NORKI.DOC

NOTICE: United Vaccine to end ADV testing

It has come to our attention that, for reasons that are not quite clear, as of January 25, 2006, United Vaccines will cease accepting blood samples for their CEP test for the Aleutian disease virus.

Introduction

Formally considered a disease of mink, the parvovirus that causes Aleutian disease is becoming increasingly prevalent in ferrets. A fulminant lethal infection in mink, the Aleutian disease virus appears to be a chronic latent infection in ferrets (often referred to as "hypergammaglobulinemia" of ferrets), which causes clinical disease over a period of one to two years. While the parvovirus itself causes little or no harm to the ferret host, the marked inflammatory response generated by the ferret may cause life-threatening hematopoietic and urinary derangement. The large number of antigen-antibody complexes associated with the humoral response to the presence of the virus results in a systemic vasculitis (with the most prominent lesions being in the glomerular capillaries, resulting in eventual renal failure and death). The marked lymphoplasmacytic response interferes with bone marrow hematopoiesis, and local accumulations of plasma cells may result in organ derangement.

In 1997, a rescue facility in San Antonio, Texas experienced an epizootic of AD which was unlike any previously documented. In contrast to the 40% or less morbidity seen in other facilities, 61/65 animals tested positive on CIEP testing for AD virus antibodies. Early signs of infection noted in a number of animals were an ascending paralysis and respiratory signs such as sneezing or coughing. Post-mortem on one of these animals showed characteristic plasmacytic infiltrates in the spinal cord and other organs. Similarly affected animals who received supporting treatment recovered from the paralysis, but have since gone on to succumb two years later of more characteristic disease (glomerulonephritis, hypergammaglobulinemia, etc.) Similar facility outbreaks have been reported in Wisconsin, Alabama, Maryland, and Virginia.

Clinical Signs

Most infected ferrets remain asymptomatic until shortly before death. Non-specific signs include lethargy, anorexia. Ataxia and paraparesis may be seen classically in chronic cases (but occasionally in very acute cases as a premonitory sign) due to accumulations of inflammatory cells within the spinal cord.. Anemia, thrombocytopenia, and/or leukopenia, cutaneous hemorrhages, and secondary infections may be seen in various combinations in end stage disease.

Diagnosis

An elevated globulin (defined as >20% of the total protein) is strongly indicative of this disease. Ferrets with total proteins over 7.5, especially those with mildly decreased albumins should be immediately suspect. Additionally, a CIEP test is commercially available from United Vaccines, and may be run on a hematocrit tube of blood. Biopsy or necropsy specimens from infected ferrets (particularly kidney, spleen, and liver) often yield a presumptive diagnosis.

Gross Lesions

Gross lesions are seen only late in the course of disease. Splenomegaly and lymphadenopathy are the most common gross lesions with this disease; splenic infarction as a result of marked splenomegaly may complicate the clinical and pathologic picture.. Enlarged, brown-tan kidneys may be present. In terminal cases, clotting abnormalities resulting from vasculitis and the marked hypergammaglobulinemia may result in petechial hemorrhage and hematuria.

Microscopic Lesions

Several characteristic microscopic findings are seen in ferret AD as well as in the mink disease. Prominent plasmacytic infiltrates are seen in numerous organs, most prominently in the renal interstitium, hepatic portal areas, and in the splenic red pulp, where an almost pure population of plasma cells expands the red pulp. Additionally, there may be marked plasmacytosis of numerous lymph nodes and the bone marrow. In most cases, there will be marked membranous glomerulonephritis and numerous ectatic protein-filled tubules as a result. (Note: Glomerulosclerosis is commonly seen in chronic interstitial nephritis in this species - but there is little evidence of tubular protein casts or plasmacytic infiltrate in uncomplicated CIN). Vasculitis may be seen in almost any organ.

Treatment

There is no current treatment for Aleutian disease in the ferret, nor is there a vaccine for this disease. Supportive therapy may prolong life; however most cases are not diagnosed until late in the course of disease, and infected animals may serve as a source of infection for other ferrets.

References

Alexandersen S et al. Acute interstitial pneumonia in mink kits inoculated with defined isolates of Aleutian mink disease parvovirus. Vet Pathol 31:216-228, 1994.

Daoust PY, Hunter DB. Spontaneous Aleutian disease in ferrets. Can Vet J 19:133-135, 1978.

Ohshima K et al. Comparison of the lesions of Aleutian disease in mink and hypergammaglobulinemia in ferrets. Am J Vet Res 39:653-657, 1978.

Oxenham M. Aleutian disease in the ferret. Vet Rec 126:585, 1990.

Palley LS et al. Parvovirus-associated syndrome (Aleutian disease) in two ferrets. JAVMA 201:100-106, 1992.

Porter HG et al. Aleutian disease in ferrets. Infect Immun 36:379-386, 1982.

Welchman E, et al. Aleutian disease in domestic ferrets: diagnostic findings and survey results. Vet Rec 132:479-484, 1993.

Une Y et al. Spontaneous Aleutian diseases in a ferret. J Vet Med Sci 62(5): 553-555, 2000.

ALEUTIAN MINK DISEASE

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]

'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 retu...