SNAP® 4Dx® Plus 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.
• Count on flexibility to test your way, because ELISA is equally effective on anticoagulated 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® 4Dx® Plus Test identifies an antibody that is produced only as a result of a Borrelia burgdorferi infection (unlike IFA, which isn’t specific for B. burgdorferi infection). The difference is the unique, revolutionary C₆ ELISA technology.

Canine anaplasmosis is a disease that is caused by the organisms Anaplasma phagocytophilum, which is transmitted by the deer tick and black-legged tick, and Anaplasma platys, transmitted by the brown dog tick. Although A. phagocytophilum infection is generally more severe than A. platys, both forms of the disease pose serious risks to canine patients.

Dogs exposed to A. phagocytophilum and A. platys 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 Plus 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.

Canine monocytic ehrlichiosis is caused by infection with Ehrlichia canis bacteria transmitted by the brown dog tick (Rhipicephalus sanguineus). Ehrlichia canis is a gram-negative bacterium that infects and multiplies in white blood cells (primarily monocytes).

Canine granulocytic ehrlichiosis is caused by infection with Ehrlichia ewingii bacteria transmitted by the lone star tick (Amblyomma americanum). Ehrlichia ewingii is a gram-negative bacterium that infects and multiplies in white blood cells (predominantly neutrophils). Infection with E. ewingii may lead to a decrease in the dog’s platelets, which are important in the formation of blood clots, and the infection may produce marked joint pain or neurologic signs.

Any color development in the sample spots indicates the presence of heartworm antigen or Borrelia burgdorferi, Anaplasma phagocytophilum/Anaplasma platys or Ehrlichia canis/Ehrlichia ewingii antibodies. 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.

Refer to package insert (PDF) for test storage information.

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

The SNAP 4Dx Plus Test requires 3 drops of sample and 4 drops of conjugate.

The test result must be read 8 minutes after the device is snapped. The test does not contain stop solution, and after 8 minutes, color development may occur that is not related to the sample. Do not report results read after 8 minutes.

Serum, plasma and anticoagulated whole blood can be used.

The SNAP 4Dx Plus Test, and any other SNAP® test, must be used within 2 hours of removing it from the foil package.

The SNAP 4Dx Test is no longer available to purchase because the SNAP 4Dx Plus Test provides all the benefits of the SNAP 4Dx Test and more. With the SNAP 4Dx Plus Test, you can detect heartworm antigen and antibodies to B. burgdorferi, A. phagocytophilum and E. canis plus antibodies to A. platys and E. ewingii. The SNAP 4Dx Plus Test allows you to perform one comprehensive test for the vector-borne diseases in your area.

Sensitivity and Specificity data are available through Customer Service upon request.

The heartworm portion is an antigen test, and the B. burgdorferi, A. phagocytophilum/A. platys and E. canis/E. ewingii portions are antibody tests.

Yes, with the introduction of software version 1.40, the SNAPshot Dx Analyzer can read the SNAP 4Dx Plus 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 they 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 10–14 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 2–3 months and then enter the heart and pulmonary arteries, where they reach adult size in another 3 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 5–7 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.

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 preventive and tell the pet owner how often and how long that preventive 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 and Companion Animal Parasite Council’s guidelines encourage annual testing, testing in between prevention product changes and year-round prevention to manage heartworm disease in dogs and cats. “Annual testing is an integral part of ensuring that prophylaxis is achieved and that more timely treatment can be provided to dogs that test positive in order to minimize pathology.”¹

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

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 tick (or deer tick, Ixodes scapularis) and the Western black-legged tick (Ixodes pacificus), and it 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, 1996–2010

Source: Centers for Disease Control and Prevention. Reported Cases of Lyme Disease by Year, United States, 1996–2010. www.cdc.gov/lyme/stats/chartstables/casesbyyear.html. Accessed March 1, 2012.

As reported by the CDC:

Lyme disease is the most commonly reported vector-borne illness in the U.S. In 2009, it was the 5th most common Nationally Notifiable disease.

In 2010, 94% of Lyme disease cases were reported from 12 states:

• Connecticut
• Delaware
• Maine
• Maryland
• Massachusetts
• Minnesota
• New Jersey
• New Hampshire
• New York
• Pennsylvania
• Virginia
• Wisconsin²

View the most recent prevalence map for Lyme disease, heartworm disease, Anaplasma and Ehrlichia (interactive maps require Adobe® Flash® version 8 or later). Just click the tab for the map you want to view.

