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BackgroundSelecting 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.ResultsThe 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).ConclusionPrevious 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.

The order Piroplasmida contains a diverse group of intracellular parasites, many of which can cause significant disease in humans, domestic animals, and wildlife. Two piroplasm species have been reported from raccoons (Procyon lotor), Babesia lotori (Babesia sensu stricto clade) and a species related to Babesia microti (called B. microti-like sp.). The goal of this study was to investigate prevalence, distribution, and diversity of Babesia in raccoons. We tested raccoons from selected regions in the United States and Canada for the presence of Babesia sensu stricto and Babesia microti-like sp. piroplasms. Infections of Babesia microti-like sp. were found in nearly all locations sampled, often with high prevalence, while Babesia sensu stricto infections had higher prevalence in the Southeastern United States (20–45% prevalence). Co-infections with both Babesia sp. were common. Sequencing of the partial 18S rRNA and cytochrome oxidase subunit 1 (cox1) genes led to the discovery of two new Babesia species, both found in several locations in the eastern and western United States. One novel Babesia sensu stricto sp. was most similar to Babesia gibsoni while the other Babesia species was present in the ‘western piroplasm’ group and was related to Babesia conradae. Phylogenetic analysis of the cox1 sequences indicated possible eastern and western genetic variants for the three Babesia sensu stricto species. Additional analyses are needed to characterize these novel species; however, this study indicates there are now at least four species of piroplasms infecting raccoons in the United States and Canada (Babesia microti-like sp., Babesia lotori, a novel Babesia sensu stricto sp., a novel western Babesia sp.) and a possible fifth species (Babesia sensu stricto) in raccoons in Japan., Graphical abstract Image 1, Highlights • Raccoons in all locations tested were infected with piroplasms. • Babesia microti-like sp. was commonly found in raccoons throughout North America. • Babesia sensu stricto spp. were less common throughout North America. • Four, possibly five, distinct species of piroplasms in raccoons. • Possible spatial genetic variation within the two raccoon piroplasm species.

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.

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, CatSperβ, 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.

Aleutian disease (AD) is one of the most important health problems in the mink industry worldwide, leading to economic losses. We used a set of single nucleotide polymorphisms (SNPs) to detect the genomic regions potentially under selection for response to Aleutian mink disease virus (AMDV) infection in black American mink. A total of 191 mink which were inoculated with a local strain of AMDV and survived until pelting were genotyped using genotyping-by-sequencing technique. The presence of viral DNA in the spleen samples were tested by polymerase chain reaction. After filtering, 47,800 SNPs at 171 individuals were used for further analyses. Signatures of selection for response to AMDV infection were detected using fixation index (FST) and nucleotide diversity (θπ) statistics measured between negative and positive groups. The overlap of top 1% SNPs obtained from both FST and θπ scores were considered as potential selection signs. This measurement identified a total of 21 candidate regions containing 11 genes which were likely subjected to selection for viral clearance. Several identified genes were those that modulate immune system (TCF4), reproductive process (CATSPERB, MAS1 and IGF2R), response to stimulus (WNT11 and MAS1), and functions of heart (TENM4 and WNT11) and liver (IGF2R). In addition, gene ontology showed that 63.6% of detected genes (seven) were involved in binding activities (GO:0005488). These genes can be used in molecular assessment of viral clearance in American mink. The results indicated that selection for viral clearance and thus AD tolerant animals can be a feasible strategy to deal with the global AMDV infection on mink farms.

BackgroundFeed 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).ResultsNo 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.ConclusionKelp supplementation improved kidney function of mink infected with AMDV with no effect on liver function, immune response to infection by AMDV or virus replication.

Farmed American mink (Neovison vison (Schreber, 1777)) pose a risk to biodiversity owing to escape and release from farms. Feral mink may affect native species in locations where American mink are not endemic, such as Europe. In contrast, escaping domestic mink may hybridize with wild mink in North America, leading to introgression of domestic traits via hybrid-mediated gene flow. We tested this idea in eastern Canada, which has a history of mink farming. We sampled known domestic and free-ranging mink, and profiled 508 individuals at 15 microsatellite loci. We found that 33% of free-ranging mink were either escaped domestic individuals, domestic–wild hybrids, or were introgressed to domestic or wild parental groups. The greatest prevalence of free-ranging domestic, hybrid, or introgressed mink (59%) occurred in Nova Scotia, which also had the most mink farms. Historic (1980s or earlier) mink sampled from museums had higher allelic richness and private allelic richness than contemporary wild mink. Domestic mink are artificially selected for traits desired by farmers, and as such, introgression with wild mink may lead to a loss of local adaptation. Our findings demonstrate that continued escape and release of mink could pose risks to the maintenance of genetic integrity in wild mink.

