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      [Used with written permission
      "Compendium on Continuing Education for the Practicing Veterinarian 2001;23(2):178-185"]

      
                                 		Universal City Animal Hospital
      University of Georgia			Universal City, Texas
      M. A. McCrackin Stevenson DVM, PhD	Leo Gates III, DVM

			                        Rocky Mountain Laboratories
      Animal Clinic of Farmers Branch	        National Institutes of Health
      Dallas, TEXAS			        Hamilton, Montana
      Jerry Murray, DVM			        Marshall E. Bloom, MD


      

        
ABSTRACT:  Aleutian mink disease parvovirus (ADV) causes disease in mink and ferrets and can infect such related animals as raccoons, weasels, fishers, martens, and striped skunks. Severe Aleutian disease (AD) in adult mink is characterized by viral persistence, high levels of antiviral antibody (which is ineffective at eliminating the virus), and immune-complex disease. Death results when virus-antibody immune complexes deposit in the kidneys, producing im-mune-mediated glomerulonephritis. Ferrets infected with ADV are usually asymptomatic and maintain a low antibody titer, but severe disease can occur. Ferrets with clinical signs of AD may have chronic wasting disease (similar to that seen in mink) or neurologic disease (most often manifested as posterior paresis or paralysis).

        
Aleutian mink disease parvovirus (ADV) can infect ferrets and is capable of negatively impacting the companion ferret population. Several reports of Aleutian disease (AD) in companion [1] and research [2,3] ferrets have been published during the past decade. In recent years, outbreaks have occurred in a ferret shelter [4] and multiple-ferret homes.[5] Despite the perceived increase in disease incidence in ferrets, little is known about incubation, transmission, interpretation of diagnostic tests, pathogenesis, treatment, and prevention of AD in ferrets. This article summarizes information about AD in mink and ferrets and provides suggested management options for affected companion ferrets.
        
HISTORY OF ALEUTIAN DISEASE IN MINK
        
Aleutian disease has long been considered primarily a disease of ranch mink
(Mustela vison). Highly susceptible Aleutiain mink (aa) carry two autosomal recessive genes for dilute coat color (producing a gun-metal gray pelt) that is associated with Chediak-Higashi syndrome, [6] an inherited disorder of the immune sysrem. ADV was named after Aleutian mink because of their unique susceptibility to AD. [8]  However, dark-coated mink that are hererozygous (Aa) or homozvgous dominant (AA) are also susceptible to ADV infection but tend to have lower morbidity and mortality compared with Aleutian mink. [9,10] AD was recognized as a disease syndrome in 1946 when mink ranchers realized the economic value of the gray pelts and began actively breeding Aleutian mink for their desirable coat color. The first published description of AD in mink appeared in 1956.[11]
        
Before the identification of ADV, distemper and botulism were the primary disease concerns of mink ranchers. Ranchers commonly made their own autogenous distemper vaccines by homogenizing spleen from distemper-infected mink, making suspensions, and injecting all the mink on their ranch. This practice led to a severe outbreak of AD on a Connecticut ranch, with a mortality rate of almost 100% within 6 months.[12] Over the ensuing decade, suspicions rose that AD was caused by a "filterable agent" (i.e., a virus). ADV was isolated and studied during the early I 970s [13,14] but was not correctly characterized as a parvovirus until l98O.[15] Extensive molecular characterizations of ADV and studies of its pathogenesis in mink have been published over the past 20 years. [16,17] Although very distantly related to parvoviruses that cause acute gastrointestinal disease (e.g.. canine parvovirus, feline parvovirus, mink enreritis virus), ADV is antigenically distinct from these members of the feline subgroup of parvoviruses.[18]
        
ALEUTIAN DISEASE IN FERRETS
        
Many ferrets (Mustela putorius furo) were probably naturally exposed to ADV on mink ranches because some farmers raised mink and ferrets on the same property.
Ferrets were also experimentally infected with ADV during the 1960s.[19,20] Researchers believed that AD could be developed as an animal model for human immune-mediated diseases but sought an animal that was easier to handle than the sometimes ferocious mink,[21] Ferrets were infected with tissue homogenates from infected mink[20] or were closely housed with infected mink and ferrets)[19] Although the ferrets did not develop clinical illness, they did acquire periportal lymphocyric cellular infiltrates in their livers,[19,20] thymic hypertrophy,[19,22] hypergammaglobulinemia, splenomegaly, and mesenteric lymphadenopathy.[19] Glomerulonephritis and arteritis, hallmarks of AD in highly susceptible Aleutian mink, occurred in naturally infected ferrets[19] but not in ferrets experimentally infected with material from mink.[20-22] Mink ADV appeared to persist in the ferrets for 136[20] to 180 days.[21]
        
