Long-term antibody production and viremia in American mink (Neovison vison) challenged with Aleutian mink disease virus.

BACKGROUND: Selecting American mink (Neovison vison) for tolerance to Aleutian mink disease virus (AMDV) has gained popularity in recent years, but data on the outcomes of this activity are scant. The objectives of this study were to determine the long-term changes in viremia, seroconversion and survival in infected mink. Mink were inoculated intranasally with a local isolate of Aleutian mink disease virus (AMDV) over 4 years (n = 1742). The animals had been selected for tolerance to AMDV for more than 20 years (TG100) or were from herds free of AMDV (TG0). The progenies of TG100 and TG0, and their crosses with 25, 50 and 75% tolerance ancestry were also used. Blood samples were collected from each mink up to 14 times until 1211 days post-inoculation (dpi) and were tested for viremia by PCR and for anti-AMDV antibodies by counter-immunoelectrophoresis (CIEP). Viremia and CIEP status were not considered when selecting replacements. Low-performing animals were pelted and the presence of antibodies in their blood and antibody titer were measured by CIEP, and viremia and viral DNA in seven organs (n = 936) were tested by PCR. RESULTS: The peak incidences of viremia (66.7%) and seropositivity (93.5%) were at 35 dpi. The incidence of viremia decreased over time while the incidence of seroconversion increased. The least-squares means of the incidence of PCR positive of lymph node (0.743) and spleen (0.656) were significantly greater than those of bone marrow, liver, kidneys, lungs and small intestine (0.194 to 0.342). Differences in tolerant ancestry were significant for every trait measured. Incidences of viremia over time, terminal viremia, seropositivity over time, AMDV DNA in organs and antibody titer were highest in the susceptible groups (TG0 or TG25) and lowest in the tolerant groups (TG100 or TG75). CONCLUSION: Previous history of selection for tolerance resulted in mink with reduced viral replication and antibody titer. Viremia had a negative effect and antibody production had a positive effect on survival and productivity.

Serum Analytes of American Mink (Neovison Vison) Challenged with Aleutian Mink Disease Virus.

Black American mink (Neovison vison), which had been selected for tolerance to Aleutian mink disease virus (AMDV) for more than 20 years (TG100) or were from herds that have been free of AMDV (TG0), along with their progeny and crosses with 50% and 75% tolerance ancestry, were inoculated with a local isolate of AMDV. Blood samples were collected from 493 mink between 120 and 1211 days post-inoculation, and concentrations of 14 serum analytes were measured. Distributions of all analytes significantly deviated from normality, and data were analyzed after Box-Cox power transformation. Significant differences were observed among tolerant groups in the concentrations of globulin (GLO), total protein (TP), alkaline phosphatase, urea nitrogen, and calcium. Concentrations of GLO and TP linearly and significantly decreased with an increasing percentage of tolerance ancestry. Eleven analytes had the smallest values in the tolerant groups (TG100 or TG75), and eight analytes had the greatest values in the non-selected groups (TG0 or TG50). Antibody titer had the greatest correlation coefficients with GLO (0.62), TP (0.53), and creatinine (0.36). It was concluded that selection for tolerance decreased the concentrations of most serum analytes, and TP and GLO were the most accurate biomarkers of tolerance to AMDV infection. Males had significantly greater values than females for phosphorus and total bilirubin concentrations, but females had significantly greater amylase, cholesterol, and BUN concentrations than males.

Detection of selection signatures for response to Aleutian mink disease virus infection in American mink.

Aleutian disease (AD) is the most significant health issue for farmed American mink. The objective of this study was to identify the genomic regions subjected to selection for response to infection with Aleutian mink disease virus (AMDV) in American mink using genotyping by sequencing (GBS) data. A total of 225 black mink were inoculated with AMDV and genotyped using a GBS assay based on the sequencing of ApeKI-digested libraries. Five AD-characterized phenotypes were used to assign animals to pairwise groups. Signatures of selection were detected using integrated measurement of fixation index (FST) and nucleotide diversity (θπ), that were validated by haplotype-based (hap-FLK) test. The total of 99 putatively selected regions harbouring 63 genes were detected in different groups. The gene ontology revealed numerous genes related to immune response (e.g. TRAF3IP2, WDR7, SWAP70, CBFB, and GPR65), liver development (e.g. SULF2, SRSF5) and reproduction process (e.g. FBXO5, CatSperß, CATSPER4, and IGF2R). The hapFLK test supported two strongly selected regions that contained five candidate genes related to immune response, virus-host interaction, reproduction and liver regeneration. This study provided the first map of putative selection signals of response to AMDV infection in American mink, bringing new insights into genomic regions controlling the AD phenotypes.

