SNAP® 4Dx® Test

With the superior accuracy of ELISA technology, you can:

• Find low worm-burden cases sooner and more often, and begin treatment when you can do the most good. It’s the only test proven to be more sensitive than other in-house tests in detecting low worm-burden.¹
• Count on flexibility to test your way, because ELISA is equally effective on whole blood, serum or plasma.
• Interpret the test results easily every time—it’s the only pet-side test with a built-in wash step and amplification of a positive reaction, making positives easier to detect.

The SNAP® 3Dx®/4Dx® Test identifies an antibody that is produced only as a result of a B. burgdorferi infection (unlike IFA, which isn’t specific for B. burgdorferi infection). The difference is the unique, revolutionary C₆ ELISA technology.

Canine granulocytic anaplasmosis is caused by the bacterium Anaplasma phagocytophilum and is transmitted by the deer tick or black-legged tick. Coinfection of Anaplasma species with other vector-transmitted pathogens may lead to more complex disease presentations and a slower response to therapy.

Dogs exposed to Anaplasma phagocytophilum are at risk of becoming sick. Unfortunately, pet owners aren’t aware of infection until their dogs become seriously ill. Testing with the SNAP 4Dx Test helps you detect exposure and infection, and reinforces the importance of tick control.

Early detection is the most effective way to protect your patients because the prognosis is good for early-stage infection, but variable-to-guarded for chronic infections.

The SNAP 3Dx/4Dx Test allows for inexpensive in-house E. canis screening, helping you diagnose sooner. Therefore, treatment can be initiated to prevent the dog from progressing into later stages, where the illness is more difficult to manage.

Ehrlichia specificity is high on the SNAP 3Dx/4Dx Test. The SNAP 3Dx/4Dx Test uses an E. canis-specific peptide for accurate, reliable confirmation of exposure to E. canis, with a specificity of at least 97%.⁴

Any color development in the sample spots indicates the presence of heartworm antigen, or B. burgdorferi, A. phagocytophilum or E. canis antibody. If no color develops in the positive control spot, repeat the test.

Any color development in any of the sample spots indicates a positive result. The amount of antigen or antibody produced by individual dogs differs. So it’s not possible to correlate the color intensity of the sample spot and the level of infection. Keep in mind that every dog is different.

As stated in the package insert (PDF), the SNAP 4Dx Test can be stored in the refrigerator at 2°–7°C (35°–45°F) until its expiration date or it can be stored at room temperature (15°–25°C/59°–77°F) for a period of up to 90 days.

After the test kit or any of its components have been out of the refrigerator for a period of 24 hours or more, the expiration date is now 90 days or the printed expiration date, whichever occurs first.

The locations of the positive control and sample spots on the SNAP 4Dx Test have been researched for optimal performance. In the current configuration, the spots allow for maximum sensitivity and specificity of each test.

The SNAP 4Dx Test requires four drops of conjugate and three drops of sample.

The test result must be read eight minutes after the device is snapped. The test does not contain stop solution, and after eight minutes, color development may occur that is not related to the sample. Do not report results read after eight minutes.

Serum, plasma and anticoagulated whole blood can be used.

The SNAP 4Dx Test, and any other SNAP® test, must be used within two hours of removing it from the foil package.

Currently, IDEXX distributes and sells both the SNAP 4Dx Test and the SNAP 3Dx Test kits.

The heartworm portion is an antigen test, and the Ehrlichia canis, Anaplasma phagocytophilum and Lyme portions are antibody tests.

Yes. With the introduction of software version 1.11, the SNAPshot Dx® Analyzer can read the SNAP 4Dx Test.

Heartworm disease (also called dirofilariasis) is a preventable, but serious and potentially fatal, parasitic disease that primarily affects dogs and cats. The heart and lungs are the major organs affected by heartworms in dogs.

Adult heartworms (Dirofilaria immitis) can be up to 14 inches long. They live in the right side of the heart and the pulmonary arteries, which connect the heart to the lungs. Heartworms cause blockage and injury that may lead to heart failure and may damage other organs, including the liver and kidneys. A dog may harbor several hundred heartworms, but in most cases the number is much lower.

