Aleutian Mink Disease Virus in Free-Ranging Mink from Sweden

Aleutian mink disease (AMD) is a chronic viral disease in farmed mink and the virus (AMDV) has been found in many free-ranging mink (Neovison vison) populations in Europe and North America. In this study, AMDV DNA and AMDV antibodies were analysed in 144 free-ranging mink hunted in Sweden. Associations between being AMDV infected (defined as positive for both viral DNA and antibodies) and the weight of the spleen, liver, kidneys, adrenal glands and body condition were calculated and the sequences of ten AMDV isolates were analysed in order to characterize the genetic relationships. In total, 46.1% of the mink were positive for AMDV antibodies and 57.6% were positive for AMDV DNA. Twenty-two percent of the mink tested on both tests (n = 133) had dissimilar results. The risk of having AMDV antibodies or being positive for AMDV DNA clearly increased with age and the majority of the mink that were two years or older were infected. Few macroscopic changes were found upon necropsy. However, the relative weight of the spleen was sexually dimorphic and was found to be slightly, but significantly (p = 0.006), heavier in AMDV infected male mink than uninfected. No association between AMDV infection and body condition, weight of the kidneys, liver or adrenal glands were found. Several different strains of AMDV were found across the country. Two of the AMDV sequences from the very north of Sweden did not group with any of the previously described groups of strains. In summary, AMDV seems to be prevalent in wild mink in Sweden and may subtly influence the weight of the spleen.     

Evaluation of two enzyme-linked immunosorbent assays for serodiagnosis of Aleutian mink disease virus infection in mink

Background

Aleutian disease in mink is caused by infection with Aleutian mink disease virus (AMDV). In Sweden, the infection most commonly causes classical Aleutian disease in which the immune system fails to neutralize the virus and the infection becomes persistent. Diagnosis of AMDV infection is based on serological methods that detect virus-specific antibodies. Traditionally counterimmunoelectrophoresis (CIEP) has been the preferred method, but in order to enable automation interest has been paid to other antibody detecting systems. Recently, at least two different ELISA systems that detect antibodies to AMDV have been manufactured; one is based on an in vitro grown AMDV as antigen, and the other system is based on the AMDV capsid protein VP2 as antigen. The aim of this study was to evaluate the two ELISA systems for detection of antibodies to AMDV using CIEP as the gold standard.

Results

When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the ELISA with the VP2 antigen had a sensitivity of 99.7% and a specificity of 98.3% compared to CIEP (n?=?364). Analysis of samples with the AMDV-G antigen based ELISA employing an assay cut-off value based on the negative control samples, as suggested by the manufacturer, resulted in a sensitivity of 54.3% and a specificity of 93.2% with reference to CIEP as the gold standard (n?=?359). When employing the mean optical density of the samples from CIEP negative mink plus three standard deviations as cut-off value, the AMDV-G ELISA had a sensitivity of 37.6% and a specificity of 98.3%.

Conclusions

The ELISA system based on VP2 antigen had high sensitivity and specificity, and was concluded to be an alternative to the CIEP as a diagnostic tool for AMDV antibodies. In contrast, the AMDV-G ELISA suffered from low sensitivity when compared to CIEP.

     

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

Background

Aleutian 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.

Methods

Samples from 13 different countries were collected and partial NS1 gene was sequenced and subjected to phylogenetic analyses.

Results

The 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.

Conclusion

To 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.

     

Aleutian mink disease parvovirus in wild riparian carnivores in Spain

Serious declines in populations of native European mink (Mustela lutreola) have occurred in Europe. One responsible factor may be infectious diseases introduced by exotic American mink (Mustela vison). In order to investigate a possible role for Aleutian mink disease parvovirus (ADV), we surveyed native riparian carnivores and feral American mink. When serum samples from 12 free-ranging European and 16 feral American mink were tested, antibodies to ADV were detected from three of nine European mink. ADV DNA was detected by polymerase chain reaction in whole cell DNA from four of seven carcasses; two American mink, one European mink and a Eurasian otter (Lutra lutra). Lesions typical of Aleutian disease were present in one of the American mink. A portion of the ADV VP2 capsid gene was sequenced and the results suggested that two sequence types of ADV were circulating in Spain, and that the Spanish ADVs differed from other described isolates from North America and Europe. Future conservation and restoration efforts should include measures to avoid introduction or spread of ADV infection to native animals.     