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 36–48 hours before they can transmit the Lyme disease bacterium.¹

It is possible. 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 3–4 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® 4Dx® Plus Test, veterinarians can tell if a dog has been infected with B. burgdorferi. Then the veterinarian and pet owner can discuss further diagnostic/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. 

Several broad-spectrum antibiotics can effectively treat Lyme disease, especially in its early stages. In early stages, response to antibiotics is usually seen within 3–5 days and is often dramatic. Patients should be monitored for clinical response to treatment according to the algorithm in the SNAP® 4Dx® Plus Test Clinical Reference Guide (PDF).

No, Lyme disease 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, which means that people sharing the same habitat as the pet 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.

Anaplasma phagocytophilum is a bacterium that infects granulocytes and is the causative agent of canine granulocytic anaplasmosis. The bacterium is transmitted by deer ticks, including 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 vectors that carry Borrelia burgdorferi, which is the causative agent of Lyme disease. A. phagocytophilum can be transmitted to dogs, cats, horses and humans.

Anaplasma platys (formerly Ehrlichia platys) is an intracellular bacterium that infects canine platelets. It is most likely transmitted through the bite of an infected tick Rhipicephalus sanguineus (brown dog tick). A. platys is the cause of infectious cyclic thrombocytopenia in dogs. A. platys is frequently seen as a coinfection with Ehrlichia canis.

Yes. In the late 1990s, several species—Ehrlichia equi, Ehrlichia phagocytophila and the organism responsible for human granulocytic anaplasmosis (HGA) were classified as a single species and renamed Anaplasma phagocytophilum.

The Anaplasma portion (A. phagocytophilum/A. platys) of the SNAP 4Dx Plus 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.

IFA and the SNAP 4Dx Plus Test are two different assays. Measurement of the antibody response on these two types of tests is very different. IFA is a whole-cell preparation that contains A. phagocytophilum organisms. Interpretation of an IFA test result is more subjective and, therefore, requires substantial expertise by the testing laboratory. The SNAP 4Dx Plus Test is an ELISA that uses a well-defined peptide to detect antibodies to A. phagocytophilum/A. platys. In general, a sample testing positive for A. phagocytophilum/A. platys on the SNAP 4Dx Plus Test has a good correlation with a positive IFA test result. During the early stages of an A. phagocytophilum/A. platys infection, the Anaplasma spot on the SNAP 4Dx Plus Test may not report a positive result as the dog’s immune system may not have mounted sufficient antibody respose to the peptide used in this assay.

When screening for exposure to A. phagocytophilum/A. platys, an IFA titer of approximately 1:100 or greater will be positive on the SNAP 4Dx Plus Test.

An A. phagocytophilum antibody response occurs 7–21 days postinfection. An A. platys antibody response occurs 10–21 days postinfection.1

Antibody responses differ from dog to dog. Some dogs generate antibodies to a particular antigen quickly, while others take longer. Because of this variation in antibody response time, it is possible to have a dog that is positive for Anaplasma morulae and negative on the SNAP 4Dx Plus Test. If you suspect Anaplasma infection, but the SNAP 4Dx Plus Test is negative, it is recommended to retest the animal in 1–2 weeks or consider testing by PCR.1

As the test for A. phagocytophilum/A. playts 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 Plus Test. In some cases of acute infection, specific antibodies have not yet reached a level that is detectable by the SNAP 4Dx Plus Test.

If you suspect Anaplasma infection, but the SNAP 4Dx Plus Test is negative, it is recommended to retest the animal in 1–2 weeks or consider testing by PCR.1

A. platys cases usually show minimal clinical signs. Some dogs may have fever, thrombocytopenia, uveitis, petechia and ecchymoses or epistaxis.

The A. phagocytophilum organism infects the granulocytes (primarily the neutrophils), while the A. platys organism primarily infects the platelets. 

It is possible for BOTH infections, A. phagocytophilum and A. platys, to induce thrombocytopenia. 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 southern and southwestern regions of 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, orally two times daily for one month
• Tetracycline—22 mg/kg, orally three times daily for one month (not recommended for young animals)

It is difficult to diagnose anaplasmosis based on clinical signs alone because many tick-borne infections induce similar clinical signs. Some cases can be subclinical, thereby reinforcing the importance of screening all dogs.

The SNAP 4Dx Test is an ELISA that identifies the presence of antibodies to A. phagocytophilum/A. platys. 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 Plus Test.