Cats usually have smaller and fewer heartworms than dogs, and often don’t exhibit clinical signs until the disease is considerably advanced. Occasionally, heartworms are found in other animals such as foxes, wolves and ferrets. Heartworms can also lodge in the lungs of people and form nodules, but their presence has not been associated with clinical disease.

Mosquitoes transmit heartworms.

• Adult female heartworms release microfilariae into the bloodstream of infected animals. When a mosquito bites an infected animal, it takes up blood containing these microfilariae.
• The microfilariae incubate in the mosquito for 10 to 14 days, during which time they become infective larvae. When the mosquito bites another animal, the infective larvae are passed on to the second animal through the wound.
• Infective larvae migrate through the tissues of the body for two to three months, and then enter the heart and pulmonary arteries, where they reach adult size in another three months. If both sexes are present, the mature worms will mate and produce new microfilariae, and the cycle begins again.
• Adult heartworms may survive for five to seven years in dogs. The mosquito is the only natural agent of transmission for heartworms. Microfilariae cannot mature into adult heartworms without passing through a mosquito.

Cats rarely develop microfilaremia. When present, microfilariae are usually short-lived. Some cats rid themselves of heartworm infections spontaneously, whereas infective larvae in other cats may mature into adult heartworms that can cause serious disease.

According to the American Heartworm Society:

Pets recently or lightly infected with heartworms may show no signs of disease. In later stages, dogs with heavy or persistent infection may cough, become lethargic, lose their appetites or have difficulty breathing. Owners may first notice that their dogs seem to tire rapidly after only moderate exercise. Fluid may accumulate in the abdomen (ascites) as a result of advanced heartworm infection. Another serious, but less common, manifestation is caval syndrome (a form of liver failure). Animals affected by caval syndrome rapidly become weak and their urine turns dark brown. Caval syndrome requires prompt surgical removal of the heartworms.

Veterinarians can detect heartworm infection in its early stages by examining a dog’s blood for the presence of circulating microfilariae or by performing laboratory tests to look for heartworm antigen (a protein produced by adult heartworms). Radiography of the chest and electro- or echocardiography are also helpful in making a diagnosis, and may indicate the severity of the infection.

Clinical signs in cats are similar. However, most cats never show signs of disease and heartworm infection may be a postmortem diagnosis. While the diagnostic approach to heartworm disease in cats is similar to that used for dogs, diagnosis is much more difficult because cats usually harbor very few adult worms.

Yes, heartworm disease is almost 100% preventable with oral (daily or monthly), topical (monthly) or injectable (biannually) medications.

Prior to beginning a prevention program, a blood test is recommended to detect or rule out the presence of heartworms. Then prescribe an appropriate preventative and tell the pet owner how often and how long that preventative should be administered. You can determine the patient’s risk for heartworm disease on the basis of its species, lifestyle and geographic location.

The American Heartworm Society’s 2007 treatment guidelines encourage annual testing, testing in between prevention product changes, and year-round prevention to manage heartworm disease in dogs and cats. “Annual retesting is an integral part of ensuring that [prevention] is achieved and that more timely treatment can be provided to dogs that test positive in order to minimize pathology.”¹

One of the most common tick-transmitted diseases in the United States and worldwide, Lyme disease, or borreliosis, is a multifaceted infectious disease syndrome that can cause serious problems for dogs. Although it’s most frequently reported in people and dogs, Lyme disease has also been seen in cats, horses and cattle.

Lyme disease is caused by the corkscrew-shaped bacterium, or spirochete, Borrelia burgdorferi. The bacterium lives in the gut of the black-legged (or deer tick, Ixodes scapularis) and the Western black-legged tick (Ixodes pacificus), and can be transmitted when an infected tick feeds on a dog, person or other mammal. Black-legged ticks are extremely small, ranging from the size of a grain of sand to the size of a sesame seed.

