Canine Lyme Disease: One-Year Duration of Immunity Elicited With a Canine OspA Monovalent Lyme Vaccine
R.E. Wikle, MS1 B. Fretwell2 M. Jarecki, LAT3 J.C. Jarecki-Black, PhD3
1Perkin-Elmer Wellesley, Massachusetts
2AviGenics, Inc. Athens, Georgia
3Merial Limited Duluth, Georgia
ABSTRACT
Canine Lyme disease is characterized by various clinical signs ranging from subclini- cal disease to acute arthritis and arthralgia. Previous reports demonstrate significant protection against Lyme disease is elicited by a vaccine containing the major outer sur- face protein (OspA) of the causative agent, Borrelia burgdorferi. The purpose of the present study was to determine the safety and duration of immunity of protection offered by a recombinant OspA vaccine (RECOMBITEK® Lyme, Merial Limited, Duluth, GA). Twenty beagles, aged 9 to 10 weeks, were vaccinated per label subcuta- neously with 2 doses of RECOMBITEK Lyme vaccine 3 weeks apart, and 11 beagles served as unvaccinated controls. The inves- tigators, including those providing daily health assessments post-vaccination, and post-challenge spirochete isolation and clin-
ical signs assessment, were blinded to the vaccination status of the animals. Dogs were challenged 1 year (366 days) after vaccina- tion by exposure to naturally infected ticks. Spirochete reisolation from biopsies was used to assess vaccine efficacy at monthly intervals for 3 months after challenge. All dogs were monitored for clinical signs of Lyme disease. No adverse reactions were noted following vaccination at any time dur- ing the study. One-year duration of immuni- ty results demonstrated that the vaccine offered significant protection against Lyme disease as no vaccinated animals showed clinical signs, and no spirochetes were reisolated from vaccinated animals from biopsies performed for 3 months post-chal- lenge. By contrast, 18% of control animals experienced clinical signs, and spirochetes were reisolated from 82% of the non-vacci- nated dogs. In conclusion, the canine OspA vaccine is both safe and efficacious with long-lasting protection from clinical signs and spirochete proliferation for at least 1 year (366 days) after vaccination.
KEY WORDS: recombinant vaccine, Lyme disease, Borrelia burgdorferi, duration of immunity
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23INTRODUCTION
Lyme disease, caused by infection with the spirochete Borrelia burgdorferi transmitted by ixodid ticks, occurs mostly in dogs and humans and to a lesser extent in horses, cat- tle, and cats.1 It remains the most commonly reported vector-born disease in people in North America and Europe. More than 90% of Lyme disease in the United States occurs in the northeastern, mid-Atlantic, and upper Midwest regions, with reported cases in sev- eral counties in California.2
In dogs, infection with B. burgdorferi may cause recurrent arthritis with fever, myalgia, anorexia, and lethargy.3–6 More rarely, dogs may also experience renal fail- ure, heart block, and neurologic disease. Diagnosis of canine Lyme disease can be challenging due to the variability in clinical signs, nonspecific clinicopathology, and serology using enzyme-linked immunosor- bent assay, which may not distinguish between exposure and prior vaccination. The C6 peptide test (Canine SNAP® 3Dx® Test, IDEXX Laboratories, Inc., Westbrook, Maine) and Western blot assays are fre- quently used to distinguish post-vaccination titers from those of infection.3–5 Treatment for Lyme disease includes appropriate antibiotic therapy; however, chronic Lyme disease may be unresponsive. Non-steroidal anti-inflammatory therapy may be indicated but may also obscure the animal's observ- able response to antibiotic therapy.
Due to the challenges of diagnosis and therapy, vaccination of dogs is recommended in endemic areas.7–9 However, there is evi- dence that whole cell bacterins may illicit clin- ical signs of Lyme disease in dogs showing no evidence of infection with B. burgdorferi.4,8 Therefore, the focus in animal medicine has been the development of a vaccine containing the major outer surface protein (OspA) of B. burgdorferi that is also non-adjuvanted, as part of a continual quest for more defined and less aggressive vaccines. The anti-OspA antibody produced by the vaccine enters the tick upon feeding and kills the spirochete in the tick's midgut preventing transmission to the host.10
Previously, investigators have conducted several short-term studies to investigate the efficacy of a non-adjuvanted recombinant OspA vaccine (RECOMBITEK® Lyme, Merial Limited, Duluth, GA) in protecting dogs against challenge with B. burgdorferi- infected ticks. Results demonstrated vacci- nated dogs were protected against both spirochete proliferation and clinical disease. The present study was designed to evaluate the duration of this protective response elicited in dogs vaccinated with the OspA vaccine. Vaccine-induced immunity was evaluated using dogs challenged with a nat- ural infection model using B. burgdorferi- infected ticks at 366 days post-vaccination.