Limitations of these early studies included the inability to detect and differentiate viral strains[19-22] and easily test for preexisting antibody in the acquired research animals.[19,20] The overall conclusions of studies conducted before 1985 were that there were distinct mink and ferret strains of ADV,[21,22] the disease progressed more slowly in ferrets than in mink)[21,22] and the disease and microscopic tissue changes were less severe in ferrets than in mink.[20-22]
        
CLINICAL DISEASE
        
Manifestations of clinical disease are likely determined by virus strain and host genotype and immune status. Thus a broad array of clinical signs--ranging from clinical normalcy to nonspecific signs (e.g., lethargy, anorexia) to specific problems (e.g., uremia; neurologic dysfunction; frank hemorrhage of the digestive tract, including the mucosa of the oral cavity and intestines)--can be seen.
        
Mink
        
Aleutian disease was first manifested as a chronic wasting disease of adult Aleutian mink.[11] Weight loss, poor pelts, lethargy, anorexia, polydipsia, anemia, and melena were common clinical signs in affected mink.[6] Infertility, small litters, and high stillborn rates were also noted.[23] Necropsy examinations of end-stage infected animals classically showed small, shriveled kidneys; splenomegaly; mesenteric lymphadenopathy: hepatomegaly; and blood in the intestinal tract.[6] Aleutian mink experimentally infected with virulent strains of ADV tested positive for anti-ADV antibodies (end-point titers were 1024 or greater using counterimmu-noelectrophoresis [CIEP]), tested persistently positive when polymerase chain reaction (PCR) was used to detect nucleic acid in the serum, were hypergammaglobulinemic (i.e., more than 20% of total serum proteims were y-globulins), and were azotemic in end-stage disease.[24,25] Virus was found in the cytoplasm of such phagocytic cells as macrophages and dendritic cells[26,27] and, in renal tubular epithelial cells.[28]
        
Adult non-Aleutian mink may develop one of three general types of ADV infection: progressive AD as described for Aleutian mink [9]; persistent nonprogresssive infection; or nonpersistent, nonprogressive infection with eventual clearance of the virus.[10] Whether these three categories apply to ferrets is unknown.
        
The target cells for viral replication are different in newborn kits compared with adult mink.[17] In contrast to the protracted infection of macrophages and dendritic cells in adult mink, kits infected within the first 2 weeks of life developed rapid viral replication in the alveolar type II epithelial cells of the lungs.[17,29,30] Direct viral damage rather than immune-mediated disease caused severe, fulminant, and often fatal pneumonitis. [17,29,30] The behavior of this acute infection of neonatal mink is more reminiscent of feline subgroup parvovirus infections (e.g., canine parvovirus, feline parvovirus, mink enteritis virus) than the persistent infection of adult mink with ADV.
        
Ferrets
        
Aleutian disease in ferrets was originally considered primarily a subclinical problem. One 1978 report and all studies of AD in ferrets published since 1990, however, have described overt clinical disease.[1-3,31] Clinical syndromes included chronic wasting disease[3,31] and neurologic disease consisting of posterior paresis or paralysis.[1-3] Virus isolated from the spleen of an infected ferret (ADV-F') was amplified by PCR for DNA sequencing.[?] The DNA sequence of a small segment of the capsid protein that makes up the virus "shell" showed that ADV-F was 88% to 89% identical to some previously sequenced pathogenic strains of mink ADV.[32] DNA sequence differences confirmed that ADV-F was dissimilar to isolates identified in mink.[32]
        
Clinical syndromes seen during a 1998 outbreak of AD in a ferret shelter in San Antonio, Texas, included generalized wasting and respiratory, neurologic, and cardiac forms of disease in ADV-positive ferrets [4] Ferrets with chronic wasting disease had small kidneys on necropsy and glomerulonephritis on microscopic examination. Respiratory disease in affected ferrets included severe coughing, right middle lung lobe consolidation and collapse, and serosanguineous pleural effusion. Microscopic examination of necropsy tissue samples revealed hemorrhagic interstitial pneumonia. Neurologic signs usually followed respiratory signs by several weeks but occurred alone in some ferrets. Neurologic dysfunction started as posterior paresis and either remained stable or progressed to ascending paralysis accompanied by urinary and fecal incontinence. A few ferrets developed heart disease reminiscent of ferret cardiomyopathy, but necropsy samples showed arteritis in the cardiac muscle (suspected to have resulted from immune-complex deposition) and lymphoplasmacytic infiltrates. Severe anterior uvettis also occurred in some ferrets. Uveitis has also been described in AD-affected mink.[33]
        
