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This page last updated 7-6-01






[Used with written permission
Please do not copy without written permission from Dr. Stephon]
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Testing for Aleutian Disease Virus in Ferrets

Introduction

1.What is Aleutian Disease Virus

ADV is a member of the class of viruses known as Parvoviruses.

Parvoviruses are among the smallest, simplest viruses in nature. Some other examples are Canine Parvovirus, Feline Parvovirus and Mink Enteritis Virus. Each of these, though genetically related, are distinct with regard to protein sequence changes on their outer protein shells.

Parvoviruses like ADV are composed mainly of proteins and DNA. DNA, as you probably know, is the sequence of biochemicals that is the “code” that actually tells your cells which proteins are to be synthesized. Ultimately, the sequence of DNA determines the physical and psychological characteristics of a living organism.

The proteins of ADV consist of the capsid proteins VP1 and VP2 which encapsulate and protect its DNA, and another group of “non-structural” proteins called NS1, NS2 and NS3 which are expressed during viral replication and play numerous roles in the assembly of new viruses within the host cell.

ADV infects and multiplies according to the following scheme:

A. The virus is recognized by receptors on the cellular surface and its DNA is internalized by receptor transport mechanisms. The viral DNA is then transported to the nucleus of the cell, where replication occurs with the use of the host cell DNA-replication machinery, or the enzymes and other biochemicals that the host cell uses to typically grow and replicate on its own.

B.It appears that the section of ADV DNA that codes for the non-structural proteins is replicated first. Then, the section of ADV DNA that codes for the VP proteins is replicated, and the non-structural proteins combine and interact with the VP proteins to form a new viral capsid.

The newly duplicated ADV DNA is inserted into the nascent virus, and the viral shell of the newly created ADV is closed.

C.The accumulation of new ADVs within the host cell causes the cell to burst, releasing the daughter viruses into the circulation where they can seek out other host cells to replicate further.

ADV is distinct from other parvoviruses in that it appears to lack the ability to stimulate the host animal’s immune system to produce Neutralizing antibodies – that is, as a foreign body, it does cause a host immune response, but the virus gathers in clumps with host antibodies known as “immune complexes” that physically deposit within tissue structures, rather than being “cleared” from the host by disposal through the reticuloendothelial system.

This is the crucial difference of ADV. Deposition of immune complexes within tissues such as the kidney, liver, arteries and lymph nodes leads to a state of chronic inflammation which, over time, can cause the discontinued ability of a specific organ to function, or compromise the general health of the host animal and lead to a general inability of the animal’s immune system to function due to constant overstimulation. This may allow other opportunistic infections to intercede in the animal’s life. The degree of severity of these effects is most likely determined by the genetic makeup of the host animal, its age and overall health, and the strain of ADV involved.




2.Different Strains of ADV

The phrase “different strains of ADV” refers mainly to the differences in the DNA sequence of a particular region of the DNA known as the hypervariable region. The hypervariable region is a short section of the ADV viral DNA that has a different sequence of the molecules (called bases in DNA) that make up the DNA, in each strain. The rest of the ADV viral DNA appears to be identical, or nearly identical between strains. Some research indicates that the strains are between 92% – 99% identical with each other. It is the differences in this region that gives rise to the ideas of different strains and, along with the other factors involved in ADV infection already mentioned, gives rise to the different physiological responses to ADV in mink, ferrets and other animals such as raccoons, skunks and otters. It is generally thought now that there is a distinct strain, or perhaps strains, of ADV in ferrets.

A “wasting” disease was first recognized in ranch mink almost 60 years ago, particularly those being breed for a distinct pelt color termed “Aleutian.” These mink appeared to be most susceptible to this syndrome, hence the name as its been historically called. Subsequently it was found that other types of mink were also susceptible to this disease to some degree, albeit with lower rates of dysfunction and mortality.

During the past 40 years or so, ferrets have been injected with tissue homogenates from infected mink and have also been injected with urine, saliva and blood from infected mink in order to determine if this “wasting” disease was transmissible to ferrets. Some ferrets were also apparently housed with infected mink or other infected ferrets on occasion, and may have developed natural infections from this exposure. Mutations, or slight changes in DNA sequence of the ADV viral DNA may have taken place in ferrets over time, perhaps due to the distinct cellular machinery of these mammals. These changes, while not disabling to the virus or its ability to cause infection, may have lead to an altered form of ADV as its manifested in ferrets.