According to the Centers for Disease Control and Prevention (CDC), “Lyme disease was first recognized in the United States in 1975, after an unusual outbreak of arthritis near Lyme, Connecticut. Since then, reports of Lyme disease have increased dramatically, and the disease has become an important public health problem in some areas of the United States.”¹

Reported Cases of Lyme Disease by Year, United States, 1995-2009

Source: Centers for Disease Control and Prevention. Reported Cases of Lyme Disease by Year, United States, 1995–2009. Available at: http://www.cdc.gov/lyme/stats/chartstables/casesbyyear.html. Accessed September 7, 2011.

As reported by the CDC, “Lyme disease is the most commonly reported vector-borne illness in the U.S. In 2009, 95% of Lyme disease cases were reported from 12 states:

• Pennsylvania
• New Jersey
• New York
• Massachusetts
• Connecticut
• Wisconsin
• Maryland
• Minnesota
• New Hampshire
• Delaware
• Maine
• Virginia”²

View the most recent prevalence map for Lyme, heartworm, Anaplasma, and Ehrlichia (interactive maps require Flash).

The CDC brochure, Lyme Disease: A Public Information Guide, states:

“The Lyme disease bacterium, Borrelia burgdorferi, is spread through the bite of infected ticks. The black-legged tick (or deer tick, Ixodes scapularis) spreads the disease in the northeastern and north-central United States, and the western black-legged tick (Ixodes pacificus) spreads the disease on the Pacific coast. These ticks are usually found in wooded areas and have complex life cycles. In some regions, black-legged ticks can spread other diseases in addition to Lyme disease, including babesiosis and anaplasmosis.

In general, ticks need to be attached for 36 to 48 hours before they can transmit the Lyme disease bacterium.”¹

Possibly. Where and how your clients live may increase their risk of tick exposure—especially if they garden, hike, camp, hunt, work outdoors or spend time in wooded, brushy or overgrown areas and their dogs accompany them. Black-legged or deer ticks prefer to hide in shady, moist ground litter. But they can be found above ground, clinging to tall grass, brush, shrubs and low tree branches. They also inhabit gardens and lawns, particularly at the edges of woodlands and around old stone walls, where deer and white-footed mice, the ticks’ preferred hosts, thrive.

In most areas of the country, people and their pets are at a moderate to high risk of exposure from April to November. Ticks are most active during these months and people and their pets are spending more time enjoying outdoor activities, but disease onset can occur at any time of the year. Lyme disease transmission is a high concern in both spring and fall. In spring, deer ticks must feed to progress from larvae to nymphs—and then again—to mature into adult ticks.

While a variety of signs may appear, the most common signs of Lyme disease are hidden. Other common signs are recurrent arthritis and lameness that last for only three to four days, sometimes with appetite loss and depression. Dog owners should be aware of these warning signs:

• Sudden occurrence of lameness
• Reluctance to move or a stiff, painful gait
• Warm, swollen joints
• Pain in the legs or throughout the body
• Fever
• Fatigue
• Loss of appetite
• Swollen lymph nodes

Dogs can also develop fatal kidney disease, although rare. Signs of Lyme disease may come and go, vary in intensity from mild to severe, and can mimic many other conditions.

Lyme disease is diagnosed by testing for B. burgdorferi antibodies, noting the presence of clinical signs, ruling out other causes of these signs and observing a response to antibiotic therapy. Experts agree that annual testing in endemic regions provides the best first line of defense against Lyme disease.

Lyme disease is challenging to diagnose. The signs can be very subtle and can be easily mistaken for other medical problems. But with the SNAP® 3Dx®/4Dx® Test, veterinarians can tell whether a dog has been infected with Lyme disease. Then the veterinarian and pet owner can discuss treatment options.

Unfortunately, vaccines aren’t 100% effective. If a dog were infected prior to vaccination, the vaccine would not stop disease from occurring. Having a dog tested adds the benefit of knowing whether or not it has been infected. The SNAP® 3Dx®/4Dx® Test is the only test that can definitively distinguish between vaccinated and infected dogs.

Several broad-spectrum antibiotics can effectively treat Lyme disease, especially in its early stages. In early stages, response to antibiotics is usually seen within three to five days and is often dramatic. Patients should be monitored for clinical response to treatment according to the SNAP® 3Dx®/4Dx® Test Lyme disease treatment protocol (PDF).