Dietary supplementation of Ascophylum nodosum improved kidney function of mink challenged with Aleutian mink disease virus.

BACKGROUND: Feed additives which can ease the negative effects of infection by the Aleutian mink disease virus (AMDV) are of interest to mink farmers. The effects of kelp meal (Ascophylum nodosum) supplementation on immune response, virus replication and blood parameters of mink inoculated with AMDV were assessed. AMDV-free black mink (n = 75) were intranasally inoculated with a local strain of AMDV and fed a commercial pellet supplemented with kelp meal at the rates of 1.5% or 0.75% of the feed or were kept as controls (no kelp) for 451 days. Blood was collected on days 0 (pre-inoculation), 31, 56, 99, 155, 366 and 451 post-inoculation (dpi). RESULTS: No significant difference was observed among the treatments for the proportion of animals positive for antibodies against the virus measured by the counter-immunoelectrophoresis (CIEP), viremia measured by PCR, antibody titer measured by quantitative ELISA, total serum protein measured by a refractometer or elevated levels of gamma globulin measured by iodine agglutination test at the sampling occasions. At the termination of the experiment on 451 dpi, there were no differences among treatments for antibody titer measured by CIEP, total serum protein, albumin, globulins, albumin:globulin ratio, alkaline phosphatase, gamma-glutamyl transferase, and proportions of PCR positive spleen, lymph node or bone marrow samples, but blood urea nitrogen and creatine levels were significantly lower in the 1.5% kelp supplemented group than in the controls. CONCLUSION: Kelp supplementation improved kidney function of mink infected with AMDV with no effect on liver function, immune response to infection by AMDV or virus replication.

Linkage Disequilibrium, Effective Population Size and Genomic Inbreeding Rates in American Mink Using Genotyping-by-Sequencing Data.

Knowledge of linkage disequilibrium (LD) patterns is necessary to determine the minimum density of markers required for genomic studies and to infer historical changes as well as inbreeding events in the populations. In this study, we used genotyping-by-sequencing (GBS) approach to detect single nucleotide polymorphisms (SNPs) across American mink genome and further to estimate LD, effective population size (Ne), and inbreeding rates based on excess of homozygosity (FHOM) and runs of homozygosity (ROH). A GBS assay was constructed based on the sequencing of ApeKI-digested libraries from 285 American mink using Illumina HiSeq Sequencer. Data of 13,321 SNPs located on 46 scaffolds was used to perform LD analysis. The average LD (r 2 ± SD) between adjacent SNPs was 0.30 ± 0.35 over all scaffolds with an average distance of 51 kb between markers. The average r 2 < 0.2 was observed at inter-marker distances of >40 kb, suggesting that at least 60,000 informative SNPs would be required for genomic selection in American mink. The Ne was estimated to be 116 at five generations ago. In addition, the most rapid decline of population size was observed between 100 and 200 generations ago. Our results showed that short extensions of homozygous genotypes (500 kb to 1 Mb) were abundant across the genome and accounted for 33% of all ROH identified. The average inbreeding coefficient based on ROH longer than 1 Mb was 0.132 ± 0.042. The estimations of FHOM ranged from -0.44 to 0.34 among different samples with an average of 0.15 over all individuals. This study provided useful insights to determine the density of SNP panel providing enough statistical power and accuracy in genomic studies of American mink. Moreover, these results confirmed that GBS approach can be considered as a useful tool for genomic studies in American mink.

Dose response of black American mink to Aleutian mink disease virus.