MATERIALS AND METHODS
Dogs
Thirty one male and female Beagle puppies, 9 to 12 weeks of age, were obtained com- mercially (Harlan Sprague Dawley, Indianapolis, IN). These puppies were tested negative for Lyme and Leptospira vaccina- tion as well as Lyme disease. The puppies were randomly split into 2 groups: 20 dogs were vaccinated subcutaneously with the OspA vaccine, and 11 dogs served as unvaccinated controls.
Vaccine Preparation and Monitoring
The OspA monovalent vaccine used in this study was prepared utilizing a stock concen- tration of the OspA vaccine and diluting the vaccine with sterile diluent to a concentra- tion exactly equal to the minimum release dose allowed for the commercially available product. The preparation was placed into single-use vials under sterile conditions (1 mL each) and was deemed satisfactory by quality control.
Vaccination Protocol
Two doses of vaccine (1 mL/dose) were administered subcutaneously to dogs at a 3- week vaccination interval. Concurrently, the control dogs received a placebo injection. All assessments were performed and recorded by trained individuals who were blinded to vac- cination status. Dogs were monitored for
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signs of anaphylaxis, including labored breathing, pruritis, and edema, for 15 minutes following each injection. Animal technicians observed dogs continuously for 1 hour fol- lowing vaccination and then at regular daily intervals during the 14 days after each injec- tion for clinical signs, including swelling, pain, tenderness, and scratching at the injec- tion site. The injection site was palpated for signs of tenderness and swelling prior to administration of the second injection.
Challenge
All dogs were challenged with 50 Ixodes scapularis nymphs infected with B. burgdor- feri at 366 days after vaccination according to a previously demonstrated procedure.1 Nymphs were used due to the scarcity of adult ticks as a result of an unseasonably dry summer. Dr. Thomas Mather (Director, Center for Vector Borne Diseases, University of Rhode Island) determined the nymph infection rate to be 100%.
Skin Biopsy and Spirochete Reisolation
Upon challenge, tick attachment sites were marked with indelible ink to allow identifi- cation and future biopsy. At 1, 2, and 3 months postchallenge, site-specific skin biopsies were performed. Tick attachment sites were shaved, prepped with surgical scrub, anesthetized with 2% lidocaine injected intradermally, and biopsied using a Baker skin punch. Skin samples were placed in tubes containing Barbour- Stoenner-Kelly culture medium (BSK media) with heat-inactivated rabbit serum and antibiotics and transported to the labo- ratory. Samples were supplemented with additional BSK media, placed in a candle jar, and incubated for 6 weeks. The investi- gator was blinded to the dog's vaccination status for the samples submitted. The tubes were examined weekly for spirochetes using dark field microscopy. When 10 or more fields were examined under the 40× objec- tive with no evidence of spirochetes, the sample was considered negative.
Clinical Signs
All dogs were monitored daily for any signs
of reaction or illness, including those con- sistent with Lyme disease, such as fever, lameness, ataxia, depression, anorexia, and pain and tenderness at the infection site. Again, all health assessments were per- formed and recorded by trained individuals who were blinded to vaccination status. Due to the variable manifestation of Lyme dis- ease, the absence of clinical signs was not considered to be an indication of protection; however, the presence of clinical signs would lend support to a positive diagnosis, and be confirmed by spirochete reisolation.
RESULTS
Vaccine Safety
No general adverse reactions to the vaccine were noted at any time during the study. This included a lack of any detectable pain, swelling, tenderness, or itching at the injec- tion site, which was examined daily for 2 weeks following vaccination.
