An Overview of Aleutian Disease in Ferrets

Jennifer Schuler, DVM; Kate E. Pennick, BS, MS; Kenneth S. Latimer, DVM, PhD

Class of 2007 (Schuler) and Department of Pathology (Latimer), College of Veterinary Medicine, The University of Georgia Athens, GA 30602-7388 and Class of 2010, College of Veterinary Medicine, Michigan State University, East Lansing, MI 44824 (Pennick).

Introduction

Aleutian Disease Virus (ADV) is a parvovirus that was first discovered in farm raised mink in the 1950’s. It was so named because mink that were homozygous for the Aleutian (blue) gene were most severely affected by the illness.¹ In addition to mink, ADV has been found to infect other mustelids such as skunks and ferrets. Aleutian disease (AD) has been a problem in farm raised mink populations for many years; however, it has been observed in pet ferret populations more recently. The increasing popularity of ferrets as companion animals makes AD an important emerging disease in this species. This article summarizes the most recent information available about AD in ferrets including pathophysiology, viral transmission, clinical signs of disease, pathologic findings, diagnostic testing, treatment, and disease prognosis.

Pathophysiology of Disease

In adult mink, AD has been classified into three categories:  1) progressive infection, 2) persistent nonprogressive infection, and 3) nonpersistent nonprogressive infection with clearance of the virus. Thus far ADV infection in ferrets has been similar to that in mink.⁵ In adults, the virus targets macrophages and dendritic cells for replication and sequestration. The greatest amount of viral antigen is usually found in phagocytic cells of the liver and lymphoid organs.⁶ Once the ferret becomes infected with ADV, a strong antibody response and associated hypergammaglobulinemia result from the antiviral immune response. Antibodies are directed against both structural and nonstructural proteins, but are unable to clear the virus from the body. Long term infection with ADV subsequently causes immune complex disease.^7,8  The deposition of the immune complexes in various organs including the kidneys, liver, arteries, and nervous tissue accounts for the clinical signs and pathologic abnormalities observed. It is possible for ferrets to be infected for years with ADV before any clinical signs are observed.

In newborn mink kits, ADV replicates rapidly in type II alveolar cells of the lung causing an interstitial pneumonitis. This disease presentation is more typical of other parvoviral infections including feline panleukopenia, canine parvoviral enteritis, and mink enteritis. It is unknown why these cells are targeted by ADV in young ferrets and not in adults, but viral infection appears to be more severe in young ferrets than in adults.⁹ Little investigation has been done into the disease process in mink kits, but some studies have shown that natural or passive immunity to the virus in kits can prevent fulminate infection. It is interesting that the same immune response in adults is the major contributor to progressive disease.⁷

Currently, there is little information concerning shedding of the virus. It is suspected that the virus is shed only during times of replication. One case report from The University of Georgia detected viral DNA in the blood, urine, and feces of one asymptomatic ferret that exhibited persistently high antibody titers.¹⁰ Since asymptomatic carriers of the disease have been identified, control and management of ADV infection through routine testing has become even more important.

Clinical Signs of Disease

The clinical signs of ADV infection in most ferrets are compatible with chronic wasting and include progressive weight loss, weakness, ataxia, and posterior paresis.¹ Patients also may present with fecal and urinary incontinence as well as severe coughing and dyspnea. Clinical signs seen in AD are not specific for the disease but usually are related to the organ system affected by the immune complex deposition. Clinical signs related to renal failure, liver failure, or intestinal involvement (melena) may be observed. Abnormalities that may be noted on physical examination include hepatomegaly and splenomegaly. Central nervous system signs with ADV infection can include tremors, convulsions, and paralysis. There also are instances where ferrets die suddenly without apparent clinical signs. The immune response to ADV varies from animal to animal. This may be related to the variable pathogenicity of different ADV strains coupled with the specific immune response by the host against the virus.¹¹

Clinicopathologic Findings

ADV infection is characterized by a hypergammaglobulinemia. In most ferrets diagnosed with AD, globulins comprised greater than 20% of the over all protein concentration  Thus, hyperglobulinemia in any ferret should warrant further laboratory investigation with the aid of serum protein electrophoresis. Anemia also can be seen and is usually related to chronic inflammation. Clinical pathologic findings suggestive of specific organ damage can include azotemia and increased activity of liver enzymes. Hypoalbuminemia may be present secondary to decreased albumin production by the liver or increased renal loss in the urine.

Post Mortem Gross Lesions

Nonspecific gross lesions can be seen in many organ systems on post mortem examination. The kidneys can appear pale and enlarged or small and shrunken, depending upon the chronicity of the disease. The liver is usually enlarged and pale with scattered white nodules. Lesions in the small intestine include scattered mucosal ecchymoses with intraluminal melena. Mild to moderate enlargement of the spleen and lymph nodes may be observed. Lesions involving the respiratory system may include diffuse pulmonary congestion, serosanguineous pleural effusion, pulmonary edema, pulmonary ecchymoses, and lung lobe consolidation and collapse. Gross lesions usually are not seen in subclinical infections with ADV.