3.Why is ADV a Danger to Ferrets?

There have been stories related to me personally of ADV-antibody positive ferrets living in close proximity with ADV-antibody negative ferrets for long periods of time without the negative ferrets becoming positive, or without the ADV-positive ferrets showing outward signs of ill health. There have also been journal reports of outbreaks of highly virulent ADV, and subsequent sickness and deaths attributed to ADV in ferrets over the past 25 years or so.

Some researchers who concentrate on the study of ferrets believe that an ADV outbreak in ferrets is likely over the next decade, similar to the escalation of HIV that has occurred in the human population over the last 20 years.

Perhaps there is a danger that an outbreak of highly virulent ADV in ferrets similar to the ones that have been documented in certain regions of the U.S. may repeat. And certainly there are those who shelter ADV-positive ferrets and know the debilitating effects of this disease on a day-to-day basis. Here’s the old tired phrase again, “Until more is known about ADV in ferrets,” perhaps its best to err on the side of caution. If you have an ADV-positive ferret that appears healthy, that is a good thing and hopefully they will stay healthy.




4.How does ADV Spread?

While not yet studied thoroughly in ferrets, in mink transmission of ADV can occur both “horizontally” and “vertically.”

Horizontal transmission can occur by the physical exchange of body fluids such as saliva, urine, feces and blood between animals in close proximity. In addition, it has been postulated that transmission can occur through the air at distances of up to 1 meter (via viral particles from expectoration, sneezing, etc.). As a group parvoviruses are very robust and may persist in the environment for extended periods of time on articles of clothing, furniture, fixtures, etc. The ubiquitous use of parvocides is highly recommended in ferret households.

Vertical transmission between infected dams and kits has been demonstrated in mink. In many of these cases the kits have been aborted or, if carried to term have shown signs of acute interstitial pneumonia and high mortality rates. Infected hobs may be a risk in breeding.




Diagnosing ADV in Ferrets

1.Physical Symptoms and Signs

Some overt symptoms observed in ferrets that are ADV-positive and suspected of having clinical ADV-related disease are general lethargy, chronic weight loss, kidney and liver dysfunction, respiratory symptoms and hind leg paralysis or other neurological signs.

Although these are generalized symptoms and may be considered hallmarks of many other disease states, it is perhaps best to consider ADV as a differential diagnosis. In other words, if ADV can be ruled out as a factor, efforts can be concentrated in other areas. If the ferret is ADV positive and has these symptoms as well, it is wise to consider ADV as a contributing, or major, factor.

2.General Serology – Total Protein and Hypergammaglobulinemia

Elevated Total Protein and concomitant hypergammaglobulinemia have been observed in ferrets with clinical ADV disease and also in those without overt symptoms. Total Protein greater than 7.5 g/dL and a gammaglobulin fraction in excess of 20% are certainly indicators of chronic ADV infection.

3.Gross Examination and Histopathology

Necropsy results on ferrets thought to have expired from ADV have shown enlarged spleens and livers, small kidneys and blood in the intestinal tract. Microscopic examination has indicated lesions associated with plasmacytosis, glomerulonephritis and arteritis. Examination of lung tissues has revealed hemorrhagic interstitial pneumonia.

4.Specialized Serological Testing – PCR, CIEP and ELISA/POCT (Point of Care Test)

The currently available diagnostic tests that can be useful in the determination of ADV in ferrets include Polymerase Chain Reaction (or PCR) for ADV viral DNA, Counterimmunoelectrophoresis (or CIEP) and the ELISA and Point of Care Test for ADV Antibodies.

I will now address the mechanisms of all, but first would like to address the use of the phrase “shedding the virus” since it is being used quite liberally by ferret owners interested in testing.