INTRODUCTION: Aleutian mink disease virus (AMDV) causes a serious health problem for mink globally. The disease has no cure nor an effective vaccine and selection for tolerance using antibody titer is adopted by many mink farmers. The objective of this study was to investigate the effects of various doses of a local AMDV isolate on the response of black American mink to infection with AMDV. METHODS: Eight black American mink were each inoculated intranasally with 0.5 mL of eight serial 10-fold dilutions (100 to 10-7 ) of a 10% spleen homogenate containing a local AMDV isolate. Blood samples were collected on days 0, 20, 35, 56, 84, 140, and 196 postinoculation (dpi). Anti-AMDV antibodies and viral DNA were tested by counter-immunoelectrophoresis (CIEP) and PCR, respectively. Animals that were PCR or CIEP positive at 196 dpi (n = 41) were killed at 218 dpi, and samples of blood and seven organs were tested by CIEP and PCR. RESULTS: Antibody production persisted in all seroconverted mink until the termination of the experiment, whereas 71.1% of the mink showed short-lived viremia. Significant associations were observed between inoculum dose and the incidence of viremia until 84 dpi which disappeared thereafter, whereas associations between inoculum dose and the incidence of seropositive mink were significant on all sampling occasions. Antibody titer at 218 dpi significantly decreased with decreasing inoculum dose. AMDV DNA was detected in the bone marrow, lymph nodes, and spleen samples of almost all mink inoculated at every dose but was not detected in other organs of some mink. CONCLUSIONS: CIEP is more accurate than PCR for detecting AMDV infection in mink. Using antibody titer in naturally infected mink may not be accurate for the identification of tolerant mink.

A comparison between intraperitoneal injection and intranasal and oral inoculation of mink with Aleutian mink disease virus.

Intranasal, with (INS) and without (IN) sedation, and oral inoculation were compared with intraperitoneal (IP) injection for establishing infection with a local isolate of Aleutian mink disease virus (AMDV) in 35 American mink. Blood samples were collected on 0, 21, 36 and 56 day post-inoculation (dpi). Antiviral-antibodies and viral DNA in plasma and tissues were measured by counter-immunoelectrophoresis (CIEP) and PCR, respectively. The presence of AMDV DNA was tested by PCR in saliva, rectal and fecal samples collected on 0, 6, 10, 15, 21, 28, 36 and 56 dpi. Animals were killed at 56 dpi, samples of six organs were tested for antibody and AMDV DNA, and samples of the lungs, liver, kidneys and heart were subjected to histology. Viral DNA was detected in the spleen, lungs and lymph nodes of all inoculated mink on 56 dpi, indicating that all inoculation routes caused infection in mink. Viral DNA and antibodies were detected in plasma of all IP and INS inoculated mink by 36 dpi, but some animals which were inoculated orally or via IN remained seronegative by 56 dpi. It was concluded that INS route was the most effective method for establishing infection in mink without breaking the integrity of the animals' anatomical barriers. Viremia was short-lived in some mink, whereas antibody production persisted in seroconverted animals during the duration of the experiment. Saliva, rectal and fecal samples did not accurately detect infection. Histologic lesions of AD were observed on the four organs of most mink.

Reduced severity of histopathological lesions in mink selected for tolerance to Aleutian mink disease virus infection.

The objective of this study was to measure the effect of selection for tolerance on the severity of the Aleutian disease (AD) lesions in mink. Sensitivity and specificity of antibody detection in the blood by counter-immunoelectrophoresis (CIEP) relative to the presence of Aleutian mink disease virus (AMDV) in the spleen by PCR in naturally infected farmed mink were also estimated. Carcasses of 680 sero-positive (CIEP-P) black mink from 28 farms in Nova Scotia, Canada, and from 132 sero-negative (CIEP-N) mink from 14 of these farms were collected at pelting time. A total of 116 of the CIEP-P mink were from three farms where animals have been selected for tolerating AD for almost 20years. The severity of the AD lesions was assessed by histopathological examination of kidneys, lungs, heart, brain and liver on a scale of 0 to 4. Sensitivity and specificity of CIEP relative to PCR were 0.97 and 0.85, respectively, and 16.5% of CIEP-N mink were PCR positive, which could be one of the reasons for the failure of virus eradication by CIEP in Canada. The CIEP-N and tolerant CIEP-P animals had 9.39 and 6.23 greater odds of showing lower lesion severity, respectively, than the CIEP-P animals (P<0.01). The CIEP-N mink had a slightly higher chance (P=0.07) of showing lower lesion severity (odds ratio 1.51) compared with tolerant CIEP-P mink. The results suggested that tolerant mink had significantly reduced severity of AD lesions despite having anti-viral antibodies and carrying the virus.