Serologic survey for selected viral pathogens in free-ranging endangered European mink (Mustela lutreola) and other mustelids from south-western France

To investigate the possible role of selected pathogens in the decline of endangered European mink (Mustela lutreola) populations and the potential for these pathogens to affect mink survival, a serologic survey was conducted using serum samples collected from March 1996 to March 2003 in eight departments of south-western France. In total, 481 free-ranging individuals of five mustelid species (including the European mink) were tested. Sympatric mustelids can serve as sentinels to determine the presence of antibodies to viruses in the study area that could potentially infect mink. Antibodies to Canine distemper virus (CDV) were detected in all species; 9% of 127 European mink, 20% of 210 polecats (Mustela putorius), 5% of 112 American mink (Mustela vison), 33% of 21 stone marten (Martes foina) and 5% of 20 pine marten (Martes martes). Antibody prevalence was significantly higher in stone marten and polecats, possibly because their ranges overlap more closely with that of domestic species than that of the other species tested. Antibodies to Canine adenovirus were detected in all species but the pine marten; antibody prevalence estimates ranging from 2% to 10%. Antibodies to canine parainfluenza virus were detected in 1% of European mink, 1% of American mink and 5% of tested polecats but were not detected in Martes species. Antibodies to Rabies virus (RV) were detected in three animals, possibly because of interspecies transmission of bat lyssaviruses as the sampling area is considered to be free of RV, or to a lack of test specificity, as antibody titers were low. The high antibody prevalence to potentially lethal CDV suggests that this pathogen could have significant effects on the free-ranging populations and has implications for the conservation efforts for the endangered European mink.     

Leptospirosis in free-ranging endangered European mink (Mustela lutreola) and other small carnivores (Mustelidae, Viverridae) from southwestern France

To study the possible role of disease in the decline of endangered European mink (Mustela lutreola), we conducted a survey of antibody prevalence and renal carriage of pathogenic leptospira (Leptospira interrogans sensu lato) using serum and kidney samples collected from 1990 to 2007 from several free-ranging small carnivores and farmed American mink (Mustela vison) in southwestern France. An indirect microscopic agglutination test using a panel of 16 serovars belonging to 6 serogroups (Australis, Autumnalis, Icteroh?morrhagi?, Grippotyphosa, Panama, Sejroe) revealed antibodies in all species, with significant differences in antibody prevalences: 74% in European mink (n=99), 65.4% in European polecats (Mustela putorius, n=133), 86% in American mink (n=74), 89% in stone martens (Martes foina, n=19), 74% in pine martens (Martes martes, n=19), 35% in common genets (Genetta genetta, n=79), and 31% in farmed American mink (n=51). Serogroups Australis and Icteroh?morragi? were dominant in most free-ranging species; serogroup Grippotyphosa had high prevalences in European mink. Such high antibody prevalences have never been reported. They are probably related to the large number of known reservoirs, rats (Rattus spp.), muskrat (Ondatra zibethicus), and coypu (Myocastor coypu), in the study area. The polymerase chain reaction test specific for pathogenic leptospiral DNA detected renal carriage in 23% of 34 European mink, 22% of 18 polecats, and 15% of 33 free-ranging American mink, with no significant differences. Renal carriage shows that mustelids may shed leptospira for short periods, but their epidemiologic role is probably limited. High antibody prevalences suggest that the disease is unlikely to be highly pathogenic for these species; however, chronic forms of the disease (abortions, renal lesions) could reduce the reproductive success or life span of infected animals. Further studies on the pathogenicity of leptospirosis in these populations are needed to measure its impact on the population dynamics of these rodent predators.     

