pathogens to Propose Tentative Epidemiological Cut-Off Values: A Step Toward Rationalizing Antimicrobial Use in Mink.

Optimizing antimicrobial dosage regimens and development of breakpoints for antimicrobial susceptibility testing are important prerequisites for rational antimicrobial use. The objectives of the study were (1) to produce MIC data for four mink pathogens and (2) to employ these MIC data to support the development of tentative epidemiological cut-off values (TECOFFs), which may be used for future development of mink-specific antimicrobial dosages and breakpoints. Broth microdilution was used to establish MIC distributions for 322 mink bacterial isolates of clinical origin from six European mink-producing countries. The included species were E. coli (n = 162), S. delphini (n = 63), S. canis (n = 42), and P. aeruginosa (n = 55). Sixty-four E. coli isolates and 34 S. delphini isolates were whole-genome sequenced and analyzed for antimicrobial resistance genes. No EUCAST MIC data are available on S. delphini and S. canis, hence tentative ECOFFs were suggested for the majority of the tested antimicrobials. For E. coli and P. aeruginosa, the wildtype distributions were in accordance with EUCAST data. Overall, the genotypes of the sequenced isolates were in concordance with the phenotypes. These data constitute an important piece in the puzzle of developing antimicrobial dosages and clinical breakpoints for mink. Until pharmacokinetic and clinical data become available, the (tentative) ECOFFs can be used for monitoring resistance development and as surrogates for clinical breakpoints.

Comparing the treatment effect of narrow spectrum antimicrobial, probiotic and fluid with amoxicillin in mink kits (Neovison vison) with pre-weaning diarrhea.

Pre-weaning diarrhea in mink kits (PWD), also known as "sticky kits" is a multifactorial syndrome of considerable concern in the mink production. Evidence based treatment protocols are not available, and treatment is therefore empirical and often based on the use of antimicrobials. The purpose of the study was to test the effect of 3 alternative treatments to a standard antibiotic treatment, to characterize the study groups microbiologically, and finally to compare the intestinal microbiota of the different treatment groups at the age of 42 days. In total, 226 one to three week old mink kits with PWD from 36 litters were treated with either 1) Lactobacillus reuteri, 2) benzylpenicillin, 3) Ringer lactate or 4) amoxicillin (controls). Effects of the treatments were measured as weight gain from day 0 to day 15 and mortality. Multivariable linear mixed model regression showed no significant difference in weight gain between probiotic-, penicillin or fluid-treated mink kits and the amoxicillin treated controls. There was also no significant difference in mortality risk between the treatment groups. Bacterial culture and next generation sequencing of the viral contents showed that the study groups were uniform with a high frequency of Staphylococcus intermedius group (SIG) bacteria, Escherichia coli, Enterococcus hirae, Mamastrovirus and Sapovirus which were representative for mink kits with PWD. 16S sequencing results of the bacterial microbiota, when the kits were 42 days old were dominated by clostridia in all groups and showed no clear differences in the bacterial composition between the different treatment groups.

Low concentration of serum immunoglobulin G is associated with pre-weaning diarrhea in young mink kits (Neovison vison).

BACKGROUND: Pre-weaning diarrhea (PWD) is a severe syndrome, with world-wide occurrence, affecting farmed mink (Neovison vison) kits during the lactation period. Kits affected by PWD often display clinical signs such as: yellow-white diarrhea, greasy skin, and dehydration. In severe cases the kits eventually die. It is common practice to treat PWD using antimicrobials; however the effect is not well documented. Due to the multifactorial etiology of PWD vaccine development is not feasible. The role played by the immune status of the mink kits with respect to their susceptibility to PWD is not well studied. To elucidate the possible association between PWD and total IgG serum concentration in young kits we analyzed blood collected from kits from 100 litters on two mink farms during the same breeding period, one farm being a case farm with high prevalence of PWD, and the other being a control farm with no cases of PWD. RESULTS: Kits affected by PWD had a significantly reduced weight gain compared to unaffected control kits. Litters born later in the breeding period came down with PWD at an earlier age than litters born at the start of the breeding period. We found that PWD affected kits had significantly lower concentrations of serum IgG compared to unaffected kits at 13-15 days of age (the last blood sampling point of the study). CONCLUSION: The results in this study suggest that PWD affected kits less efficiently absorbed IgG from maternal milk or had a lower intake of maternal milk, potentially contributing to the exacerbation of disease. A lower intake of IgG and/or less absorption from maternal milk could also pre-dispose kits for PWD. Future studies will be needed to elucidate if the circulating level of IgG is directly related to protection against disease and to investigate if administration of IgG could be helpful in alleviating and/or preventing PWD in mink kits.