No, Lyme disease is not spread by person-to-person contact or by contact with infected animals. However, infected dogs serve as sentinels to indicate the presence of infected ticks in the area, which means that you or the pet owner may also be at risk.

Lyme disease may be prevented through vaccination and tick control. Since ticks carry other devastating diseases, such as Rocky Mountain spotted fever, anaplasmosis and ehrlichiosis, it’s important to keep dogs tick-free.

The SNAP® 3Dx®/4Dx® Test identifies infection. The test’s C₆ peptide is highly specific for Borrelia burgdorferi and is only present in the face of active infection. In addition, antibodies from currently available Lyme vaccines have been shown not to cross-react with SNAP® Lyme antibody detection, so you can identify infection in vaccinated dogs.¹

References

Anaplasma phagocytophilum is a bacterium that is transmitted by ticks and is the causative agent for canine anaplasmosis. These ticks include Ixodes scapularis and Ixodes pacificus in the United States, and Ixodes ricinus in Europe. These tick species, more commonly known as deer ticks, are the same organisms that carry Borrelia burgdorferi, which is the causative agent of Lyme disease. A. phagocytophilum can be transmitted to dogs, cats, horses and humans.

Yes. In the late 1990s, several species—E. equi, E. phagocytophila and the organism responsible for human granulocytic ehrlichiosis (HGE)—were classified as a single species and renamed A. phagocytophilum.

The A. phagocytophilum portion of the SNAP 4Dx Test detects antibodies to an Anaplasma species that can be pathogenic in dogs, A. phagocytophilum. A positive result supports exposure to A. phagocytophilum.

The test has also been shown to cross-react with another Anaplasma species known as A. platys. In an internal IDEXX study involving dogs infected with a laboratory strain of A. platys, the SNAP 4Dx Test was reactive with serum from 10 out of 10 dogs.

A. phagocytophilum portion of the SNAP 4Dx Test detects both IgM and IgG antibodies. As with most infections, the immune system will produce high concentrations of IgM antibodies during the early acute phase of infection. Later in the course of infection, the immune system will begin to produce IgG antibodies, which can remain elevated in the serum for months to even years.

The SNAP 4Dx Test is a screening test recommended for all dogs. It detects the presence of antibodies to A. phagocytophilum, E. canis and B. burgdorferi. It also detects heartworm antigen.

IFA and the SNAP 4Dx Test are two different types of assays. IFA uses a whole-cell preparation containing A. phagocytophilum organisms grown in cell culture. The SNAP 4Dx Test is an ELISA assay that uses a well-defined peptide to detect antibodies to A. phagocytophilum. Measurement of the antibody response on these two types of tests is very different. Interpretation of an IFA test result is more subjective and, therefore, requires substantial expertise by the testing laboratory. In general, a sample testing positive for A. phagocytophilum on the SNAP 4Dx Test has a good correlation with a positive IFA test result.

The SNAP 4Dx Test has also been shown to cross-react with another Anaplasma species, known as A. platys. In an internal IDEXX study involving dogs infected with a laboratory strain of A. platys, the SNAP 4Dx Test was reactive with serum from 10 out of 10 dogs.

An IFA titer of 1:80 or greater should be positive on the SNAP 4Dx Test.

The sensitivity and specificity (compared to IFA/Western blot) of the A. phagocytophilum portion of the SNAP 4Dx Test for the population tested are:

• Sensitivity = 99.1%
• Specificity = 100%

Sensitivity and Specificity of the SNAP 4Dx Test

Antibody response occurs 7–21 days post-infection. Anytime between 7–21 days post-infection, you could expect to see a positive result on the SNAP 4Dx Test.

In experimentally infected dogs (n=2), the SNAP 4Dx Test showed a positive result on day 8 post-inoculation.

Antibody response differs from dog to dog. Some dogs generate antibodies to a particular antigen quickly, while others take longer. Due to this variation in antibody response time, it is possible to have a dog that is positive for A. phagocytophilum morulae and negative on the SNAP 4Dx Test.

If you suspect A. phagocytophilum infection, but the SNAP 4Dx Test is negative, it is recommended to retest the animal in one to three weeks. Please keep in mind that the clinical signs of A. phagocytophilum closely resemble those of Lyme disease and E. canis infection.

