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.     

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.     

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).     

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.     

Vesical Hemorrhages in Male Aleutian Mink Caused by Hypersensitivity to Sulphadimidine

During an outbreak of Salmonella abortion in mink farms receiving food from a central feed plant, sulphamezathine (a 16 % solution of sulphadimidine sodium) was added to the food to combat the infection. After 3 days of medication, some males of the Aleutian type developed severe urinary bleedings. The serum concentration of the drug was not above the recommended value in 2 severely affected animals (1.5 and 1.7 mg/100 ml, respectively). Screening tests for the extrinsic (Thrombotest and Normotest) and intrinsic (cephalin time) coagulation mechanism, fibrinogen assay, fibrinolysis (plasma clot lysis time), and platelet count were not much different from normal. Coagulation or platelet defects did not therefore seem to be the cause of the bleedings. Some of the diseased animals died, and the only necropsy finding was a greatly distended urinary bladder filled with clotted blood. Histologically, hemorrhages and necrotic changes of varying severity were found in the vesical wall. In several cases, the arteries were the structures most evidently affected, indicating that the hemorrhages were due to vascular injury (Fig. 1). The damaged vessels were sporadically occluded by thrombi. The lesions were often most evident in subserosal arteries and in the relatively large arteries situated between the inner circular and the outer longitudinal muscular layer, whereas the submucosal structures were obscured by massive extravasations of red blood cells. Occasionally, the necrotic arteries were surrounded by incipient circumferential cellular accumulations, predominantly mononuclear cells, but some eosinophils were also present (Fig. 2). Thus, in these cases the vascular damage was similar to vascular lesions frequently accompanying viral plasmacytosis (periarteritis nodosa). The possibility exists that the animals were in an early developmental stage of plasmacytosis, but no extravesical changes suggesting plasmacytosis were discovered during the microscopic examination. Although other sulphonamides have occasionally shown toxic properties when administered to mink, this preparation has not, to the authors’knowledge, previously been recorded as injurious to this species. The following experiment was performed to elucidate the toxicity of sulphadimidine sodium to male Aleutian mink.     

Three-Dimensional Structure of Aleutian Mink Disease Parvovirus: Implications for Disease Pathogenicity

The three-dimensional structure of expressed VP2 capsids of Aleutian mink disease parvovirus strain G (ADVG-VP2) has been determined to 22 A resolution by cryo-electron microscopy and image reconstruction techniques. A structure-based sequence alignment of the VP2 capsid protein of canine parvovirus (CPV) provided a means to construct an atomic model of the ADVG-VP2 capsid. The ADVG-VP2 reconstruction reveals a capsid structure with a mean external radius of 128 A and several surface features similar to those found in human parvovirus B19 (B19), CPV, feline panleukopenia virus (FPV), and minute virus of mice (MVM). Dimple-like depressions occur at the icosahedral twofold axes, canyon-like regions encircle the fivefold axes, and spike-like protrusions decorate the threefold axes. These spikes are not present in B19, and they are more prominent in ADV compared to the other parvoviruses owing to the presence of loop insertions which create mounds near the threefold axes. Cylindrical channels along the fivefold axes of CPV, FPV, and MVM, which are surrounded by five symmetry-related beta-ribbons, are closed in ADVG-VP2 and B19. Immunoreactive peptides made from segments of the ADVG-VP2 capsid protein map to residues in the mound structures. In vitro tissue tropism and in vivo pathogenic properties of ADV map to residues at the threefold axes and to the wall of the dimples.     

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.     

Viral Plasmacytosis (Aleutian Disease) of Mink Resembling Human Collagen Disease

A disease in mink has been discovered that has many of the features of collagen diseases in man. Affected animals suffer from wasting with leukopenia and thrombocytopenia as well as plasma cell infiltration, hypergammaglobulinemia, glomerulonephritis, arteritis and amyloidosis. Cell-free filtrates and ultracentrifugates from diseased animals induced the disease in normal mink, and aleutian genotypes were unusually susceptible to infection. This genotype was characterized by abnormal lysosomal structures in all the granule-forming cells, resembling the Chediak-Higashi syndrome of man. Anti-γ-globulin factors similar to human rheumatoid factors were reported, although tests for antibodies such as ANF and LE factors have been negative. Arteritis and glomerulonephritis lesions stained positively for γ-globulin, and Coombs-type sensitized red cells have been detected in the majority of affected mink. Some mink develop a monodispersion of hypergammaglobulinemia resembling the serum protein changes in human myeloma. These studies highlight genetic, immunological and microbiological causative factors in a mink disorder resembling human collagen disease.     

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.     

Purification and characterization of Aleutian disease virus

Virus was isolated from infected mink organs by a combination of tissue homogenization, fluorocarbon extraction and ultracentrifugation. The final preparation was analysed by crossed immunoelectrophoresis and electronmicroscopy. Virions had a capsid diameter of 22 nm. Preparative agarose electrophoresis separated virions from contaminating ferritin. Crossed immunoelectrophoresis of virus gave a single precipitate with sera from infected mink. Crossed immunoelectrophoretic analysis with intermediate gels showed that a part of the virus preparation was complexed with antibody. Serum from a certain mink was found to contain precipitating antibody to (poly)nucleotid. Virus and virus-antibody complexes were found to focus at pH 4.0-4.4 in isoelectric focusing. In SDS-polyacrylamide gel-electrophoresis the main virus protein was found to have a molecular weight of 69000. This study gives further support to the classification of aleutian disease virus as a parvovirus.     