5.The Phrase “Shedding of the Virus”

ADV can be sequestered within immune complexes, which can cause some physiological ills as I have mentioned. The phrase “shedding of the virus” indicates that the ADV is in a replicating mode, that is to say, the virus is actively replicating, a condition that is not always in motion, but in this mode is most likely to cause transmission under the right circumstances. When the virus is shed, it can most likely be detected by PCR or some other DNA amplification technique.

Determining exactly when the virus is “shedding” is not always easy, especially when no overt clinical symptoms are observed.

The ADV viral genome (the DNA sequence) is about 5,000 bases in length. This means that there are 5,000 individual molecules that, when attached together, form the string of molecules, or sequence, that is particular to ADV, as opposed to a DNA sequence from another species. This is a rather small genome, which is good as far as ease of analysis is concerned. As I mentioned, there is also a hypervariable region that distinguishes each ADV strain, and the rest of the DNA in all the strains is mostly identical.

PCR , as its used in ADV viral DNA analysis, is a technique that can specifically identify this hypervariable region of ADV DNA, regardless of the strain, and amplifies it, or increases its concentration, so that it can be detectable. DNA, either in tissues or in circulation is typically present in very small quantities; to small to be detected by simpler chemical or biological means.

Because all strains of ADV DNA share a mostly common structural motif outside of the hypervariable region, chemicals known as primers are added to a sample containing suspected ADV DNA, along with enzymes and other promoters of DNA binding. The primers bind to the sequence regions of ADV DNA that border on the hypervariable region, and through successive rounds of heating and cooling in an instrument called a thermocycler, actually make multiple copies of the hypervariable region exclusively.

If ADV viral DNA is not present, there is nothing for the primers to bind to, no amplification of the hypervariable region can therefore occur, and the sample would be considered “negative.” If ADV viral DNA is present, the primers do bind, amplified hypervariable region DNA is subsequently detected, and the sample is said to be “positive” for ADV viral DNA.

What can be done with a positive result for ADV viral DNA? Two things: As I just mentioned, the specimen is positive so the ferret is shedding ADV and can be said to have actively replicating virus, and should be considered a danger to other ferrets. Secondly, this information can be used in research in the sense that the DNA sequence of the particular amplified hypervariable region can then be determined (by other means) and a comparison of the sequence of the hypervariable region of that particular strain can be compared to other strains. This is the usefulness of the PCR technique. Finding out the DNA sequence of ferret ADV hypervariable regions, and comparing them with the hypervariable regions of mink, or other mammalian ADV strains. This knowledge may potentially be useful in vaccine research.

To accomplish this important task is not easy: One must identify a specimen from which the ferret is currently shedding, and the concentration of ADV viral DNA must be in sufficient amounts to be detectable. This is often performed in tissue specimens obtained after the ferret has expired. It is relatively rare to find detectable amounts in blood from a live ferret, and one must draw the specimen when the ferret is shedding, usually a “hit or miss” proposition.

If you have a ferret you suspect of shedding the virus, I suggest you send a blood specimen to Dr. Stevenson at the University of Georgia. Likewise if you have a ferret that has expired due to suspected ADV, the spleen or kidney would be a good candidate for PCR analysis.

1.Historical

When ADV infects a ferret, mink or other susceptible species, the animal’s host immune response is to raise antibodies to the foreign invader, as I have mentioned. Unlike ADV viral DNA, the antibodies thus raised against the virus are present in massive quantities, making the detection of antibodies to ADV much easier than the detection of ADV viral DNA. In addition, the antibodies to ADV persist much longer and are present in various body fluids such as blood and saliva.

In the 1950’s a procedure known as counterimmunoelectrophoresis was applied to the detection of ADV antibodies in mink on ranches in Scandinavia and later, the U.S.

In this technique a gel is impregnated with a serum sample on one side and with an extract of ADV virus at the other side. When electricity is applied to the gel the two migrate toward each other and meet at a point midway in the gel. If there are any ADV antibodies in the serum, they bind to some components in the extract, and the binding pair tends to “precipitate,” or “fall out” of the gel. This causes a “line” to be formed within the gel that is discernable by observation of a trained laboratory technician. If such a line is observed, the specimen is said to be positive for ADV antibodies. If no such line is observed the specimen is considered to be negative.