All of these sick ferrets tested positive for anti-ADV antibody by CIEP, and viral DNA was amplified by PCR using tissue from some of these ferrets.[ ] DNA sequence analysis of these PCR products was identical to that previously reported for ADV-F.[32,34] To date. ADV-F is the only isolate of ADV in ferrets to be documented with published DNA sequence [32,34]
        
A multiple-ferret home in Dallas, Texas, experienced the loss of 2 of 11 ferrets in the spring of 2OOO.[ ] Both ferrets tested positive for antibody using CIEP and had microscopic tissue changes consistent with AD. One of these ferrets (a 5-year-old male) had an endpoint anti-body titer of 256 by CIEP [35] hypergammaglobulinemia (32%), muscle twitches, and seizures; the liver and kidney showed extensive lymphoplasmacytic cellular infiltrates typical of AD. Glomerulonephritis was also identified in the kidney. Two of the remaining nine ferrets in this home also tested positive using CIEP; the other seven ferrets had not been tested when this article was written.
        
DISEASE TRANSMISSION
        
Natural horizontal transmission of ADV among mink is likely to occur by either the oral or aerosol route.[36-38] AD has been experimentally transmitted between mink by inoculation with whole blood, serum, urine,[25,37,39] feces, saliva, and bone marrow from infected mink.[37]
        
Vertical transmission of ADV has also been shown to occur in mink.[36,40] Dams with either progressive or nonprogressive subclinical infections were shown to have high numbers of infected kits. [36] The risk for ADV infection in kits born to dams with nonprogressive subclinical infections was less than that for kits born to dams with progressive AD.[36] Dams infected with ADV before mating had a higher percentage of dead and resorbed fetuses compared with dams infected after ex-pected embryo implantation.[40]
        
The natural route of transmission of AD among ferrets is unknown. Horizontal transmission is suspected. but whether infectious ADV is present in urine, feces, or saliva of infected ferrets is unknown. This information is critical to help companion ferret owners prevent transmission of the virus from infected to noninfected ferrets in their homes. It is likewise important for ferret clubs in terms of establishing rules regarding the admission of infected animals in ferret shows. Vertical transmission in ferrets has been suspected but not studied. Knowledge of the mechanisms of horizontal and vertical transmission is crucial for ferret breeders to be able to make decisions about acquiring new ferrets for breeding programs, monitoring breeding ferrets, and placing young jills and hobs in companion homes.
        
Disease transmission is an area that needs to be researched. Detection of infectious virus in blood or cellfree body fluids may help identify ferrets that are currently shedding the virus. This information would also be valuable in learning more about ADV tranmission in ferrets.
        

      TABLE I
        
Submitting Samples for Counterimmunoelectrophoresis Testing for Anti-Aleutian Mink Disease Parvovirus Antibody [a]
        

        

        Company        
        		

        Cost           
        		

        Accepted Sample
        		

        Payment        
        		

        Results        
        		

        United Vaccines, Inc.
        ATTN:Customer Service
        2826 Latham Dr.
        Madison, WI 53713
        Phone: 800-283-6465,
        608-277-2030        		
$15 first sample;
        $10 each 
        additional
        sample         		
10 uL whole
        blood or serum,
        preferably in a
        capillary tube.        		
Prepayment is
        required;
        send check or
        credit card
        information
        with sample         		
48-hr turnaround;
        results are
        reported as
        positive, negative,
        or no sample
        (if tube breaks)
        		


        
'Express overnight shipping recommended.
        

        DIAGNOSTICS
        
Counterimmunoelectrophoresis is the standard for detecting anti-ADV antibodies in mink and ferrets.[41] This test is a precipitation reaction[42] between antibody in serum samples and a commercial viral antigen[43] (Table 1). It detects antibodies directed against the capsid proteins comprising the protective "shell" around the viral genomic DNA. A simple positive or negative result is given. CIEP has primarily been used for test-and-slaughter programs on mink ranches, in which more detail than a positive or negative result has been unnecessary.
        