Histologic Lesions

The most common histologic finding in ferrets with ADV infection include lymphoplasmacytic infiltration of numerous organ systems including the brain, spinal cord, heart, lungs, digestive tract, liver, pancreas, and kidneys (Fig. 1). More specific lesions in the liver included periportal cholangiohepatitis with infiltrates of lymphocytes, plasma cells, and neutrophils; bile ductular hyperplasia; and periportal fibrosis. Renal lesions may include multifocal lymphoplasmacytic interstitial infiltrates and mild membranous glomerulopathy. Lymphoplasmacytic perivascular cuffing in the brain and spinal cord may be observed along with a lymphoplasmacytic meningitis.¹

Fig. 1. Lymphoplasmacytic infiltrates in the renal interstitium. Ferret, ADV infection, kidney, hematoxylin & eosin stain.

Disease Diagnosis

Presumptive diagnosis of ADV infection and AD is based on medical history, clinical signs, and the presence of hypergammaglobulinemia. Other evidence suggestive of ADV infection includes histologic observation of lymphoplasmacytic infiltrates in various tissues and organs. However, none of these findings are definitive for ADV infection or the presence of AD. Definitive disease diagnosis is made through tests such as counterimmunoelectrophoresis (CIEP), ELISA, PCR, and DNA in situ hybridization.

Counter Immunoelectrophoresis (CIEP)

Counter Immunoelectrophoresis (CIEP) used to be the primary diagnostic test for AD and was performed by United Vaccines, Inc.; however, this test is no longer available. This technique was originally developed for test and removal of ADV infected individuals in mink colonies, but is now being used to evaluate ferrets suspected of having AD. This test has proven to be very specific for detection of ADV antibodies, but its sensitivity is uncertain in ADV infections with very low antibody titers. Another potential deficiency of this test is that it detects the presence of ADV antibody but does not generally provide end point titers which are important for tracking disease progression.² The results of this test should be considered in light of clinical signs as some ferrets can be inapparent carriers of ADV. In one serologic survey 8.5% to 10% of pet ferrets had positive antibody titers for the ADV.¹ Finally, the presence of an ADV antibody titer indicates exposure to the virus but may not be able to determine whether the virus was eliminated from the body, is persisting in the body, or is causing progressive disease within the body.

ELISA Testing

The first commercial ELISA is still in use and detects antibodies to the ADV NS1 protein. This is a nonstructural viral protein that is only detected during viral replication. Thus, there is the potential for false negative results with this test because the ELISA test result will be negative if ADV is not actively replicating. A newer, second generation ELISA recently has been developed at The University of Georgia, but is not yet commercially available. This ELISA detects the major capsid proteins (VP1 & VP2) of the ADV. This assay will detect ADV even in times of latency when the virus is not actively replicating. This new ELISA test is much more sensitive than the CIEP test, and may one day replace it as the primary screening test for ADV. The turn around time on this new ELISA is approximately 4 hours while previous methods of testing took > 24 hours for test results.² The specificity of this new ELISA test is very high as well; however, there is a possibility of a false negative result if the host only produces antibodies to the nonstructural portion of the ADV. For this reason it may become standard practice in the future to run both ELISA tests concurrently.

DNA in situ Hybridization

ADV also can be detected in formalin fixed, paraffin embedded postmortem or biopsy tissue samples by DNA in situ hybridization (Fig. 2). This technique uses a PCR-generated, viral specific probe to localize ADV DNA in tissue sections. This technique has the ability to confirm viral infection and identify infected cells, tissues, and organs.² Used on biopsy specimens of liver, spleen, lymph node, or kidney, DNA in situ hybridization, in combination with serologic testing, can further aid in the diagnosis and prognosis of ADV.

Fig. 2. ADV virus is present at sites of formazan deposition (blue-black pigment) in splenic lymphoid follicle (left) and nuclei of renal tubular epithelial cells (arrows, right). Ferret, ADV infection, spleen and kidney, DNA in situ hybridization with fast green fcf counterstain and nitroblue tetrazolium chromagen solution.

Polymerase Chain Reaction (PCR) Testing

Polymerase Chain Reaction (PCR) testing is not currently a mainstay of ADV diagnostic testing, but it can be a useful technique to identify viremia in active ADV infection (Fig. 3). PCR may prove especially useful in detecting asymptomatic carriers, assuming that the virus is replicating. If the virus is in a latent state and sequestered from the circulation, the PCR may produce a negative test result. False positive PCR test results potentially can occur with laboratory error due to improper technique and cross-contamination of laboratory specimens. Conversely, a negative PCR test result does not indicate that ADV infection is absent. Therefore PCR should not be used alone to diagnose ADV infections. PCR testing coupled with serology and judicious use of DNA in situ hybridization can prove helpful in characterizing the disease process in individuals and offering a more precise prognosis.

Fig. 3. PCR gel demonstrating a band containing an amplicon of Aleutian Disease Virus (arrow). The molecular ladder marker is at left.