As mentioned the CIEP procedure uses an extract of the ADV virus. According to mink ADV researchers abroad, in mink, false-positive results for ADV antibody may occur using CIEP due to vaccinations against Mink Enterovirus or Canine Distemper Virus, because these viruses are grown in cell lines and the vaccine contains cellular debris, which may induce an antibody response to that cellular debris. Since the ADV viral extract used in the CIEP test also contains cellular debris, there may be a cross-reaction between the cellular debris in the ADV viral extract and “anti-debris” antibodies in the specimen.

Nevertheless, CIEP has been traditionally used in determining the presence of ADV antibodies in mink and ferrets.

An immunoassay technique called ELISA has been used for the quantitative determination of both antigens and antibodies since the 1950’s. In this technique (as used for the detection of antibodies) an antigen specific for a particular antibody is physically bound to the plastic surface of a microtiter plate well. Specimen is added, that may or may not contain the antibody of interest. If any antibody of interest is present, like ADV antibody, it will bind to the coated material, or ADV antigen, and remain fixed in position. The reaction well is then washed with a buffer to remove any interfering substances that may have been present in the sample, leaving only the ADV antibody bound to the immobilized ADV antigen. Next, a second antibody that is specific for ferret antibody (of which ADV antibody is a subset), which has been chemically attached to an enzyme called horseradish peroxidase is added. This material is specific only for ferret antibodies and will thus bind to ferret ADV antibodies present that have previously been bound to the ADV antigen on the well. Following another wash step to further purify the reaction mixture, a colorless chemical called a substrate is added that will turn blue only in the presence of the enzyme horseradish peroxidase. If a blue color forms in the reaction well, it can only be due to the presence of ADV antibodies that are binding the ferret antibody-horseradish peroxidase material. The greater the blue color, the greater the concentration of ADV antibody in the original sample.

Hence, the degree of color formation is directly proportional to the concentration of ADV antibody in the original sample. The blue color is measured by a sensitive instrument called a spectrophotometer, and the degree of color formation, as measured in spectrophotometric units called absorbance, can be reviewed, verified and downloaded to a computer for a permanent record.

In the ELISA technique, the ADV antigen used to coat the microtiter plate wells is a highly purified form of the NS1 protein mentioned in the introduction portion of my talk. We chose to use this particular protein because published reports indicated that definitive diagnosis of ADV infection cannot be made without some proof of the existence of the NS1 protein, which in turn indicates that viral replication has taken place.

The NS1 protein we use is made by recombinant techniques, that is to say without the use of ferret or mink-materials, so as to avoid the influence of cellular debris or other materials that may be contained in extracts derived from whole viruses or from animals infected with ADV. However, discussions with ADV researchers abroad indicates that the VP proteins are probably developed very soon after the statement of the NS proteins, and perhaps these would be useful in the ELISA as well.

The recently developed Point of Care Test for ADV Antibody is based on a similar principle. In this technique, sample is added and “flows” onto a sample pad in the plastic cassette of the POCT. In the sample pad is a dried solution of red microparticles coated with the same material as is coated onto the wells of the ELISA plate. There is a TEST line coated onto the membrane that is composed of anti-ferret antibodies, and any ADV antibody bound to the red microparticles by that material will be immobilized at this point, creating a red line. A point further downstream called the CONTROL line simply binds any and all red microparticles whether they are attached to ADV antibody or not. This indicates the device is working properly, and that the sample has flowed through the membrane.

Because we suggest the use of saliva as a substitute for blood in testing for ADV Antibody, I’d like to say a few words about saliva as a diagnostic fluid.

Over the past 15 years, saliva has been studied in humans as a potential replacement fluid for blood in the diagnoses of infectious diseases. The aim was to find a way to avoid the trauma associated with blood collection, especially in children and the infirm, or those with fragile veins, with a non-invasive technique that would give the same result in a clinical test.

It has since been determined that such analytes as HIV Ab, and the antibodies associated with Hepatitis A, B and C as well as smaller molecules like drugs of abuse and alcohol are very readily determined in saliva, and the results correlate with blood results close to 100%. There are several FDA-approved saliva-based tests for HIV Ab, alcohol and drugs of abuse currently on the market.

