Tina Struve

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.

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 CDVs collected from mink and 10 CDVs 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.

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

BackgroundPre-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.ResultsThe 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.ConclusionsResults 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.

Outbreak tracking of Aleutian mink disease virus (AMDV) using partial NS1 gene sequencing

BackgroundAleutian Mink Disease (AMD) is an infectious disease of mink (Neovison vison) and globally a major cause of economic losses in mink farming. The disease is caused by Aleutian Mink Disease Virus (AMDV) that belongs to the genus Amdoparvovirus within the Parvoviridae family. Several strains have been described with varying virulence and the severity of infection also depends on the host’s genotype and immune status. Clinical signs include respiratory distress in kits and unthriftiness and low quality of the pelts. The infection can also be subclinical.Systematic control of AMDV in Danish mink farms was voluntarily initiated in 1976. Over recent decades the disease was mainly restricted to the very northern part of the country (Northern Jutland), with only sporadic outbreaks outside this region. Most of the viruses from this region have remained very closely related at the nucleotide level for decades. However, in 2015, several outbreaks of AMDV occurred at mink farms throughout Denmark, and the sources of these outbreaks were not known.MethodsPartial NS1 gene sequencing, phylogenetic analyses data were utilized along with epidemiological to determine the origin of the outbreaks.ResultsThe phylogenetic analyses of partial NS1 gene sequences revealed that the outbreaks were caused by two different clusters of viruses that were clearly different from the strains found in Northern Jutland. These clusters had restricted geographical distribution, and the variation within the clusters was remarkably low. The outbreaks on Zealand were epidemiologically linked and a close sequence match was found to two virus sequences from Sweden. The other cluster of outbreaks restricted to Jutland and Funen were linked to three feed producers (FP) but secondary transmissions between farms in the same geographical area could not be excluded.ConclusionThis study confirmed that partial NS1 sequencing can be used in outbreak tracking to determine major viral clusters of AMDV. Using this method, two new distinct AMDV clusters with low intra-cluster sequence diversity were identified, and epidemiological data helped to reveal possible ways of viral introduction into the affected herds.

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.

Global phylogenetic analysis of contemporary aleutian mink disease viruses (AMDVs)

BackgroundAleutian mink disease has major economic consequences on the mink farming industry worldwide, as it causes a disease that affects both the fur quality and the health and welfare of the mink. The virus causing this disease is a single-stranded DNA virus of the genus Amdoparvovirus belonging to the family of Parvoviridae. In Denmark, infection with AMDV has largely been restricted to a region in the northern part of the country since 2001, affecting only 5% of the total Danish mink farms. However, in 2015 outbreaks of AMDV were diagnosed in all parts of the country. Initial analyses revealed that the out breaks were caused by two different strains of AMDV that were significant different from the circulating Danish strains. To track the source of these outbreaks, a major investigation of global AMDV strains was initiated.MethodsSamples from 13 different countries were collected and partial NS1 gene was sequenced and subjected to phylogenetic analyses.ResultsThe analyses revealed that AMDV exhibited substantial genetic diversity. No clear country wise clustering was evident, but exchange of viruses between countries was revealed. One of the Danish outbreaks was caused by a strain of AMDV that closely resembled a strain originating from Sweden. In contrast, we did not identify any potential source for the other and more widespread outbreak strain.ConclusionTo the authors knowledge this is the first major global phylogenetic study of contemporary AMDV partial NS1 sequences. The study proved that partial NS1 sequencing can be used to distinguish virus strains belonging to major clusters. The partial NS1 sequencing can therefore be a helpful tool in combination with epidemiological data, in relation to outbreak tracking. However detailed information on farm to farm transmission requires full genome sequencing.

A cross-sectional field study on potential associations between feed quality measures and usage of antimicrobials in commercial mink (Neovison vison)

Feed quality is generally assumed to affect health status in animal production. In previous studies, the feed producer has been found to affect the occurrence of gastrointestinal disease and antimicrobial use in Mink (Neovison vison). Mink are fed with moist, freshly produced feed, based on perishable ingredients. The objective of this study was to investigate the potential effect of specific feed parameters on antimicrobial use on herd level. The study was cross-sectional, including 1472 mink herds, responsible for 97% of oral antimicrobials prescribed for Danish mink during the study period, 2012-2014. Data were obtained from the national veterinary prescription database (VetStat), Kopenhagen Fur database, and the Voluntary Feed Control (Mink producers Organization). All feed batches subject to feed control were included. A multi-variable variance analysis was carried out analysing the effect of the feed parameters total volatile nitrogen, dry matter, crude protein and fat; total bacterial count (21°C), and counts of sulphite producing bacteria (21°C), Clostridium spp., faecal cocci (FC) (44°C), yeast, and mould; presence of Salmonella spp. and Clostridium perfringens (dichotome). Three outcome variables were applied: prescription of oral antimicrobial on herd level within time slots of 3, 5 or 7days after feeding of an included batch. Two binomial models were developed, adjusting for significant effects (p<0.0001) of Ps. aeruginosa infection, herd size, month (season) and year. Antimicrobial prescription was significantly (p<0.0001) associated with FC (all time slots, both models). A negative association (p<0.0001) with crude protein on antimicrobial prescription within a 7day slot suggested an association between low content of crude protein and antimicrobial use. The associations need to be confirmed in controlled studies, and ideally, potential causalities should be investigated. The perspective of such findings could be the development of tests for control of feed ingredients prior to use in the feed production.