Aleutian Disease (Plasmacytosis) of Mink: II. Responses of Mink to Formalin-Treated Diseased Tissues and to Subsequent Challenge With Virulent Inoculum

An experiment was carried out to examine the responses of Aleutian and standard dark types of mink to inoculations of formalintreated suspensions of tissues of mink with experimentally transmitted plasmacytosis. Control groups of mink received similar injections of normal Aleutian mink tissues or diseased tissues without formalin treatment. A second experiment was conducted to test the formalinized diseased tissue suspension for immunogenic value. Groups of mink which received one, two, or three doses of "vaccine" were later challenged with virulent inocula. Additional groups of mink served as unvaccinated and environmental controls.Treatment with 0.3% formalin with fine trituration and incubation at 37 degrees C was effective in preventing the development of plasmacytosis in inoculated mink. These mink remained susceptible to subsequent challenge with untreated diseased tissue suspensions. No immunity was demonstrated in the vaccinated mink. Mink inoculated with normal mink tissues did not develop plasmacytosis, nor did uninoculated environmental controls.     

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.     

Reduced severity of lesions in mink infected transplacentally with Aleutian disease virus.

Inoculation of mink late in the second trimester of pregnancy with Aleutian disease virus (ADV) produces a persistent infection in the offspring. When these mink were analyzed at 83 days of age and compared with adolescent mink infected for a similar length of time, the transplacentally infected mink show: 1) a marked reduction in plasmacytosis, immunoglobulin level and specific ADV antibody; 2) increased amounts of infectious ADV and numbers of cells containing viral antigen; 3) a marked reduction in immune complex glomerulonephritis and absence of immune complex arteritis; 4) free ADV antigen in the glomeruli; and 5) a striking accumulation of eosinophils in the tissues. The findings suggest that the degree of ADV expression is partially immunologically controlled.     

Cytokine profiles in adult mink infected with Aleutian mink disease parvovirus

The aim of this study was to examine the levels of gamma interferon (IFN-gamma)-, interleukin 4 (IL-4)-, and IL-8-producing cells in peripheral blood mononuclear cells from mink infected with the Aleutian mink disease parvovirus (ADV). As expected, ADV-infected mink developed high plasma gamma globulin values (hypergammaglobulinemia) and enhanced quantities of CD8-positive (CD8(+)) cells in the blood during the infection. We quantified the percentages of IFN-gamma- and IL-4-positive lymphocytes and IL-8-positive monocytes up to week 38 after virus challenge. The results clearly indicated marked increases in the percentages of IFN-gamma- and IL-4-producing lymphocytes during ADV infection. The total number of IL-8-producing monocytes in the blood of ADV-infected mink stayed fairly constant during the infection. In order to characterize the phenotype of the cytokine-producing cells, we performed double-labeling fluorescence-activated cell sorter (FACS) experiments with CD8 surface labeling in one channel and cytokine intracellular staining in the other. We found that most IFN-gamma and IL-4 in ADV-infected mink was produced by CD8(+) cells, while in the uninfected mink, these cytokines were primarily produced by a cell type that was not CD8 (possibly CD4-positive cells). We also observed that IL-8 was almost exclusively produced by monocytes. All of the above findings led us to conclude that both Th1- and Th2-driven immune functions are found in mink plasmacytosis.     

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.     

A frameshift mutation in the LYST gene is responsible for the Aleutian color and the associated Chédiak–Higashi syndrome in American mink

One of the colors of mink is Aleutian (aa)—a specific gun‐metal gray pigmentation of the fur—commonly used in combination with other color loci to generate popular colors such as Violet (aammpp) and Sapphire (aapp). The Aleutian color allele is a manifestation of mink Chédiak‐Higashi syndrome (CHS), which has been described in humans and several other species. As with forms of CHS in other species, we report that the mink CHS is linked to the lysosomal trafficking regulator ( LYST ) gene. Furthermore, we have identified a base deletion (c.9468delC) in exon 40 of LYST, which causes a frameshift and virtually terminates the LYST product prematurely (p.Leu3156Phefs*37). We investigated the blood parameters of three wild‐type mink and three CHS mink. No difference in the platelet number between the two groups was observed, but an accumulation of platelets between the groups appears different when collagen is used as a coagulant. Microscopic analysis of peripheral blood indicates giant inclusions in the neutrophils of the Aleutian mink types. Molecular findings at the LYST locus enable the development of genetic tests for analyzing the color selection in American mink.     