Transfer of amoxicillin to suckling mink (Neovison vison) kits via the milk from dams treated orally or intra-muscularly.

Treatment of mink kits with pre-weaning diarrhea (PWD) can be time-consuming and expensive for the farmer, and the efficacy of the treatment procedure may be questioned. Evidence-based treatment protocols for application on affected animals at farms with outbreaks of PWD are lacking. In Denmark, the dams are sometimes treated with amoxicillin, however, it is unknown if it is passed on to the mink kits via the milk. The aim of the present study was to investigate if amoxicillin is transferred via the milk to the kits after oral (PO) and intramuscular (IM) treatment, respectively, of the dam. Moreover, we estimated the concentrations of amoxicillin continuously in serum from the kits up to 8 h after administration. The concentration of amoxicillin was not affected by the route of administration (P = .64) and serum reached the highest level after 8 h (34 ng/mL, CI95% = [24.3-47.7]). The serum concentrations of amoxicillin in the mink kits achieved within 8 h were judged too low to exert antimicrobial impact on relevant bacterial species.

Dam characteristics associated with pre-weaning diarrhea in mink (Neovison vison).

BACKGROUND: Pre-weaning diarrhea (PWD) in mink, also known as "sticky kits", is a frequently occurring syndrome in suckling mink kits on commercial mink farms. Outbreaks of PWD result in weakened kits, increased mortality and reduced growth and welfare as well as considerable economic losses for the farmers. The syndrome is regarded as multifactorial with a complex etiology, and studies have focused on associations with environment, management and dam characteristics. The present study was conducted from May to June 2015 and included 70 dams with mink litters with and without PWD. The aims were to examine associations between PWD and mastitis (bacterial infection and histological signs of inflammation or other lesions in the mammary gland), and to examine associations between PWD and other dam-related characteristics (age, litter size, body mass index, and weight and number of active mammary glands of the dam). RESULTS: Using multivariable mixed logistic regression analyses with farm id as a random intercept, we found that the odds for PWD in the litter were significantly higher in 1 year old dams versus > 1 year old (OR = 13.3, CI 2.0-90.2, P = 0.01), higher if litter size observed after birth was > 5 kits versus ≤ 5 kits (OR = 16.5, CI 2.2-123.7, P = 0.01), higher if the number of active mammary glands per kit was ≤ 1.5 versus > 1.5 glands per kit (OR = 6.5, CI 1.2-36.0), P = 0.03), and higher in farms with high prevalence of PWD versus low prevalence (OR = 16.8, CI 2.9-97.6, P = 0.002). There were no significant associations between PWD and bacterial infection, histological signs of inflammation or other lesions of the mammary gland, body mass index or weight of mammary gland per kit. CONCLUSION: Pre-weaning diarrhea had a statistically significant association with age of the dam, litter size and the number of active mammary glands per kit. However, PWD was not associated with mastitis, body mass index and weight of mammary gland tissue per kit.

Investigation of the viral and bacterial microbiota in intestinal samples from mink (Neovison vison) with pre-weaning diarrhea syndrome using next generation sequencing.