The currently available CIEP assay is a reliable screening test, and false-positive results are not induced by vaccines against mink enteritis virus or other viruses. However, CIEP has a few limitations when used to diagnose AD in companion ferrets: It does not detect anti-bodies directed against the ADV nonstructural proteins,[41] provide endpoint antibody titers, or distinguish among immunoglobulin subclasses. Because the vast
majority of mink and ferrets infected with ADV produce antibodies against both capsid and nonstructural proteins,[41] the lack of nonstructural protein detection has not been a problem [b] An antibody titer based on the most dilute sample that continues to test positive (end-point titer) would be helpful to clinicians, particularly when retesring patients suspected of having AD. An increasing magnitude of antibody titer likely indicates active, ongoing infection. Testing additional dilutions using the CIEP assay is possible but would likely increase the expense of the test.
        
As mentioned, CIEP cannot distinguish among immunoglobulin subclasses. In ADV-infected mink, 1gM increases as soon as 6 days after inoculation and peaks at 15 to 18 days after inoculation.[8] IgG is not detected until at least 12 days after infection but is consistently increased by 30 days after infection.[8] lgG levels remain increased long after 1gM levels have returned to normal.[8] The appearance of antibodies of different sub-classes has not been studied in detail in ferrets. Needed improvements in antibody testing include reporting endpoint titers and distinguishing between 1gM and IgG subclasses. New diagnostic tests should be developed to complement the CIEP by providing these additional data, thereby helping clinicians determine the stage and severity of infection.
        
Hypergammaglobulinemia (excessive antibody production without neutralization of the virus) is a hallmark of AD in mink[6] and can develop in infected ferrets. [1-3,19,31] Serum protein electrophoresis is used to compare y-globulin levels with the rest of the serum proteins. Most commercial laboratories conduct this test and require serum. Care must be taken during sample collection because hemolysis can interfere with the test. The typical cost is $30 to $.35, and results are generally available in 2 to 5 days. A y-globulin level exceeding 20% of total serum protein is considered hypergammagloblinemia.[22] The presence of hypergammagloblinemia in a ferret that tested positive on CIEP and has clinical signs of wasting or neurologic disease strongly supports a presumptive diagnosis of AD.[ ]
        
Horizontal transmission of AD was documented in the 1960s by means of transmis-sion studies.[ ] The inoculation of uninfecied mink with whole blood, serum, saliva, Feces, and urine[ ] from infected mink was shown to cause AD.[ ] Advanced molecular biology techniques may allow the detection of viral nucleic acid by testing ferret body fluids. Serum from experimentally infected mink is routinely tested for viral DNA using PCR.[ ] PCR has also detected viral DNA in the urine of mink infected with a highly pathogenic strain of ADV as early as 14 days after inoculation. [25 ] When testing serum [ ] or urine,[ ] PCR assays may be positive weeks before mink show any clinical sign of disease. Nevertheless, mink with progressive disease can test negative when PCR is used to detect ADV DNA in serum.
        
Histopathology is perhaps the best method for confirming AD in a ferret identified as ADV-positive using CIEP. Microscopic changes in the organs of ADV-infected mink include lymphoplasmacyric cellular infiltration in the liver, kidneys, spleen, and lymph nodes.[ ] Evidence of glomerulonephritis and arteritis may be seen as well. In early reports of ferret AD,[ ] infected ferrets showed only massive periportal lymphocytic infiltrates. More recent microscopic examination of tissue from ferrets suspected of having AD, however, have included portal lymphoplasmacytic hepatitis, interstitial lymphoplasmacytic nephritis, membranoproliferative  glomerulonephritis, and lymphoplasmacytic gastritis.[ ] Ferrets with neurologic signs have been described as having nonsuppurative encephalomyelitis.[ ] and astrocytic hyperctrophy in the gray matter of the spinal cord.[ ] often without lesions consistent with glomerulonephritis.[ ] In situ hybridization has been used to identify viral DNA and replicating DNA in tissue from infected mink.[ ] This technology can be adapted to ferrets to verify the presence of ADV in necropsy specimens.
        