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 (P0.01). 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.

Linkage disequilibrium (LD) has been defined as the correlation between alleles at different loci in the genome. The LD levels can be influenced by the evolutionary processes and historical events in populations. The main objective of this study was to estimate the LD levels at different distances of American mink genome using genotyping-by-sequencing (GBS) data. A total of 285 American mink (Neovison vison) were sequenced based on GBS libraries prepared by digesting the genomic DNA with the restriction enzyme ApeKI. After quality control, 13,321 single nucleotide polymorphism (SNP) markers located on 46 Scaffolds were used to determine the extension of LD in the genome. The average r2 was computed for all syntenic SNP pairwise at inter-marker distances from 0 up to 1 Mb. The average r2 between adjacent SNPs was 0.29, ranged from 0.18 to 0.53 across all scaffolds. In addition, the average distance between adjacent markers was 51 kb. The average r2 above 0.3 was observed in less than 1 kb distances and declined with increase in distances between markers. The average r2 was estimated to be less than 0.2 for markers more than 10 kb apart. Furthermore, the average LD level was decreased to 0.08 for inter-marker distances between 0.9 and 1 Mb. The results of this study can be used to determine the optimum maker density required for obtaining enough accuracy and power in both genomic selection and genome-wide association studies.

One-hundred black female mink naturally infected with the Aleutian mink disease virus (AMDV) were monitored between Nov. 2005 and Feb. 2008. Animals were tested for antibodies against the virus by counter-immunoelectrophoresis (CIEP) and for serum globulin level by the iodine agglutination test (IAT) on nine occasions. CIEP and IAT tests were conducted twice each year on kits at 4 and 7 months of age. The prevalence of CIEP positive adult females and kits was 12% and 20.9% (n=411) in 2006, respectively, but one female and none of the kits (n=491) were seropositive in 2007. IAT positive cases ranged between 14.1% and 80.7% in adults and between 17.0% and 57.6% in kids, suggesting infection by pathogens other than AMDV. Three of the seropositive females cleared the virus and were considered to be resistant, although they continued producing antibodies until pelted at 34 months of age. The pattern of viral transmission among individuals which were in contact with one another was complex. The virus was not transmitted from two infected males to five seronegative females to which they were mated. There was a significantly higher incidence of CIEP positive kits from seropositive dams in 2006 (63.4%) compared with kits from seronegative parents (16.8%), showing transplacental transmission of the virus. All eight progeny of the only infected male in 2006 were CIEP- and PCR-positive, implying that the male possibly transmitted susceptible gene(s) to his progeny. Estimates of heritability for the CIEP-positive kits at 4 and 7 months of age were 0.573 and 0.497, respectively, suggesting the strong contribution of the host genetics to this trait. It was concluded that resistance to infection played a more important role than the survival rate of infected individuals on the herd’s health status. Measures of reproduction were not affected by the IAT scores of the dams.

In this report we present the results of the analysis of approximately 2.7 Mb of genomic information for the American mink (Neovison vison) derived through BAC end sequencing. Our study, which encompasses approximately 1/1000th of the mink genome, suggests that simple sequence repeats (SSRs) are less common in the mink than in the human genome, whereas the average GC content of the mink genome is slightly higher than that of its human counterpart. The 2.7 Mb mink genomic dataset also contained 2,416 repeat elements (retroids and DNA transposons) occupying almost 31% of the sequence space. Among repeat elements, LINEs were over-represented and endogenous viruses (aka LTRs) under-represented in comparison to the human genome. Finally, we present a virtual map of the mink genome constructed with reference to the human and canine genome assemblies using a comparative genomics approach and incorporating over 200 mink BESs with unique hits to the human genome.

A rare color variant of the American mink (Neovison vison), discovered on a ranch in Nova Scotia and referred to as the “marbled” variety, carries a distinctive pigment distribution pattern resembling that found in some other species, e.g., the Siamese cat and the Himalayan mouse. We tested the hypothesis that the color pattern in question—light-colored body with dark-colored points (ears, face, tail, and feet)—is due to a mutation in the melanin-producing enzyme tyrosinase (TYR) that results in temperature-sensitive pigment production. Our study shows that marbled mink carry a mutation in exon 4 of the TYR gene (c.1835C > G) which results in an amino acid substitution (p.H420Q). The location of this substitution corresponds to the amino acid position that is also mutated in the TYR protein of the Himalayan mouse. Thus, the marbled variant is more aptly referred to as the Himalayan mink.