Antibodies to Aleutian mink disease parvovirus in free-ranging European mink (Mustela lutreola) and other small carnivores from southwestern France

Owing to the rapid decline of the European mink (Mustela lutreola) in France, a national conservation action plan has been initiated, in which scientific research to improve understanding of the causes of the decline is one of the primary objectives. In order to investigate the possible role of Aleutian disease parvovirus (ADV) in decline of the species, a serologic survey was conducted from March 1996 to March 2002 in 420 free-ranging individuals of six species of small carnivores distributed in eight departments of southwestern France. Antibodies to ADV were detected in 17 of 75 American mink (Mustela vison), 12 of 99 European mink, 16 of 145 polecats (Mustela putorius), four of 17 stone martens (Martes foina), one of 16 pine martens (Martes martes), and three of 68 common genets (Genetta genetta). Seroprevalence was significantly higher in American mink than in other species. Seropositive individuals with gamma globulin levels >20% were observed in four European mink, four American mink, two stone martens, and one pine marten. Geographic distribution of positive animals indicates the virus has spread to all areas where European mink are found. Furthermore, a trend of increasing prevalence seems to appear in Mustela sp. sympatric with American mink. Although further investigations are necessary to evaluate the role of ADV in decline of European mink, evidence of the virus in the wild at the levels found in our study has implications for conservation of this species.     

Development of a Peptide ELISA for the Diagnosis of Aleutian Mink Disease

Aleutian disease (AD) is a common immunosuppressive disease in mink farms world-wide. Since the 1980s, counterimmunoelectrophoresis (CIEP) has been the main detection method for infection with the Aleutian Mink Disease Virus (AMDV). In this study, six peptides derived from the AMDV structural protein VP2 were designed, synthesized, and used as ELISA antigens to detect anti-AMDV antibodies in the sera of infected minks. Serum samples were collected from 764 minks in farms from five different provinces, and analyzed by both CIEP (a gold standard) and peptide ELISA. A peptide designated P1 (415 aa–433 aa) exhibited good antigenicity. A novel ELISA was developed using ovalbumin-linked peptide P1 to detect anti-AMDV antibodies in mink sera. The sensitivity and specificity of the peptide ELISA was 98.0% and 97.5%, respectively. Moreover, the ELISA also detected 342 early-stage infected samples (negative by CIEP and positive by PCR), of which 43.6% (149/342) were true positives. These results showed that the peptide ELISA had better sensitivity compared with CIEP, and therefore could be preferable over CIEP for detecting anti-AMDV antibodies in serological screening.     

Royal pastel mink respond variously to inoculation with Aleutian disease virus of low virulence.

Information was sought on the varied responses of royal pastel mink (a non-Aleutian genotype) to Aleutian disease virus of low virulence. Thus, of 20 yearling female pastel mink inoculated subcutaneously with a large amount of the Pullman strain of Aleutian disease virus, only 3 succumbed to the disease. Of the other 17 mink, 3 had neither viremia nor a rise in level of serum gamma globulin during the 24 weeks after inoculation. The other 14 mink were viremic for variable periods during the first 12 weeks. In only five mink was the viremia accompanied by elevated levels of serum gamma globulin, usually from week 8 on. Of the 16 subclinically infected mink that did not succumb to intercurrent disease and otherwise remained healthy, 9 were examined at 19 to 31 months for persisting virus. In only one mink, small amounts were detected in the mesenteric lymph node and spleen nearly 28 months after inoculation. The other seven mink that survived the infection were not protected when challenged 31 months later with a small amount of the highly virulent Utah-1 strain. Even though still poorly understood, these varied responses of the royal pastel mink to infection with Aleutian disease virus of low virulence have important pathogenetic and epidemiological implications.     

Inbreeding affects fecundity of American mink (Neovison vison) in Danish farm mink

Inbreeding is an increasing problem in farmed mink, because of limited exchange of individuals between farms. In this study, genetic relatedness within seven American mink (Neovison vison) colour strains originating from 13 different mink farms in Denmark was analysed using 21 polymorphic microsatellite loci. We detected large differences in the level of relatedness (range 0.017-0.520) within colour strains. Moreover, a very strong and highly significant negative correlation between the level of relatedness and fecundity was observed (r = 0.536, P < 0.001) [Correction added after online publication on 9 March 2011: r(2) has been changed to r]. To our knowledge, this is the first time that such a correlation has been demonstrated for commercially farmed mink.     

Analysis of experimental mink enteritis virus infection in mink: in situ hybridization, serology, and histopathology.