Pre-weaning diarrhea (PWD) in mink kits is a common multifactorial syndrome on commercial mink farms. Several potential pathogens such as astroviruses, caliciviruses, Escherichia coli and Staphylococcus delphini have been studied, but the etiology of the syndrome seems complex. In pooled samples from 38 diarrheic and 42 non-diarrheic litters, each comprising of intestinal contents from 2-3 mink kits from the same litter, the bacterial populations were studied using Illumina Next Generation Sequencing technology and targeted 16S amplicon sequencing. In addition, we used deep sequencing to determine and compare the viral intestinal content in 31 healthy non-diarrheic and 30 diarrheic pooled samples (2-3 mink kits from the same litter per pool). The results showed high variations in composition of the bacterial species between the pools. Enterococci, staphylococci and streptococci dominated in both diarrheic and non-diarrheic pools. However, enterococci accounted for 70% of the reads in the diarrheic group compared to 50% in the non-diarrheic group and this increase was at the expense of staphylococci and streptococci which together accounted for 45% and 17% of the reads in the non-diarrheic and diarrheic group, respectively. Moreover, in the diarrheic pools there were more reads assigned to Clostridia, Escherichia-Shigella and Enterobacter compared to the non-diarrheic pools. The taxonomically categorized sequences from the virome showed that the most prevalent viruses in all pools were caliciviruses and mamastroviruses (almost exclusively type 10). However, the numbers of reads assigned to caliciviruses were almost 3 times higher in the diarrheic pools compared the non-diarrheic pools and Sapporo-like caliciviruses were more abundant than the Norwalk-like caliciviruses. The results from this study have contributed to the insight into the changes in the intestinal microbiota associated with the PWD syndrome of mink.

Mink (Neovison vison) kits with pre-weaning diarrhea have elevated serum amyloid A levels and intestinal pathomorphological similarities with New Neonatal Porcine Diarrhea Syndrome.

BACKGROUND: Pre-weaning diarrhea (PWD) is a syndrome affecting farm-raised neonatal mink kits. Apart from diarrhea it causes greasy skin exudation, dehydration, and distressed behavior and can ultimately lead to death. No specific causative agents have been identified and the syndrome is regarded as multifactorial. The aim of the present study was to investigate a possible inflammatory state in mink kits with PWD, as indicated by raised serum concentrations of the acute phase protein serum amyloid A (SAA) and by changes in intestinal pathomorphology and intestinal contents of bacteria. Samples collected from 20 diarrheic mink kits with PWD and 20 age-matched non-diarrheic control mink kits from two commercial Danish farms during the pre-weaning period (April-May) in 2016 were analyzed. RESULTS: Concentrations of SAA in serum samples from mink kits with PWD were significantly higher (up to 1000-fold) compared to non-diarrheic control mink kits. Significant features of enterocytic vacuolization, atrophy and fusion of villi in jejunum and mucosal atrophy of the colon of kits with PWD were found. Moreover, attachment of coccoid bacteria to enterocytes was more often found in kits suffering from PWD, while intra-cytoplasmic eosinophil bodies were more frequently observed in control kits. Cellular infiltrations with mononuclear and neutrophil leukocytes were not associated with disease status. Bacteria from the Staphylococcus intermedius group, such as Staphylococcus delphini, were more frequently cultivated from control mink kits, whereas Enterococcus spp. dominated in mink kits with PWD. Escherichia coli was cultivated from both control and mink kits with PWD, but with a higher frequency from mink kits with PWD. CONCLUSION: A significant increase in circulating concentrations of SAA was found in PWD affected mink kits from 6 to 23 days old compared to controls. The histopathological changes in PWD mink kits suggest that the type of diarrhea is secretory. Attachment of coccoid bacteria, therefore, might be responsible for an enterotoxic effect causing a loss of balance in movements of ions and water leading to the vacuolization and swelling of the enterocytes. The slight to moderate infiltrations of neutrophils irrespectively of diarrheic status and the attachment of coccoid bacteria to enterocytes are comparable to observations found in piglets suffering from New Neonatal Porcine Diarrhea Syndrome. Mechanisms behind the correlation between increased SAA levels and the observed pathological intestinal features remain obscure.

Quantitative immunoassay for mink immunoglobulin in serum and milk.