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.

Presence of Aleutian mink disease virus (AMDV) was tested in 466 wild animals of 12 furbearing species that were trapped in 10 Nova Scotia counties. DNA was extracted from cell-free spleen homogenates by magnetic beads and presence of the virus was tested for by PCR using two primer pairs, each with three DNA volumes. Anti-viral antibodies in spleen homogenates were also tested by counterimmunoelectrophoresis (CIEP) in duplicate. Positive PCR or CIEP samples were detected in 96.9% of 64 mink, 70.5% of 61 weasels, 25.0% of 8 skunks, 18.2% of 11 otters, 10.6% of 85 raccoons and 10.0% of 20 bobcats. Samples of six fishers, 24 coyotes, 25 red foxes, 58 beavers, 45 squirrels and 59 muskrats were negative. Anti-viral antibodies were detected in 16 (25%) of the mink and one of the skunk samples, possibly due to the low antibody titer in spleen homogenates. All CIEP positive animals were also PCR positive, except for three mink and one skunk which were PCR negative. This observation could be the manifestation of persistent antibody production and short-lived viral replication which are signatures of AMDV pathogenesis. The numbers of samples amplified by the two primer pairs were significantly different, suggesting that PCR amplification failure of CIEP positive animals could be the result of differences in the sequence of viruses circulating in different animals. The results imply that the combination of CIEP and PCR using multiple primers would increase the accuracy of detecting AMDV exposure in wild animal species. Infected animals were present in all counties from which samples were collected. The high proportion of animals of so many species across the province carrying AMDV has important epidemiological ramifications and could pose a serious threat to ranched mink as well as susceptible wild animal species.

The present study aimed to identify single-nucleotide polymorphism (SNP) in coding and non-coding regions of interleukin-6 (IL-6) gene of Pakistani sheep. The IL-6 gene of 205 animals from nine sheep breeds were sequenced for screening of SNP. Characterizing the IL-6 gene revealed thirteen SNP sites within the intronic region of IL-6 gene. The novel SNPs found in the present study can serve as genetic marker for association studies with susceptibility/resistance to parasite infection in sheep. This is first report of SNP polymorphism of IL-6 gene of Pakistani sheep.

Amino acid polymorphisms in the prion protein gene (PrP) affect the susceptibility of sheep to scrapie, a transmissible spongiform encephalopathy (TSE). In particular, amino acid substitutions at codons 136, 154 and 171 of the ovine PrP gene are associated with different degrees of susceptibility to the classical form of scrapie, caused by ‘typical’ scrapie strains. Existing genotyping tests for scrapie susceptibility normally interrogate only the single nucleotide polymorphisms (SNPs) most relevant to ‘typical’ strains. Recently, however, a number of novel variants of the scrapie agent have been discovered. The ability of these new, ‘atypical’ scrapie variants to infect sheep that are resistant to ‘typical’ variants has raised concerns about the reduction in genetic variability that may result from intense selection for resistance to classical scrapie. Furthermore, a growing interest in a potential role for specific PrP genotypes in modulating performance traits is also driving a move toward more extensive characterization of haplotypes at the PrP locus. Here, we describe a single-tube method for the interrogation of eight SNPs within seven codons (112, 136, 141, 154, 171, 231 and 241) of the ovine PrP gene. This method is as accurate as sequencing, yet more affordable, and can easily be automated for high-throughput sample screening. Moreover, it can be modified to accommodate genetic variations that are found in local and heritage breeds.

Primary reasons for disposal of 26,651 dairy cows of the Ayrshire, Guernsey, Holstein and Jersey breeds were collected in Canadian Record of Performance herds from September, 1967 to August, 1968. Reproduction and low milk production were major causes of voluntary herd removals. The percentages culled annually for these problems ranged from 13.4 to 24.4% and from 15.5 to 28.3%, respectively. The Guernsey breed was highest (24.4%) in the percentage culled because of reproduction. The Holstein was lowest (15.5%) in the percentage culled because of low production, but highest (10.2%) in the percent removed because of udder problems. Disposal patterns changed with the lactation number of the cow. Young cows were culled more heavily for low production than their older herdmates. This was offset by increases in the percentages of older cows removed for reproduction, diseases and weaknesses in udders. No major monthly trends were observed in these data, although there was a statistically significant increase in summer sales for dairy purposes with a parallel decrease in beef sales.