Detection of Aleutian mink disease virus DNA and antiviral antibodies in American mink (Neovison vison) 10 days postinoculation.

Early detection of infection by the Aleutian mink disease virus (AMDV; Carnivore amdoparvovirus 1) by polymerase chain reaction (PCR) or counterimmunoelectrophoresis (CIEP) has important ramifications in virus eradication programs. A spleen homogenate containing a local isolate of AMDV was injected intraperitoneally into black (n = 44) and sapphire (n = 12) American mink (Neovison vison). Animals were euthanized 10 days postinoculation and anti-AMDV antibodies and AMDV DNA were tested in plasma and 7 organs by CIEP and PCR, respectively. Viral DNA was detected in the plasma, spleen, lymph nodes, bone marrow, and lung samples of all inoculated mink, but was not detected in some small intestine, kidney, and liver samples. In contrast, antibodies were detected in the plasma of 3 sapphire (25.0%) and 19 black (43.2%) mink but not in any of the organs. The sensitivity of the CIEP test on plasma samples was 39.3%, implying that low levels of antibodies during the early stages of virus exposure resulted in failure to detect infection by the CIEP test. We concluded that CIEP is not a reliable test for early detection of AMDV infection in mink and that there were considerable differences among mink of each color type for production of detectable levels of antibodies. PCR tests on samples of saliva, rectal swabs, and feces did not produce consistent and reliable results.

Aleutian mink disease virus in furbearing mammals in Nova Scotia, Canada.

BACKGROUND: Aleutian mink disease virus (AMDV) is widespread among ranched and free-ranging American mink in Canada, but there is no information on its prevalence in other wild animal species. This paper describes the prevalence of AMDV of 12 furbearing species in Nova Scotia (NS), Canada. METHODS: Samples were collected from carcasses of 462 wild animals of 12 furbearing species, trapped in 10 NS counties between November 2009 and February 2011. Viral DNA was tested by PCR using two primer pairs, and anti-viral antibodies were tested by counterimmunoelectrophoresis (CIEP) on spleen homogenates. RESULTS: Positive PCR or CIEP samples were detected in 56 of 60 (93.3%) American mink, 43 of 61 (70.5%) short-tailed weasels, 2 of 8 (25.0%) striped skunks, 2 of 11 (18.2%) North American river otters, 9 of 85 (10.6%) raccoons, and 2 of 20 (10.0%) bobcats. Samples from six fishers, 24 coyotes, 25 red foxes, 58 beavers, 45 red-squirrels and 59 muskrats were negative. Antibodies to AMDV were detected by CIEP in 16 of 56 (28.6%) mink and one of the 8 skunks (12.5%). Thirteen of the mink were positive for PCR and CIEP, but three mink and one skunk were CIEP positive and PCR negative. Positive CIEP or PCR animals were present in all nine counties from which mink or weasel samples were collected. CONCLUSIONS: The presence of AMDV in so many species across the province has important epidemiological ramifications and could pose a serious health problem for the captive mink, as well as for susceptible wildlife. The mechanism of virus transmission between wildlife and captive mink and the effects of AMDV exposure on the viability of the susceptible species deserve further investigation.

A survey of Aleutian mink disease virus infection of feral American mink in Nova Scotia.

Spleen samples from 14 mink that were trapped in 4 counties of Nova Scotia were tested for the presence of the Aleutian mink disease virus (AMDV) by polymerase chain reaction. Viral DNA was not detected in samples from Kings County (n = 2), but was detected in all the mink sampled from Colchester (n = 2) and Halifax (n = 6) counties, and 3 of 4 mink from Yarmouth County. The high level of AMDV-infected mink in Colchester and Halifax counties may pose a serious threat to the captive mink and wild animal populations. Because treatment of infected free-ranging mink is not an option, AMDV control strategies for the captive mink should be primarily focused on bio-security to protect clean ranches.