RT-qPCR assay for detection of mink astrovirus in outbreaks of diarrhea on Danish mink farms

Diarrhea in mink kits is a major cause of disease and mortality in the mink production. The etiology remains unknown in most outbreaks due to a lack of diagnostic assays. In the current study we present an RT-qPCR method to detect mink astrovirus in fecal samples from mink kits with diarrhea. All sampled animals were classified based on age and patoanatomical evaluation as having pre-weaning diarrhea, diarrhea in the growth period or as having no macroscopic signs of diarrhea. Fecal samples were analyzed for MiAstV with RT-qPCR, next generation sequencing and electron microscopy in parallel. Mink astrovirus was detected with RT-qPCR in 92 out of 203 samples. This detection was confirmed by next generation sequencing in a high proportion of samples (22/27), and by visualization of astrovirus particles with EM in some of the samples. Mink astrovirus was highly prevalent (68%) among kits in the outbreaks of pre-weaning diarrhea, in particular outbreaks from May, while less prevalent in outbreaks in June. Mink astrovirus was detected in outbreaks of diarrhea in the growth period, though in a much lesser extent than in the pre-weaning period. The role of mink astrovirus in the diarrhea disease complex of mink remain to be investigated, and for that purpose this sensitive and robust RT-qPCR can be a valuable tool in the future.

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 (CISH) 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.

The effect of antimicrobial treatment on mortality associated with urinary tract disease in mink kits (Neovison vison)

Mink urinary tract disease (MUTD) often presents as urolithiasis and/or cystitis and is known as an important cause of mortality in mink kits during the early growth season. Antimicrobial flock treatment has been routinely applied as preventive/therapeutic protocol on Danish mink farms with increased mortality associated with MUTD. The therapeutic effect of this treatment strategy has not previously been investigated. In this study, we applied controlled parallel group treatment trials to assess the effect of sulfadiazine/trimethoprim and amoxicillin treatment on mortality associated with MUTD in mink kits. On farm A, eight mink kits were diagnosed with MUTD post mortem in the treatment group (n = 1920, sulfadiazine/trimethoprim treatment: 30 mg/kg, q 24 h, P.O for 5 days) compared to 16 in the untreated control group (n = 1920). No significant difference in mortality associated with MUTD were found between the treatment and the control group using the Fisher’s exact test (P = 0.15). Treatment group 2 (n = 1920, amoxicillin treatment: 14 mg/kg q 24 h, P.O for 5 days) and treatment group 3 (n = 2088, amoxicillin treatment: 7.5 mg/kg q 24 h, P.O for 5 days) were investigated on farm B. Eight and four mink kits were diagnosed with MUTD post mortem in group 2 and 3, respectively. No difference between occurrence of MUTD were found between the control group and treatment group 2 (P = 0.42) or treatment group 3 (P = 0.75). No significant difference between final body weights or weight gain were found between treatment and control weighing groups on farm A or B. In conclusion, antimicrobial treatment administered in the feed showed no significant effect on weight gain or mortality associated with MUTD on the farms included in this study.

Absorption of α-tocopheryl acetate is limited in mink kits (Mustela vison) during weaning

Bioavailability of α-tocopherol varies with source, dose and duration of supplementation. The effect of source and dose of α-tocopherol on response of α-tocopherol stereoisomers in plasma and tissues of mink kits during the weaning period was studied. Twelve mink kits were euthanised in CO2 at the beginning of the experiment, and 156 mink kits (12 replicates per treatment group) were randomly assigned to thirteen treatment groups: no added α-tocopherol in the feed (0 dose) or four different doses (50, 75, 100 and 150 mg/kg of diet) of RRR-α-tocopherol (ALC), RRR-α-tocopheryl acetate (ACT) or all-rac-α-tocopheryl acetate (SYN). Six mink kits per treatment group were euthanised 3 weeks after initiation of the experiment, and the remaining six were euthanised 6 weeks after initiation of the experiment. The RRR-α-tocopherol content in plasma, liver, heart and lungs was affected by interaction between source and dose (P < 0.01 for all). The highest RRR-α-tocopherol content in plasma (13.6 µg/ml; LS-means for source across dose and week), liver (13.6 µg/mg), heart (7.6 µg/mg) and lungs (9.8 µg/mg) was observed in mink kits fed ALC. The RRR-α-tocopherol content in plasma and tissues depended on source and dose interaction and increased linearly with supplementation. In conclusion, the interaction between source and dose reveals a limitation in hydrolysis of ester bond in α-tocopheryl acetate in mink kits around weaning as the likely causative explanation for the higher response of ALC at the highest doses. Thus, considerable attention has to be paid to the source of α-tocopherol during weaning of mink kits fed a high dose of α-tocopherol.