The Rip Van Wrinkler, Volume XV, Issue 3, August 2011

Pages 10 & 11

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"A new "multiplex" test for Lyme disease in horses and dogs will speed diagnosis and pinpoint time of infection, opening possibilities for more effective treatment plans."  Cost listed as $36.../ submitted by Donna Hess

Note from Natalie Culver:
My experiment with the new Lyme test by Cornell, as described in the August Wrinkler.
This new Lyme disease test distinguishes between (i) active acute infection and (ii) positive test for a dog that had previously been infected, and (iii) a dog who was vaccinated.  The old snap test would show the same thing (positive) for all three of these dogs - no information about vaccine vs old infection that was treated vs new acute infection. The caveat to get the best information for a dog that was previously infected is that you must test no later than 6 to 8 weeks after your dog is  re-infected if your dog is unlucky enough to get re-infected.  If it's after 6 to 8 weeks the "resent infection proteins" the test distinguishes between old/chronic infection and new/acute infection will have dissipated in the dogs system and the long term / chronic proteins will be present.  The presence of "chronic infection" proteins does not mean the dog is sick and has symptoms.  Once infected I believe dogs will always show these proteins in their system; even dogs successfully treated with antibiotics.

In Kunjo's case, he had Lyme disease in 2007, was successfully treated, and therefore always will test positive for the presence of these long-term/chronic Lyme proteins.  I think Kunjo likely was re-infected 3 months ago, in March as he started having very slight symptoms (sore joints, giving a little yip when jumping off high furniture, and being stiff in the joints in the morning).  It was three months after this started that this new test came out on 15 June.  As soon as I learned of the test I had him tested, and of course he shows only chronic, no evidence of recent re-infection.  However my vet and I agreed to treat him, as if he was re-infected in March, as too much time has elapsed to show recent infection for him on this test. We decided it won't hurt him to have the course of antibiotics, and it could help him.  If there’s no improvement then he could just be getting sore joints from older age.  I hope its Lyme though, and therefore treatable.

Kunjo gives the photographer "the eye".

For a dog who was vaccinated and then may have become infected, you need to use this test.  For a dog who was previously infected and treated and then re-infected, please test right away (before 6 to 8 weeks elapse), so you can get the best information out of this test.  For a dog with first time infection, also test before 6 to 8 weeks have passed so you can see the acute / new infection results.

New Lyme Test for Dogs and Horses /Cornell University College of Veterinary Medicine

As of June 15, 2011, the Serology/Immunology laboratory at the AHDC offers a new assay for the detection of antibodies to B. burgdorferi, the causative agent of Lyme disease. The new assay (Lyme multiplex assay) is available for testing samples from dogs and horses.

The Lyme multiplex assay combines the advantages of the previous ELISA and Western blot testing. It is a fully quantitative assay that simultaneously detects antibodies to different specific antigens of B. burgdorferi as indicators of acute or chronic infection. The assay also distinguishes between antibodies induced by infection with the spirochetes or vaccination against Lyme disease.

Both serum and/or cerebrospinal fluid samples can be submitted for multiplex testing using the regular AHDC sample submission form. The fee for the Lyme multiplex assay is $36. Further information can be obtained on the AHDC website ( or you can call (607-253-3900). Serological  Lyme testing by ELISA and Western blot will be discontinued after June 15, 2011.

Animal Health Diagnostic Center

Lyme Disease Multiplex Testing for Dogs

Background on Lyme disease and Lyme diagnostics

Lyme disease is induced by the spirochete B. burgdorferi. The bacteria are transmitted to the host by infected ticks. Typical clinical signs in dogs are fever, acute arthritis, arthralgia and lameness1,2. Clinical signs of lameness often develop 2-5 months after exposure. B. burgdorferi can persist for at least one year in clinically recovered dogs2. Serum antibodies to antigens of B. burgdorferi are commonly used to identify dogs that were exposed to the pathogen and are at risk of developing Lyme disease 2-5. Current ELISA based diagnostic tests identify antibodies as early as 4-6 weeks after exposure. High antibody levels were found in serum of experimentally infected dogs for at least 17 months2.

How does the new multiplex test work?

The new Lyme multiplex assay was developed at the Animal Health Diagnostic Center at Cornell University. It detects antibodies to three B. burgdorferi antigens in canine serum. The multiplex test is based on fluorescent bead technology that allows the simultaneous measurement of antibodies to different B. burgdorferi antigens in a single sample6,7.

Which B. burgdorferi antigens are used and how is the test interpreted?

The new multiplex test is based on three different outer surface proteins (Osp) of B. burgdorferi. Various research studies have shown that Osp antigen expression on the bacterial surface changes in response to tick feeding and after infection of a warm-blooded host, such as dogs, horses, or humans. In response to infection, dogs develop antibodies to these Osp proteins and testing for antibodies to specific Osp antigens can assist in the diagnosis of Lyme infection and disease.

Figure 1:

Lyme multiplex assay to detect antibodies to B. burgdorferi in canine serum. Osp-antigen specific antibodies in serum bind to the multiplex beads and are detected by a fluorescent conjugate. The assay values are expressed as median fluorescent intensities (MFI).