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 they can be less sensitive than antibody detection tests for screening. As a sick dog test, PCR may be helpful in dogs with acute clinical disease. The PCR test is the best test available to differentiate between A. phagocytophilum and A. platys. PCR testing is recommended for dogs with clinical signs consistent with tick-borne illness.

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

View the most recent prevalence map for Anaplasma, Lyme disease, heartworm disease and Ehrlichia (interactive maps require Adobe® Flash® version 8 or later). Just click the tab for the map you want to view.

A positive on the Ehrlichia spot indicates that the dog has antibodies to Ehrlichia canis and/or Ehrlichia ewingii. Refer to the product insert.

Ehrlichia canis is an intracellular bacterium that is transmitted by the brown dog tick (Rhipicephalus sanguineus). This is the only tick that can survive indoors.

Ehrlichia ewingii is an intracellular bacterium that is transmitted to dogs and humans by Amblyomma americanum (lone star tick). This tick can also transmit Ehrlichia chaffeensis, the bacteria that causes human monocytic ehrlichiosis (HME).

The initial immune response will produce IgM (immunoglobulin M molecule) antibodies early in an infection. Later in the infection, the body will begin to produce IgG (immunoglobulin G) antibodies. The Ehrlichia portion of SNAP 4Dx Plus Test could detect both IgM and IgG.

It is the second-most common canine infectious disease in the United States (after parvovirus).1

View the most recent prevalence map for Ehrlichia, Lyme disease, heartworm disease and Anaplasma (interactive maps require Adobe® Flash® version 8 or later). Just click the tab for the map you want to view.

In most areas of the country, people and their pets are at a moderate to high risk of exposure during April–November. Ticks are most active during these months, and people and their pets are spending more time enjoying outdoor activities. Ehrlichia ewingii is of high concern in both spring and fall. E. canis infections can occur year-round.

While a variety of signs may appear, oftentimes, the signs of E. canis infections are hidden. 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.

Clinical signs for E. ewingii infection can be highly variable among dogs, and most often develop within 7–14 days from the start of infection. Clinical manifestations include fever, lethargy, lameness and neurological manifestations, including ataxia, paresis, proprioceptive deficits and vestibular dysfunction.

Veterinarians can perform a simple blood test to determine if a dog has been exposed to E. canis/E. ewingii. The SNAP 4Dx Plus Test screens dogs simultaneously for E. canis, E. ewingii, as well as Lyme disease, A. phagocytophilum, A. platys and heartworm disease, in just 8 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 4Dx Plus Test, veterinarians can tell if a dog has been exposed to and may be infected with E. canis/E. ewingii and then provide treatment options.

A positive result should be followed by a complete blood count (CBC) to check thrombocyte levels. If thrombocytopenia is evident, treatment may be prescribed. (For treatment monitoring algorithms, see the ehrlichiosis protocol.)

Several broad-spectrum antibiotics can effectively treat ehrlichiosis, especially in its early stages. Response to antibiotics is usually seen within 2–3 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, which means that people sharing the same habitat as 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.

E. canis
Antibody response to E. canis infection occurs 7–21 days (1–3 weeks) postinfection. Most dogs are seropositive by 3–4 weeks following exposure.

E. ewingii
Most dogs are seropositive about 3–4 weeks after exposure to infection.1

As the test for Ehrlichia species is an antibody test, it is possible for your dog to be infected with the organism and not show up positive on the SNAP 4Dx Plus Test. In some cases of acute infection, the antibody titer level has not yet reached a level that is detectable by the test. If you suspect Ehrlichiosis and the SNAP 4Dx Plus Test is negative, it is recommended to retest the animal in 1–3 weeks or consider testing by PCR.1

Both E. canis and E. ewingii are gram-negative bacteria that infect white blood cells. E. canis infects monocytes and E. ewingii infects granulocytes (neutophils).

Canine monocytic ehrlichiosis is caused by infection with E. canis bacteria transmitted by the brown dog tick (Rhipicephalus sanguineus). Canine monocytic ehrlichiosis can be mild or severe, acute or chronic, with varying clinical signs that include:

• Fever, anorexia, lethargy
• Bleeding disorders
• Polyarthritis, lameness
• Lymphadenomegaly
• Neurological signs

Canine granulocytic ehrlichiosis is caused by infection with E. ewingii bacteria transmitted by the lone star tick (Amblyomma americanum). Canine granulocytic ehrlichiosis can be inapparent, mild or severe and typically acute, with varying clinical signs that include:

• Fever, anorexia, lethargy
• Polyarthropathy, lameness
• Neurological signs