Summary of Diagnostic Testing

All diagnostic test results should be considered in light of the individual patient history and clinical signs.² The current gold standard of testing and screening used to be the CIEP; however, this test is no longer commercially available. Although this test was good for detection of ADV antibodies, it did not give end point antibody titers and its sensitivity in infections with low antibodies counts was questionable. The NP1 ELISA test will only detect viral infections when viral replication is taking place and is not sensitive or specific enough to stand alone. The new VP1/VP2 ELISA test developed at The University of Georgia is more sensitive in that these viral structural proteins are present in active and latent ADV infections. This test is more specific than the NP1 ELISA; however, false negative test results can occur if the host only produces antibodies to the non-structural ADV proteins. For this reason, it is recommended that both ELISAs be used concurrently to detect structural and nonstructural ADV proteins (antigens). DNA in situ hybridization can be used to detect the presence of the ADV in formalin fixed, paraffin embedded biopsy or necropsy tissues. PCR, along with the results of the ELISA tests and DNA in situ hybridization, can help characterize patients as to whether ADV infection is recent, persistent and progressive, persistent and nonprogressive, or nonpersistent with impending recovery.

Treatment and Prognosis

Definitive treatment for ADV currently does not exist. Supportive therapy may include administration of fluids, electrolyte supplementation, anti-inflammatory drugs, and antibiotics to prevent secondary infections. Treatment with immunosuppressive drugs has been tried to help decrease the deleterious effects of the immune response. These treatments have shown short-term improvements in clinical signs; however, mortality rates remain high in ferrets with clinical signs of AD.

Levamisole, cyclophosphamide, and interferon inducer have been tested in mink to decrease the hypergammaglobulinemia and immune complex deposits. These therapeutic agents have shown variable response in mink and are currently of limited benefit in ferrets due to their unknown effectiveness. Treatment with gamma globulin-containing anti-ADV antibody has been shown to decrease mortality rates in mink kits, but has not shown any beneficial effects in adult animals.

Currently, a commercial vaccine does not exist to protect ferrets against the ADV infection.¹ Some experimental vaccines have been shown to decrease disease severity upon challenge with virulent ADV; however, much more work is needed to document the actual efficacy of these vaccines and reveal any potential side effects.

Since little information on ADV shedding and transmission is currently available regarding ferrets, the disease is best control through prevention. Mink farms currently use the CIEP test for screening and removal of individuals suspected of having ADV infection. This practice has helped to manage and control of outbreaks of ADV in mink colonies, but has not eradicated the disease. Although a test and removal program is helpful in commercial mink colonies, it is not an ideal solution for pet ferret owners. Until effective vaccines and anti-viral treatments are developed, management of ADV will need to be carried out through proper hygiene and quarantine protocols.

References

1. Quesenberry KE, Carpenter JW. Ferrets, Rabbits and Rodents Clinical Medicine and Surgery, 2nd ed. St. Louis: W B Saunders Co., 2004.

2. Pennick KE:  Development of an ELISA to Determine Aleutian MinkDisease Prvovirus (ADV) Capsid Protein Antibody Titers. Master’s of Science thesis, The University of Georgia, Athens, GA, 2005.

3. Alexandersen S. Pathogenesis of disease caused by Aleutian mink disease parvovirus. Acta Pathol Microbiol Immunol Scand1990;98 (Suppl 14):1-32.

4. Hadlow WJ, Race RE, Kennedy RC. Comparative pathogenicity of four strains of Aleutian disease virus for pastel and sapphire mink. Infect Immun 1983;41:1016-1023.

5. Bloom ME, Kanno H, Mori S, Wolfinbarger JL. Aleutian mink disease: Puzzles and paradigms. Infect Agents Dis1994;3:279-301

6. Porter DD, Larsen AE, Porter HG. The pathogenesis of Aleutian disease of mink: in vivo viral replication and the host antibody response to viral antigen. J Exp Med 1969;130:575-589.

7. Aasted B, Alexandersen S, Christensen J. Vaccination with Aleutian mink disease parvovirus (AMDV) capsid proteins enhances disease, while vaccination with the major non-structural AMDV protein causes partial protection from disease. Vaccine1998;16:1158-1165.

8. Bloom ME, Race RE, Wolfinbarger JB. Identification of a non-virion protein of Aleutian disease virus: mink with Aleutian disease have antibody to both virion and nonvirion proteins. J Virology1982;43:608-616.

9. Stevenson MA, Gates III L, Murray J, Bloom M. Aleutian mink disease parvovirus: implications for companion ferrets. Compend Cont Edu Pract Vet2001;23:178-185.

10. Pennick KE, McCrackin Stevenson MA, Latimer KS, Ritchie BW, Gregory CR. Persistent viral shedding during asymptomatic Aleutian mink disease parvoviral infection in a ferret. J Vet Diagn Invest2005;17:594-597.

11. Porter DD. Aleutian disease:  A persistent parvovirus infection of mink with a maximal but ineffective host humoral immune response. Prog Med Virol1986;33:42-60.

Acknowledgement

The image detail from "Country Kitchen Capers" The Ferrets © 2003 by Lora S. Irish is from the Fine Art Dog Prints website and is used with permission.

^  Top of page