Aleutian disease of mink: the antibody response of sapphire and pastel mink to Aleutian disease virus.

The specific antiviral antibody response of sapphire and pastel mink to Pullman strain of ADV has been examined. Sapphire mink inoculated with from 300,000-3 LD50 developed high levels of specific antibody and AD. Pastel mink inoculated with parallel doses of ADV also produced antibody but did not develop AD. The low incidence of AD in pastel mink inoculated with Pullman strain of ADV is probably related to factors other than antiviral antibody.     

Treatment of Neonatally Aleutian Disease Virus (ADV) Infected Mink Kits with Gammaglobulin Containing Antibodies to ADV Reduces the Death Rate of Mink Kits

Aleutian disease virus (ADV) can cause pneumonitis in newborn kits up to 3 weeks old. In many cases the pneumonitis is fatal, but can be reduced by treatment with antibodies to ADV. The present report describes antibody therapy in both experimentally infected mink kits and in mink kits from a farm, where an ADV epidemic developed during the whelping period in the spring of 1987. In both cases the antibody treatment was found to have a beneficial effect on the survival rate of the mink kits. One hundred percent survival rate was found for the experimentally infected mink kits. The most pronounced effect for the naturally infected mink was found in the wildtype mink kits, where the death rate was 9.6 % for the antibody treated group versus 16.9 % for the untreated group (p < 0.001). In general the success rate of the gammaglobulin treatment seemed to correlate with the ADV-infection level in the mink sheds. The highest success rate was found in the sheds with the highest ADV-infection level (the standard and wildtype mink), while no effect whatsoever was found for the pearl mink, which were placed in a shed with a low ADV-infection level.     

水貂阿留申病毒VP2蛋白主要抗原表位基因原核表达及其检测应用

将构建好的重组质粒pMD-VP2a、pMD-VP2b分别经双酶切获得基因片段VP2a、VP2b,分别将其定向插入到原核表达载体pMAL-c2中,构建原核表达载体pMAL-VPa和pMAL-VPb。阳性重组质粒转化宿主菌TB1,经IPTG诱导,两段基因均获得表达;Westernblot分析表明表达蛋白具有良好的抗原性。用KCI染色后,切胶回收的方法对表达蛋白进行纯化,以纯化的目的蛋白作为诊断抗原初步建立了水貂阿留申的VP2-CIEP诊断方法。结果表明该方法具有较高的灵敏性和特异性,与传统的CIEP方法的检出符合率达94.3%。     

Molecular cloning of the Aleutian disease virus genome: expression of Aleutian disease virus antigens by a recombinant plasmid

Three nonoverlapping segments representing approximately 80% of the 4.8-kilobase pair Aleutian disease virus (ADV-G) duplex genome were molecularly cloned into either bacteriophage M13mp9 (M13bm2 = 0.07 to 0.15 map unit; M13bm1 = 0.15 to 0.54 map unit) or plasmid pUC8 (pBM1 = 0.54 to 0.88 map units). In addition the 0.54- to 0.88-map unit segment of a Danish isolate of ADV (DK ADV) was also cloned into pUC8 (pBM2). The recombinant plasmids pBM1 and pBM2 induced expression of several polypeptides in Escherichia coli JM103 that were specifically recognized by sera from mink infected with ADV. The same three proteins with approximate molecular weights of 55,000, 34,000, and 27,000 were detected both by immune blotting and by immunoprecipitation of [35S]methionine-labeled JM103 (pBM1). None of these proteins were recognized in JM103 or JM103 (pUC8), nor were they detected by sera from normal mink. Purified pBM1 and pBM2 DNA appeared identical in size by gel analysis and contour length measurement, and electron microscopic heteroduplex mapping revealed no visible areas of heterology. However, restriction endonuclease mapping showed that pBM2 was different from pBM1, indicating that this segment of the ADV genome was similar but not identical for two strains of ADV (ADV-G and DK ADV). Furthermore, when cloned DNA from ADV-G was labeled with [32P]dCTP by nick translation, DNA relatedness to several field strains of ADV (Utah I, Pullman, and DK), but not to mink enteritis virus or cellular DNA, was shown by Southern blot hybridization.     

Homogenate factor of minks with Aleutian disease]

Antibodies to the factor present in the homogenate of the organs of minks experimentally infected with aleutian disease (AD) were revealed in the sera of minks with AD and also in the sera of humans suffering from systemic lupus erythematosus, hepatitis and cirrhosis, scleroderma and dermatomyositis. Sera of sick minks and humans failed to interact with control homogenates of the organs of healthy minks. In turn, sera of practically healthy minks and humans did not react with the AD-homogenate.     

DNA and antibodies to DNA in Aleutian mink disease]

Free DNA and various types of antibodies to DNA were determined in the blood serum and plasma of minks which contracted Aleutian disease (AD) spontaneously and of minks experimentally infected with this disease. Healthy minks and other animals served as controls. It was found that a higher incidence of antibodies to DNA of the 2nd and 3rd types in high titres (1: 80-1: 2560) was characteristic of the experimental group of animals. Besides, a free polymeric DNA was more frequently revealed in the experimental group of animals.     

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.