TREATMENT
        
Test-and-slaughter programs have been used to decrease the incidence of AD on mink ranches and are extremely effective when used conscientiously.[48] Treatment for individual mink infected with ADV has been limited to experimental reports of immunosuppressive therapy using levamisole,[49]  cyclophosphamide.[50] and an interferon inducer (polyinosinic-polycytidilic acid [poly lC][51][49] Body weight may have increased slightly in the treated group, and hypergammaglobulinemia was reduced in some treated animals.[49] No statistical analysis was done in this study, and the variation within the groups suggested that statistical significance was unlikely. However, no dernimental side effects associated with levamisole were mentioned.[49]
        
In contrast, cyclophosphamide (10 mg/kg intraperitoncally [IP], three times weekly for 13 weeks) was very effective in suppressing host antibody responses and deposition of immune complexes in the kidneys.[ ] Cyclophosphamide was also used at 10 mg/kg IP twice weekly for 8 weeks.[ ] This lower dosage was not as effective as was the higher dosage, but both were associated with significant negative side effects, including depression, anorexia, cyanosis, and leukopenia in treated mink.[50]  Levels of virus replication were not affected by cyclophosphomide treatment, indicating that direct viral damage to the host is not the cause of classic AD.
        
Poly IC (30 mg/kg IP twice weekly for 4 weeks and then once weekly for 3 weeks) was given to experimentally infected mink)[ ]  Y-Globulin levels were decreased in poly IC-treated mink compared with control animals 6 weeks after infection, but this beneficial effect was no longer seen at 12 weeks postinnoculation.[ ] Microscopic changes in target organs appeared to be blunted in poly IC-treated mink, but study results were not statistically significant. [ ]  Although the use of these drugs has not been reported in ADV-infected ferrets, it may be reasonable to consider using immunosuppressive oral prednisone therapy to decrease immune-mediated consequences of AD in carefully selected companion ferrets. A prospective clinical trial should be done to determine whether this treatment would be helpful for ferrets with AD.
        
Treatment of AD in ferrets will continue to be limited to supportive care until more definitive therapy is determined. Intravenous or subcutaneous fluid therapy may be necessary to maintain hydration. Maintaining a positive nutritional plane via temporary syringe or tube feeding may be needed in sick ferrets. Studies have shown that ADV-infected mink have a decreased ability to mount a humoral immune response.[50] Thus monitoring ADV-infected ferrets for opportunistic infections and treating with appropriate antibiotics are important.
        
DISEASE PREVENTION
        
No vaccine against AD in mink or ferrets is currently available. In fact, the presence of additional antibodies directed toward capsid proteins exacerbates the chronic immune-mediated form of AD in mink.[52,53] Despite this negative data, anticapsid antibodies have been shown to blunt the acute form of AD seen in mink kits,[54] and antibodies generated against one of the nonstructural proteins (NSl) partially protected mink from immune-mediated AD--but not infection--in a recent vaccine trial.[53] Therefore, there is still hope that a vaccine against ADV can be developed. Vaccine strategies must avoid generating anticapsid antibodies in adult animals and thus must focus on alternative targets for stimulating protective immunity. The expansion of the field of vaccinology in the past 10 years has brought promising new technology that adds practicality to the hope of developing an effective vaccine against ADV in ferrets.
        
Until an effective vaccine becomes available, the best recommendations for preventing the spread of AD among ferrets are isolation of uninfected ferrets from ADV-infected ferrets and environmental cleaning. We believe that the CIEP assay is currently the best method to detect ferrets that may be carrying ADV. Ideally, infected and noninfected ferrets should not be housed on the same property, but this is often impossible in multiple-ferret homes. Recent experiments using mink feces contaminated with mink enteritis parvovirus and kept under outdoor conditions showed that the virus survived for 5 to 10 months.[55] Complete drying was helpful in inactivating the virus, and thus mechanical cleaning was as strongly recommended as was disinfection for environmental control.[55]  In all likelihood, ADV can survive in the environment in a similar fashion. Therefore, food and water bowls, toys, cages, carriers, litter boxes, bedding, and carpeting contaminated with virus particles--particularly when accompanied by such organic material as feces--may serve as prolonged sources of virus for uninfected ferrets.
        
Contaminated environmental conditions would especially exist at ferret shows if ADV-shedding ferrets are present. With the current lack of knowledge regarding the pathogenesis of AD in ferrets and the limitations in identifying virus-shedding ferrets, there is no way to guarantee the safety of all ferrets at ferret shows. Restricting entries to ferrets with negative CIEP tests 3 to 4 weeks before the show would be prudent. The best protection for valuable AD-free breeding stock and companion ferrets is to eliminate their exposure to other ferrets of questionable ADV status.
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