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.     

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.     

High prevalence of Aleutian mink disease virus in free-ranging mink on a remote Danish island

Aleutian mink disease virus (AMDV) causes severe disease in farmed mink (Neovison vison) worldwide. In Denmark, AMDV in farmed mink has been confined to the northern part of the mainland since 2002. From 1998 to 2009, samples from 396 free-ranging mink were collected from mainland Denmark, and a low AMDV antibody prevalence (3% of 296) was found using countercurrent immune electrophoresis. However, on the island of Bornholm in the Baltic Sea, a high prevalence (45% of 142 mink) was detected in the free-ranging mink. Aleutian mink disease virus was detected by polymerase chain reaction in 32 of 49 antibody-positive free-ranging mink on Bornholm, but not in mink collected from other parts of Denmark. Sequence analysis of 370 base pairs of the nonstructural gene of the AMDV of 17 samples revealed two clusters with closest similarity to Swedish AMDV strains.     

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.     

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.     

Impaired phagocytosis by the mononuclear phagocytic system in sapphire mink affected with Aleutian disease

The phagocytic function of the mononuclear phagocytic system (MPS) in normal sapphire mink and in sapphire mink affected with experimental Aleutian disease was compared. Clearance from blood of carbon particles or 125I-labeled microaggregated human serum albumin, and subsequent measurement of radioactivity in phagocytic organs indicated profound MPS blockade in mink affected with advanced Aleutian disease. In contrast, MPS activity in mink in the early stage of the disease was comparable to that of normal mink. It is suggested that the MPS blockade may be responsible for some pathologic changes in Aleutian disease.     

The relationship between capsid protein (VP2) sequence and pathogenicity of Aleutian mink disease parvovirus (ADV): a possible role for raccoons in the transmission of ADV infections.

Aleutian mink disease parvovirus (ADV) DNA was identified by PCR in samples from mink and raccoons on commercial ranches during an outbreak of Aleutian disease (AD). Comparison of DNA sequences of the hypervariable portion of VP2, the major capsid protein of ADV, indicated that both mink and raccoons were infected by a new isolate of ADV, designated ADV-TR. Because the capsid proteins of other parvoviruses play a prominent role in the determination of viral pathogenicity and host range, we decided to examine the relationship between the capsid protein sequences and pathogenicity of ADV. Comparison of the ADV-TR hypervariable region sequence with sequences of other isolates of ADV revealed that ADV-TR was 94 to 100% related to the nonpathogenic type 1 ADV-G at both the DNA and amino acid levels but less than 90% related to other pathogenic ADVs like the type 2 ADV-Utah, the type 3 ADV-ZK8, or ADV-Pullman. This finding indicated that a virus with a type 1 hypervariable region could be pathogenic. To perform a more comprehensive analysis, the complete VP2 sequence of ADV-TR was obtained and compared with that of the 647-amino-acid VP2 of ADV-G and the corresponding VP2 sequences of the pathogenic ADV-Utah, ADV-Pullman, and ADV-ZK8. Although the hypervariable region amino acid sequence of ADV-TR was identical to that of ADV-G, there were 12 amino acid differences between ADV-G and ADV-TR. Each of these differences was at a position where other pathogenic isolates also differed from ADV-G. Thus, although ADV-TR had the hypervariable sequence of the nonpathogenic type 1 ADV-G, the remainder of the VP2 sequence resembled sequences of other pathogenic ADVs. Under experimental conditions, ADV-TR and ADV-Utah were highly pathogenic and induced typical AD in trios of both Aleutian and non-Aleutian mink, whereas ADV-Pullman was pathogenic only for Aleutian mink and ADV-G was noninfectious. Trios of raccoons experimentally inoculated with ADV-TR and ADV-Utah all became infected with ADV, but only a single ADV-Pullman-inoculated raccoon showed evidence of infection. Furthermore, none of the ADV isolates induced pathological findings of AD in raccoons. Finally, when a preparation of ADV-TR prepared from infected raccoon lymph nodes was inoculated into mink and raccoons, typical AD was induced in Aleutian and non-Aleutian mink, but raccoons failed to show serological or pathological evidence of infection. These results indicated that raccoons can become infected with ADV and may have a role in the transmission of virus to mink but that raccoon-to-raccoon transmission of ADV is unlikely.     