As the test for A. phagocytophilum is an antibody test, it is possible for your dog to be infected with the organism and not show up as positive on the SNAP 4Dx Test. In some cases of acute infection, the antibody titer level has not yet reached a level that is detectable by the SNAP 4Dx Test.

If you suspect A. phagocytophilum infection, but the SNAP 4Dx test is negative, it is recommended to retest the animal in one to three weeks. Please keep in mind that the clinical signs of A. phagocytophilum closely resemble those of Lyme disease and E. canis infection.

Some of the signs of A. phagocytophilum infection may include fever, lethargy, anorexia, lameness, joint pain and thrombocytopenia. It is possible for these signs to overlap with those of Lyme disease and other tick-borne infections.

Many dogs with positive SNAP 4Dx A. phagocytophilum test results may have been previously exposed and will have no clinical signs at the time of testing.

The A. phagocytophilum organism infects the granulocytes (primarily the neutrophils), while the A. platys organism primarily infects the platelets. As these organisms reside in two different cell types, two different organisms may also be seen when examining a blood film.

It is possible for BOTH infections, A. phagocytophilum and A. platys, to induce thrombocytopenia.

It is important to keep in mind that in the U.S., A. phagocytophilum infection is generally acute and more severe, while A. platys infection is generally chronic and less severe. Most often, A. phagocytophilum infections can be found in the northern half of the United States, as well as the West Coast. A. platys has been found in the southeastern region of the U.S.

At this time, there is little known about the prevalence and pathogenicity of A. platys in the United States.

At this time, there is no agreement about whether or not subclinical dogs should be treated with antibiotics. Some veterinarians may choose to treat positive dogs that are not exhibiting clinical signs, while other veterinarians may choose to monitor these dogs.

Until more data is available, treatment is not recommended in dogs that are clinically and hematologically normal.

• Doxycycline—5 mg/kg, PO 2x per day for one month
• Tetracycline—22 mg/kg, PO 3x per day for one month (not recommended for young animals)

It’s not known whether or not the SNAP 4Dx Test can predict treatment efficacy in canines with A. phagocytophilum. Therefore, at this time, we cannot recommend when to retest the dog with the SNAP 4Dx Test. Based upon the expected IgG antibody kinetics, the test would likely remain positive for several months after successful treatment.

It is difficult to diagnose A. phagocytophilum based on clinical signs alone because many tick-borne infections induce similar clinical signs. A diagnosis of anaplasmosis is best made when clinical signs are used in conjunction with laboratory diagnostic tests. Some cases can be subclinical, thereby reinforcing the importance of screening all dogs.

There is currently no evidence suggesting that A. phagocytophilum can be passed from dogs to people. However, the disease is transmitted to both dogs and people by the same species of ticks.

• IFA tests are subjective and require the expertise of a skilled technician for analysis and interpretation. It is possible that the test result was truly positive, yet it was not interpreted that way.
• It is also possible that the SNAP 4Dx Test may be more specific than the IFA test that the reference laboratory is using.

• An IFA test is a highly subjective read and may cross-react with other species of Anaplasma and Ehrlichia. It is possible to have an acute infection where the IFA test is detecting a mixture of antibody responses to various organisms that may or may not be Anaplasma phagocytophilum.
• The SNAP 4Dx Test uses a highly specific immunodominant peptide from the A. phagocytophilum organism.
• Alternatively, if the infection truly is A. phagocytophilum, the host may require additional time to mount an immune response to the particular epitope that is detectable on the SNAP 4Dx Test.
• If you suspect A. phagocytophilum infection, but the SNAP 4Dx Test is negative, it is recommended to retest the animal in one to three weeks. Please keep in mind that the clinical signs of A. phagocytophilum closely resemble those of Lyme disease and E. canis infection.

The SNAP 4Dx A. phagocytophilum test uses a synthetic peptide to detect antibodies to A. phagocytophilum. The synthetic peptide mimics a specific region of an outer membrane protein found in the A. phagocytophilum organism.

The IFA test is a whole-cell preparation that may show cross-reactivity with other Anaplasma and Ehrlichia species.