Strand-specific hybridization probes were used in in situ hybridization studies to localize cells containing mink enteritis virus (MEV) virion DNA or MEV replicative-form DNA and mRNA. Following the experimental MEV infection of 3-month-old unvaccinated mink, a significant increase in serum antibodies to MEV was detected at postinfection day (PID) 6, 2 days after the onset of fecal shedding of virus. Prior to the appearance of virus in feces, viral DNA could be detected in the mesenteric lymph node and intestine. The largest percentage of cells positive for virion DNA was 10% and was detected in the intestine on PID 6. However, replication of the virus apparently peaked at PID 4. The number of MEV replicative-form DNA and mRNA molecules was found to be approximately 250,000 copies per infected lymph node cell or crypt epithelial cell. The localization, levels, and time course of viral replication have important implications for the pathogenesis of MEV-induced disease. The data presented on MEV are correlated with earlier results on the other mink parvovirus, Aleutian mink disease parvovirus, and a possible explanation for the remarkable differences in pathogenesis of disease caused by these two parvoviruses is discussed.     

Diet of free-ranging American mink and European polecat in Denmark

Stomach and intestine contents of 211 American minkMustela vison Schreber, 1777 from two areas (Thy and Bornholm) in Denmark and stomach contents of 47 polecatsM. putorius Linnaeus, 1758 from Thy were analysed. Sympatric mink (from Thy) preyed mostly on mammals (55% occurrence), followed by amphibians (36%), birds (33%) and fish (30%), whereas polecat preyed mostly on amphibians (87%) and mammals (34%), and only occasionally on birds (9%) and fish (6%). Allopatric mink (from Bornholm) preyed mostly on birds (50%), followed by mammals (42%), fish (25%) and amphibians (4%). With the possible exception of some amphibians, no endangered species were found in their diet. No differences were found in food composition between wild and escaped farm mink. The concern that mink in general might have a detrimental effect on its prey species and other mustelids in terms of food competition in Denmark may be unjustified. It cannot be ruled out, however, that mink may locally have a seriously negative effect on some specific prey species, and clearly, more data is needed on eg prey abundance and spring and summer mink diet, to make stronger conclusions.     

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.     

Identification of a nonvirion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins.

We studied Aleutian disease virus polypeptides in Crandall feline kidney (CRFK) cells. When CRFK cells labeled with [35S]methionine at 60 h postinfection were studied by immunoprecipitation with sera from infected mink, the major Aleutian disease virus virion polypeptides (p85 and p75) were consistently identified, as was a 71,000-dalton nonvirion protein (p71). The peptide maps of p85 and p75 were similar, but the map of p71 was different. p85, p75, and p71 were all precipitated by sera from Aleutian disease virus-infected mink, including those with signs of progressive disease, but heterologous sera raised against purified Aleutian disease virus did not precipitate the nonvirion p71. These results indicated that the nonvirion p71 was unrelated to p85 and p75 and further suggested that mink infected with Aleutian disease virus develop antibody to nonvirion, as well as structural, viral proteins.     

Demonstration of Aleutian disease virus-specific lymphocyte response in mink with progressive Aleutian disease: comparison of sapphire and pastel mink infected with different virus strains

Lymphocyte blastogenesis was used to study the antiviral lymphocyte response of sapphire (Aleutian) and pastel (nonAleutian) mink inoculated with Pullman or Utah 1 Aleutian disease virus (ADV). Both mink genotypes developed a virus-specific response when inoculated with Utah 1 ADV. In contrast, after inoculation of Pullman ADV, sapphire mink had a positive virus-specific response, whereas pastel mink did not. Response occurred late after infection (8 wk) and correlated with the development of progressive Aleutian disease (AD). The response to keyhole limpet hemocyanin (KLH) and concanavalin A (Con A) was also determined. Most mink of either genotype, inoculated with either virus strain, maintained an anti-KLH response during disease. Most mink also responded to Con A, although some exhibited suppressed Con A response late in the disease course. These results indicated that mink develop an anti-ADV lymphocyte response during progressive AD and are not immunosuppressed with regard to other antigens or mitogens.     

Temporal replication of the Pullman strain of Aleutian disease virus in royal pastel mink.