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.     

Studies on the Progression of Aleutian Disease in Mink

One hundred ninetyfive male ADV-negative mink, including 79 pairs of brothers, were followed in their response to natural ADV-infection caused by mating with ADV-positive females and under epidemic conditions. Special attention was drawn to the development of progressive versus non-progressive Aleutian disease. This was done by plasmaelectrophoresis, detection of antibodies to ADV, and finally by macroscopical examination of mink organs at pelting time. We found that the progression of Aleutian disease presumably is under some genetic influence. We also found indication of differences in the response to ADV depending on how the infection was introduced. Mating to positive females (low virus concentration) resulted in significantly higher proportion of non-progressive responders than infection under epidemic conditions (high virus concentration).     

Immunoenzyme Western blotting analysis of antibody specificity in Aleutian disease of mink, a parvovirus infection.

Aleutian disease virus (ADV), an autonomous parvovirus, persistently infects mink and induces very high levels of virus-specific antibody. All strains of ADV infect all mink, but only highly virulent strains cause progressive disease in non-Aleutian mink. The development of antibody to individual ADV proteins was evaluated by Western blotting by using the sera of 22 uninfected mink and 163 naturally or experimentally infected mink. ADV has virion proteins of 86,000 and 78,000 daltons that are closely related. A new, possibly nonvirion protein of 143,000 daltons was observed, as well as a known nonvirion protein of 71,000 daltons. Sera from mink experimentally or naturally infected with ADV of high or low virulence generally reacted about equally with all four proteins. The only exceptions noted were that 8 of 15 sera of mink infected transplacentally preferentially reacted with the two virion proteins and sera from mink with the monoclonal gammopathy of Aleutian disease reacted preferentially with either virion (10 of 12) or nonvirion (2 of 12) proteins.     

Aleutian Disease (Plasmacytosis) of Mink III. Propagation of the Virus in Mink Tissue Cultures

Cultures of mink testis and mink kidney cells were inoculated with 10% extracts and filtrates of tissue from plasmacytosis-affected mink. Specific morphological changes were observed in cultures of kidney and testis cells. Millipore filtration experiments suggested the size range of the agent to be from 10 to 50 mmu. Inoculation of fluids from the 2nd, 4th and 8th tissue culture passages of the agent induced plasmacytosis in adult mink.     

Replication of Aleutian mink disease parvovirus in lymphoid tissues of adult mink: involvement of follicular dendritic cells and macrophages.

By using strand-specific in situ hybridization and immunohistochemistry, evidence for replication of the Aleutian mink disease parvovirus was observed in cells resembling macrophages and cells resembling follicular dendritic cells at 10 days after infection but only in macrophages at 60 days. Sequestration of the Aleutian mink disease parvovirus in larger numbers of macrophages and follicular dendritic cells was noted at both 10 and 60 days.     

Slow viral infections of animals: experimental models for human disease

Sigurdsson's three criterions for a slow viral infection are quoted and discussed and the background of his work briefly described. A fourth criterion for a slow viral infection, is suggested, infection of the hosts lymphoid tissues, which is a common feature of slow infections that have been observed. The general properties of the maedi-visna virus, the diseases and the immune response it causes are discussed. Sheep and goats are susceptible to natural maedi-visna infection. In the goat an identical retrovirus causes arthritis. Arthritis has not been found in maedi-visna infected sheep. Two subacute spongiform encephalopathies of animals are shortly reviewed, scrapie in sheep and goats and transmissible mink encephalopathy (TME). General properties of the very unusual scrapie agent are mentioned briefly. The third type of a slow viral infection is mentioned, Aleutian mink disease, an immunopathological disorder of certain minks caused by a selective infection of lymphoid cells.