BACKGROUND: The significance of maternal immunoglobulin G (IgG) for the resistance against a number of infections affecting the health of young mink offspring is not known. Here, we present a validated immunoassay for quantification of mink IgG in serum and milk, using a commercially available polyclonal goat anti-ferret IgG antibody cross-reactive with mink IgG as both the catching and the detection antibody, in a sandwich format enzyme linked immunosorbent assay (ELISA). Using this ELISA, serum IgG concentrations was analyzed over time in both mothers and kits in order to establish a correlation between maternal IgG serum concentrations and those of the offspring. RESULTS: Intra-assay coefficient of variation (CV) for a serum sample ranged from 2.15 to 5.97% depending on the dilution, while the inter-assay CV ranged from 5.17 to 17.78%. In addition, the range of milk intra-assay CV was 2.71-5.92%, while the range of the inter-assay CV was 4.20-16.03%. Calibrating the ELISA with purified mink IgG (an in-house preparation purified from mink serum) the lower limit of detection was found to be 5 ng/mL for serum and 1 ng/mL for milk. Both serum and milk showed high precision and good linearity over a two-log dilution range. When comparing the serum IgG concentrations of the mink kits a clear within litter effect was seen, while the mean serum IgG concentrations of litters differed significantly between some of the litters (P = 0.0013). Mean maternal serum IgG concentrations correlated positively with the IgG serum concentration of the corresponding offspring sampled over a 3 week period (R2 = 0.63). CONCLUSIONS: A calibrated and reproducible sandwich ELISA for quantifying mink IgG concentrations in both milk and serum with high analytical sensitivity was developed and validated. The results in this study corroborate previous investigations supporting the usability of the ELISA, paving the way for investigations into the importance of maternal IgG in milk and in serum for the welfare and health of the offspring.

Risk factors associated with diarrhea in Danish commercial mink (Neovison vison) during the pre-weaning period.

BACKGROUND: Pre-weaning diarrhea in mink, also known as "sticky kits", is a syndrome and outbreaks occur every year on commercial mink farms in all mink producing countries. Morbidity and mortality can be considerable on a farm with huge economic consequences for the farmer as well as compromised welfare for the mink kits. Although efforts have been taken to identify etiologic agents involved in outbreaks, the syndrome is still regarded as multifactorial and recurring problems on the same farms draw attention to management and environmental risk factors. In the pre-weaning period from May to June 2015, a case control study was carried out on 30 Danish mink farms. Data concerning management, biosecurity, hygiene, feed consumption, antibacterial prescription and production efficiency were analyzed. RESULTS: The proportion of 1-year old females, farm size (total number of females), energy supply per female in the late gestation period, and dogs accessing the farm area were significantly associated with being a case farm. Case farms were prescribed almost twice the amount of antibacterials per gestational unit (female and litter) as in control farms. Farmers on case farms spent significantly more time nursing and treating the animals and experienced more females with mastitis compared to farmers on control farms. No significant differences in cleaning practices or hygienic measures between case and control farms were found and there were no differences in drinking water quality, bedding material, composition neither of color types nor in management regarding litter equalization. CONCLUSIONS: Results from this study showed an association between the occurrence of pre-weaning diarrhea on mink farms and parity profile, farm size and feeding intensity in the gestational period. The access of dogs to the farm area was a significant risk factor, but needs further clarification.

Associations between biosecurity and outbreaks of canine distemper on Danish mink farms in 2012-2013.

BACKGROUND: During 8 months from July 2012 to February 2013, a major outbreak of canine distemper involving 64 mink farms occurred on the Danish peninsula of Jutland. The canine distemper outbreak was associated with exposure of farmed mink to infected wild carnivores and could represent a deficit in biosecurity on the mink farms. The aim of this study was to investigate the extent and association of specific biosecurity measures with the outbreak. The study was carried out in an epidemiological case-control design. The case group consisted of the 61 farms, which had a confirmed outbreak of canine distemper from July 2012 to February 2013. The control group included 54 farms without an outbreak of canine distemper in 2012 or 2013, selected as the closest geographical neighbour to a case farm. RESULTS: The results showed that significantly more control than case farms had vaccinated their mink against canine distemper virus. Mortality was only assessed on the case farms, and there was a non-significantly lower mortality on vaccinated farms than on the non-vaccinated farms. Furthermore, the proportion of farms with observations of wild red foxes (Vulpes vulpes) inside the farm enclosures were larger for case farms, indicating that the control farms had a better biosecurity or were not equally exposed to canine distemper virus. Generally, all farms had very few specific precautions at the gate entrance in respect to human visitors as well as animals. The use of biosecurity measures was very variable in both case and control farms. Not using plastic boot covers, presence of dogs and cats, presence of demarcated area for changing clothes when entering and leaving the farm area and presence of hand washing facilities significantly lowered the odds of the farm having a canine distemper virus outbreak. CONCLUSIONS: The results of the study indicate that consistent use of correct vaccination strategies, implementation of biosecurity measures and limiting human and animal access to the mink farm can be important factors in reducing the risk for canine distemper outbreaks.