When we first endeavored to develop tests for ADV, we simultaneously collected blood and saliva from a group of shelter ferrets that had had their blood previously tested by CIEP. I found that these results correlated 100% with both the blood and saliva specimens analyzed in the (then) new ELISA test in my laboratory. Next, I solicited specimens of saliva and blood from several individual ferret owners, sent one tube of blood for CIEP analysis and performed the ELISA test on the other tube of blood as well as the saliva specimens, in our laboratory.

The ELISA using blood and saliva in our laboratory gave the same results, and 82 out of 86 blood specimens tested by CIEP agreed with those results. All 4 of the discrepancies were positive by CIEP, but negative by ELISA in both blood and saliva.

In any event, in ferrets, as well as many other small animals such as cats, I believe that it is far easier to obtain an oral fluid sample rather than attempting to draw blood. Additionally in the case of ferrets, where ADV Antibody analysis is the goal, the clipping of toenails is not only traumatic to the ferret, but the concern is cross-contamination of the clippers. Again, if you choose to go this route, be sure to have an ample supply of a parvocide at hand.

In my opinion, there is a need to conduct ADV controlled clinical trials in ferrets, similar to some of the studies that have been performed on mink, in order to:

A.Determine more precisely when the antibodies begin to appear in infected ferrets.

B.Determine whether antibody concentrations tend to fluctuate, and how they fluctuate, and if and how this correlates with clinical ADV disease.

C.Determine whether ferrets, unlike mink, can actually clear the virus from their bodies.

D.Determine the presence and concentration of other clinical indices such as CD8 lymphocytes, ADV viral DNA, total protein, the presence of hypergammaglobunemia as well as other biochemical and physiological changes associated with ADV infection.


I originally wanted to conduct a formal clinical trial along these lines about 9 months or so ago. The problems I encountered had to do with the fact that no research facilities, industrial or academic, were very willing to have a parvovirus in their facilities – they just did not want to deal with it. I recently returned from Copenhagen, Denmark, where I met with Drs. Jesper Christensen and Bent Aasted, two researchers who have done much work with ADV in mink, and parvoviruses in general. Dr. Aasted has agreed to perform these studies for us at the veterinary school in Copenhagen, and hopefully we should be getting started soon.

Although this promises to take some time, we will endeavor to publish the results as timely as possible in order to disseminate this important information to the academic community and ferret owners as soon as practicable.

Christensen and Aasted were the folks who most recently conducted vaccine trials against ADV in mink; their results were published in 1998.

Briefly, in this experiment, mink were separately vaccinated with both the VP capsid proteins and the non-structural protein, NS1 and then challenged with the virus in order to see if they had developed protection from the virus. While each of these proteins did elicit an antibody response, they did not prevent the mink from becoming infected.

The results indicated that injecting mink with the VP capsid protein actually increased the death rate amongst the mink thus treated. Inoculation with the NS1 protein, however, was said to provide “partial” immunity against the virus, because mink thus treated came down with a milder form of the virus, with a lower death rate. In other words, most of the mink with the VP capsid vaccine died, but most of the mink with the NS1 vaccination lived; albeit they were all found to have ADV after virus challenge.

So, what does this mean with regard to ferrets and a potential vaccine against ADV?

In the minutes of the mink ADV symposium held in Greece last Fall, I noticed that Bent Aasted was soliciting interest from fellow researchers for the further development of an ADV vaccine in mink. So I asked him point blank whether he would be interested in pursuing such a venture in ferrets, given that there are millions of companion ferrets in the U.S. and abroad.

The bottom line is that both he and Dr. Christensen are interested in pursuing this, but I want to caution everyone that it remains most uncertain whether a vaccine can be produced that actually gives immunity, or even partial immunity, to ADV. Realistically, such a vaccine, if at all possible is very many years in the future.

In the interim, my suggestion has always been to test your ferrets for ADV antibody using the technique you and your veterinarian are most comfortable with, and by all means, its still prudent to keep any ADV-positive ferrets segregated from ADV-negative ferrets. I believe this is the best way to protect your pets.

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