Information was sought on the temporal replication of Aleutian disease virus in 27 royal pastel mink. Groups of three were examined 8 to 126 days after they were inoculated subcutaneously with 10(3) 50% lethal doses of the Pullman strain. Much individual variation was noted in the onset of infection, occurrence of viremia, and extent of virus replication in the tissues. Thus, virus was detected in lymph nodes regional to the site of inoculation in only some mink during the first 14 days after inoculation. During this period, virus was often present as well in the mesenteric lymph node and spleen. First detected on day 10, viremia was present in all mink examined on day 28 but occurred irregularly thereafter, even when virus was widespread in the tissues. Except in five mink succumbing to the disease, the tissue distribution of virus after day 28 tended to be more limited, and the titers were generally lower than they had been earlier. Even though present in the lymph nodes and spleen, virus was often absent from the kidney, liver, and intestine after day 28. Specific antibody was detected on day 28 and was present in all mink thereafter, ostensibly without any adverse effect on virus replication. In most mink, the infection was considered subclinical, for it was usually not accompanied by a rise in serum gamma globulin or by morphologic evidence of the disease. The virologic findings in this study have a bearing on the relationship of subclinical infections to both horizontal and vertical transmission of the virus.     

Replication of Aleutian mink disease parvovirus in vivo is influenced by residues in the VP2 protein.

Aleutian mink disease parvovirus (ADV) is the etiological agent of Aleutian disease of mink. Several ADV isolates have been identified which vary in the severity of the disease they elicit. The isolate ADV-Utah replicates to high levels in mink, causing severe Aleutian disease that results in death within 6 to 8 weeks, but does not replicate in Crandell feline kidney (CrFK) cells. In contrast, ADV-G replicates in CrFK cells but does not replicate in mink. The ability of the virus to replicate in vivo is determined by virally encoded determinants contained within a defined region of the VP2 gene (M. E. Bloom, J. M. Fox, B. D. Berry, K. L. Oie, and J. B. Wolfinbarger. Virology 251:288-296, 1998). Within this region, ADV-G and ADV-Utah differ at only five amino acid residues. To determine which of these five amino acid residues comprise the in vivo replication determinant, site-directed mutagenesis was performed to individually convert the amino acid residues of ADV-G to those of ADV-Utah. A virus in which the ADV-G VP2 residue at 534, histidine (H), was converted to an aspartic acid (D) of ADV-Utah replicated in CrFK cells as efficiently as ADV-G. H534D also replicated in mink, causing transient viremia at 30 days postinfection and a strong antibody response. Animals infected with this virus developed diffuse hepatocellular microvesicular steatosis, an abnormal accumulation of intracellular fat, but did not develop classical Aleutian disease. Thus, the substitution of an aspartic acid at residue 534 for a histidine allowed replication of ADV-G in mink, but the ability to replicate was not sufficient to cause classical Aleutian disease.     

Mink farms predict Aleutian disease exposure in wild American mink

Background

Infectious diseases can often be of conservation importance for wildlife. Spillover, when infectious disease is transmitted from a reservoir population to sympatric wildlife, is a particular threat. American mink (Neovison vison) populations across Canada appear to be declining, but factors thus far explored have not fully explained this population trend. Recent research has shown, however, that domestic mink are escaping from mink farms and hybridizing with wild mink. Domestic mink may also be spreading Aleutian disease (AD), a highly pathogenic parvovirus prevalent in mink farms, to wild mink populations. AD could reduce fitness in wild mink by reducing both the productivity of adult females and survivorship of juveniles and adults.

Methods

To assess the seroprevalence and geographic distribution of AD infection in free-ranging mink in relation to the presence of mink farms, we conducted both a large-scale serological survey, across the province of Ontario, and a smaller-scale survey, at the interface between a mink farm and wild mink.

Conclusions/Significance

Antibodies to AD were detected in 29% of mink (60 of 208 mink sampled); however, seroprevalence was significantly higher in areas closer to mink farms than in areas farther from farms, at both large and small spatial scales. Our results indicate that mink farms act as sources of AD transmission to the wild. As such, it is likely that wild mink across North America may be experiencing increased exposure to AD, via disease transmission from mink farms, which may be affecting wild mink demographics across their range. In light of declining mink populations, high AD seroprevalence within some mink farms, and the large number of mink farms situated across North America, improved biosecurity measures on farms are warranted to prevent continued disease transmission at the interface between mink farms and wild mink populations.