Interpretation of Lyme multiplex results6:

1. OspA – positive values for antibodies to OspA are typically observed in vaccinated dogs. OspA is expressed while B. burgdorferi persists in the tmid-gut and also while the bacteria are cultured in-vitro. During infection of mammalian hosts the bacteria down-regulate OspA. Thus, antibodies to OspA are almost undetectable after natural infection in non-vaccinated dogs.

2. OspC - has been found to be a valuable indicator for early infection with B. burgdorferi. Antibodies to OspC can be detected 2-3 weeks after infection B. burgdorferi. Antibodies to OspC decline after 3-5 months of infection.

3. OspF – is an indicator of chronic infection. Antibodies to OspF are developed 6-8 weeks after infection and persist afterwards.

What are the advantages of the new multiplex test?

The new Lyme disease test at the Animal Health Diagnostic Center at Cornell University combines the results previously obtained by ELISA and Western blotting. The advantages of the new multiplex test compared to the previous procedure are:

- increased specificity and sensitivity

- quantitative measurement of individual antigen

- improved assay results distinguishing between:

(1) vaccination and infection

(2) early and chronic stages of infection

The test result provides advanced information beyond any of the current Lyme testing methods. The testing allows a better definition of the dog’s current infection status and assists in determining treatment options. The infection status can be determined in vaccinated dogs.

 Figure 2:


B. burgdorferi regulates its outer surface protein expression depending on its environment. In the tick gut, OspA is expressed. During tick feeding, the bacteria leave the tick’s mid gut and start to express OspC on their surface. OspC expression is maintained during early infection. In response to the different environment in the dog’s body, the bacteria again change their surface expression – OspC disappears and OspF is expressed in the chronic infection stage.

Special considerations for vaccinated dogs

The new Lyme multiplex assay can distinguish between vaccinated and infected dogs. All currently available vaccines for dogs induce antibodies to OspA. Thus, the results on antibodies to OspA will determine the dog’s vaccination status. However, some of the available vaccines also induce antibodies to other Osp antigens. To provide our clients with the best interpretation for each animal, we need information on the vaccine used. Please include the name of the vaccine and when the dog was last vaccinated on the accession form when a sample of a vaccinated dog is submitted for testing.

How can the multiplex test be compared to other serological Lyme assays?

Researchers at the Animal Health Diagnostic Center at Cornell University have compared the current ELISA/Western blot procedure and the commonly used C6 assays with the new multiplex test for Lyme disease. Multiplex assay and C6 results highly agreed on the identification of infected dogs. The multiplex assay provided additional information and also determined the infection stage of infected animals. The table below gives an overview about the properties of the different assays.

Sample submission

For detection of antibodies to B. burgdorferi in dogs, 2 ml of serum needs to be submitted. Serum should be collected in a red top blood tube. The entire red blood tube or isolated serum should be shipped by overnight shipment on an ice pack. For submission forms and shipping address go to the Animal Health Diagnostic Center website ( The fee for the assay is $36 per sample.

Samples are tested three days a week and results are available 2-3 days after the sample arrives at the laboratory. Consultation is available on the new testing platform by calling the Serology/Immunology laboratory at the Animal Health Diagnostic Center at Cornell University 607.253.3900.


1 Levy, S.A., Magnarelli, L.A., 1992. Relationship between development of antibodies to Borrelia burgdorferi in dogs and the subsequent development of limb/joint borreliosis. J. Am. Vet. Med. Assoc. 200, 344-347.

2 Appel, M.J.G., Allan, S., Jacobson, R.H., Lauderdale, T.L., Chang, Y.-F., Shin, S.J., Thomford, J.W., Todhunter, R.J., Summers, B.A., 1993. Experimental lyme disease in dogs produces arthritis and persistent infection. J. Infect. Dis. 167, 651-664.

3 Jacobson, R.H., Chang, Y.F., Shin, S.J., 1996. Lyme disease: laboratory diagnosis of infected and vaccinated symptomatic dogs. Semin. Vet. Med. Surg. (Small Anim.) 11, 172-182.

4 Wittenbrink, M.M., Failing, K., Krauss, H., 1996. Enzyme-linked immunosorbent assay and immunoblot analysis for detection of antibodies to Borrelia burgdorferi in dogs. The impact of serum absorption with homologous and heterologous bacteriae. Vet. Microbiol. 48, 257-268.

5 Guerra, M.A., Walker, E.D., Kitron, U., 2000. Quantitative approach for the serodiagnosis of canine lyme disease by the immunoblot procedure. J. Clin. Microbiol. 38, 2628-2632.

6 Wagner B, Freer H, Rollins H, Erb HN. 2011. A fluorescent bead-based multiplex assay for the simultaneous detection of antibodies to B. burgdorferi outer surface proteins in canine serum. Vet. Immunol. Immunopathol., 140: 190-198.

7 Wagner B, Freer H, Rollins A, Erb HN, Lu Z, Gröhn Y. Development of a multiplex assay for detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods.

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