Wildlife reservoirs of canine distemper virus resulted in a major outbreak in Danish farmed mink (Neovison vison).

A major outbreak of canine distemper virus (CDV) in Danish farmed mink (Neovison vison) started in the late summer period of 2012. At the same time, a high number of diseased and dead wildlife species such as foxes, raccoon dogs, and ferrets were observed. To track the origin of the outbreak virus full-length sequencing of the receptor binding surface protein hemagglutinin (H) was performed on 26 CDV's collected from mink and 10 CDV's collected from wildlife species. Subsequent phylogenetic analyses showed that the virus circulating in the mink farms and wildlife were highly identical with an identity at the nucleotide level of 99.45% to 100%. The sequences could be grouped by single nucleotide polymorphisms according to geographical distribution of mink farms and wildlife. The signaling lymphocytic activation molecule (SLAM) receptor binding region in most viruses from both mink and wildlife contained G at position 530 and Y at position 549; however, three mink viruses had an Y549H substitution. The outbreak viruses clustered phylogenetically in the European lineage and were highly identical to wildlife viruses from Germany and Hungary (99.29% - 99.62%). The study furthermore revealed that fleas (Ceratophyllus sciurorum) contained CDV and that vertical transmission of CDV occurred in a wild ferret. The study provides evidence that wildlife species, such as foxes, play an important role in the transmission of CDV to farmed mink and that the virus may be maintained in the wild animal reservoir between outbreaks.

Diagnosing Aleutian mink disease infection by a new fully automated ELISA or by counter current immunoelectrophoresis: a comparison of sensitivity and specificity.

Aleutian disease (AD) is a severe disease characterized by hypergammaglobulinemia causing multiple symptoms such as acute renal failure, arteritis, reduced reproductive performance and pneumonia in mink. AD is caused by the parvovirus Aleutian mink disease virus (ADV) and diagnosed primarily based on ADV serology sometimes supplemented by organ PCR analysis. In Denmark, approximately 3.5-4 million serum samples are tested every year for the presence of anti ADV antibodies as part of a national eradication program. The present study compares the diagnostic performance of the two most commonly used assays for serological screening for Aleutian disease: counter current immunoelectrophoresis (CIEP) and ELISA. In total, 3810 mink were sampled in doublets and analyzed by CIEP and a newly developed fully automated ELISA. The results show that the two assays have a comparable diagnostic performance with the ELISA having a higher sensitivity but lower specificity than the CIEP assay. The ELISA has been approved by the Danish authorities for diagnosing Aleutian disease in mink.

Prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in Danish slaughter pigs and retail meat identified by selective enrichment and association with cephalosporin usage.

OBJECTIVES: To investigate the prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in pigs at slaughter and retail meat, and possible associations with the consumption of third- and fourth-generation cephalosporins. METHODS: During 2009, faecal samples from Danish pigs (n=786) were collected at slaughter, and 866 meat samples [Danish: pork (153), broiler meat (121) and beef (142); and imported: pork (173), broiler meat (193) and beef (84)] were randomly collected in retail stores and outlets. E. coli was isolated after enrichment in MacConkey broth with ceftriaxone (1 mg/L). ESC genotypes were detected using PCR, microtube array and sequencing. The MIC of cefotaxime was determined for 150 E. coli from the pigs and 606 E. coli from meat isolated without selective enrichment. RESULTS: Eleven percent (86/786) of slaughter pigs contained ESC E. coli and a significantly higher prevalence was observed among pigs originating from farms with registered cephalosporin consumption in slaughter pigs (P=0.034). Among ESC E. coli from pigs, 66% contained bla(CTX-M-1). From meat, a high prevalence of ESC E. coli was found in imported broiler meat (36%) compared with 0.7%-3.3% in other meat types. ESC E. coli from imported broiler meat (n=69) contained bla(CMY-2) (48%), bla(CTX-M-1) (25%) and bla(SHV-12) (16%). Without selective enrichment, no ESC E. coli from pigs and only 4.1% from imported broiler meat were found. CONCLUSIONS: The usage of cephalosporins for slaughter pigs may increase the prevalence of ESC E. coli in slaughter pigs. Meat may be a source of ESCs in humans, especially imported broiler meat. Selective enrichment should be considered as a supplementary surveillance method.