A PCR test works by identifying and then amplifying the actual organism’s DNA or RNA when found in the blood. If performed correctly under proper laboratory conditions, PCR tests are highly specific, but can be less sensitive than antibody detection tests.

The SNAP 4Dx Test is an ELISA assay that identifies the presence of antibodies to A. phagocytophilum. As with any antibody test, the organism may have been eliminated, yet the antibody levels remain high enough to be detected by the SNAP 4Dx Test.

A. phagocytophilum may be eliminated from the dog by self-resolution or with antibiotics.

• If antibiotics were administered, did the antibiotic treatment fail? This can occur even when appropriate antibiotic, dose and duration are followed.
• If antibiotics were administered, was the dog properly treated with antibiotics for the proper amount of time?
• Did the owner comply with this treatment?
• Was the dog re-infected? Even after successful treatment, it is possible for a dog to be re-infected if placed back into the same environment. Tick prevention options should be explored.

As A. phagocytophilum is an antibody test, some dogs may always test positive for A. phagocytophilum on the SNAP 4Dx test. At this time, we do not have enough information to determine whether or not the result is re-infection or residual antibody, but IDEXX hopes to gain additional information and data over the next several years.

It is a potentially life-threatening disease that dogs can get from several common dog ticks.

It is the second most common canine infectious disease in the United States (after parvovirus).

View the most recent prevalence map for Ehrlichia, Lyme, heartworm, and Anaplasma (interactive maps require Flash). Just click the tab for the map you want to view.

Ehrlichiosis is spread through the bite of infected ticks. The Brown dog tick (Rhipicephalus sanguineus) carries the bacteria and bites the dog, spreading infection. All three stages—nymph, larvae, and adult—can transmit E. canis (the cause of ehrlichiosis). This is the only tick that can survive indoors.

In most areas of the country, people and their pets are at a moderate to high risk of exposure from April to November. Ticks are most active during these months and people and their pets are spending more time enjoying outdoor activities, but disease onset can occur at any time of the year. Ehrlichiosis transmission is a high concern in both spring and fall. In spring, dog ticks must feed to progress from larvae to nymphs—and then again—to mature into adult ticks.

While a variety of signs may appear, the most common signs of ehrlichia infection are hidden. Other common signs, which can range from mild to severe, include loss of appetite, depression, fever and painful joints. If left untreated, the disease can progress to a point at which it causes permanent blindness, autoimmune diseases, bleeding complications and even death. If caught early, the prognosis is usually very good for a full recovery from symptoms.

Veterinarians can perform a simple blood test to determine if a dog has been infected with E. canis. The SNAP 3Dx/4Dx Test screens dogs simultaneously for E. canis, Lyme disease and heartworm disease in just eight minutes.

Ehrlichiosis is challenging to diagnose. The signs may be very subtle and can be easily mistaken for other medical problems. But with the SNAP 3Dx/4Dx Test, veterinarians can tell whether a dog has been infected or exposed to E. canis and then provide treatment options.

A positive SNAP 3Dx/4Dx result should be followed by a CBC to check thrombocyte levels. If thrombocytopenia is evident, treatment may be prescribed (see below). Patients should be retested within 3–6 months; see the Ehrlichiosis protocol (PDF).

Several broad-spectrum antibiotics can effectively treat ehrlichiosis, especially in its early stages. Response to antibiotics is usually seen within two to three days, with the most striking changes being in behavior and attitude. Dogs experiencing severe anemia or bleeding problems may initially require a blood transfusion. However, this does nothing to treat the underlying disease. Unfortunately, the chronic form of the disease can be fatal.

No, ehrlichiosis is not spread by person-to-person contact or by contact with infected animals. Although the disease is not transmitted directly from dogs to humans, infected dogs serve as sentinels to indicate the presence of infected ticks in the area, indicating that you or the pet owner may also be at risk.

The best prevention of ehrlichiosis is to keep dogs free of ticks. This should include checking the skin daily for ticks and treating dogs with tick control. Since ticks carry other devastating diseases, such as Lyme disease, anaplasmosis and Rocky Mountain spotted fever, it's important to keep dogs tick-free.