Spirochete Reisolation From Skin Biopsies
No vaccinated dogs were positive for spiro- chetes at any time during the study (Table 1). However, 6 of the 11 control animals were biopsy positive at 1 month post-challenge, 8 of the 11 control animals were positive at 2 months post-challenge, and by the third biopsy date, spirochetes were reisolated from biopsy samples from 9 of the 11 con- trol animals (82%).
Clinical Signs of Lyme Disease
None of the 20 vaccinated dogs experienced clinical signs of Lyme disease. Two of the 11 unvaccinated controls (18%) experienced lameness attributable to Lyme disease.
DISCUSSION
Lyme disease is the most commonly report- ed tick-borne disease in humans in the United States. High infection rates are reported to occur in unvaccinated dogs, but the disease remains difficult to diagnose and treat. Research has shown that the OspA of B. burgdorferi is a strong immunogen that provides protection against B. burgdorferi in a variety of animals.11,12
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Table 1. Results of Spirochete Isolation and Clinical Signs. Spirochete Isolation (post-challenge)
Dog # Vaccine 1 Month 2 Months 3 Months
Clinical Signs Consistent With Lyme Disease
4D499 Control - + + + 4D513 Control + + + - 4D520 Control - - + - 4D557 Control - - - - 4D561 Control - - - - 4E410 Control + + + + 4E420 Control + + + - 4E441 Control - + + - 4E478 Control + + + - 4E469 Control + + + - 4E583 Control + + + - 4E482 Vaccinate - - - - 4D516 Vaccinate - - - - 4D563 Vaccinate - - - - 4D597 Vaccinate - - - - 4E408 Vaccinate - - - - 4E421 Vaccinate - - - - 4E430 Vaccinate - - - - 4E436 Vaccinate - - - - 4E442 Vaccinate - - - - 4E450 Vaccinate - - - - 4D526 Vaccinate - - - - 4E411 Vaccinate - - - - 4E417 Vaccinate - - - - 4E425 Vaccinate - - - - 4E434 Vaccinate - - - - 4E437 Vaccinate - - - - 4E440 Vaccinate - - - - 4D444 Vaccinate - - - - 4D454 Vaccinate - - - - 4D458 Vaccinate - - - -
In 1996, a recombinant non-adjuvanted OspA vaccine (RECOMBITEK Lyme) was introduced for the prevention of Lyme dis- ease in dogs. The current study demon- strates that vaccination with RECOMBITEK Lyme is safe. Moreover, RECOMBITEK Lyme is highly efficacious and provides at least 1-year duration of immunity.
Safety of the OspA vaccine was assessed via monitoring of animals for acute and subacute clinical signs following vacci- nation. No local or systemic reaction was
detected in any vaccinated animal. This vac- cine contains no adjuvant thus eliminating even mild or transient granulomatous response characteristic of vaccination with most adjuvanted preparations. This study supports previous study results demonstrat- ing the vaccine's high level of safety.
Efficacy of the vaccine was established at a 1-year period following administration of the initial vaccination protocol. Evaluation of clinical signs, as well as determination of the vaccine's ability to pre- vent spirochete dissemination, was used to
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assess protection elicited in this long-term duration of immunity trial. Dogs were moni- tored daily for clinical signs associated with Lyme disease, including fever, lameness, ataxia, depression, and anorexia. No clinical signs of Lyme disease were observed in any vaccinated dogs; however, 2 unvaccinated controls (18%) demonstrated lameness asso- ciated with spirochete infection.
Clinical signs of Lyme disease in dogs are variable, with the majority of infections being subclinical, so this study primarily focused on spirochete reisolation from skin biopsy samples as the most accurate meas- ure of the vaccine's effectiveness. In this study, with a significant challenge at 366 days, 100% of the vaccinated dogs were protected from infection. These results were verified by negative spirochete reisolation from multiple biopsies performed on 3 sepa- rate occasions. In contrast, spirochetes were reisolated from 82% of the unvaccinated controls.
These results confirm protection against infection seen in several short-term duration of immunity studies.13–15 Most notably, Conlon and colleagues13 purpose-bred mixed breed dogs 10 to 12 weeks of age that were sequentially vaccinated with 2 doses of the same OspA vaccine. Dogs were challenged with I. scapularis ticks 3 weeks after the second vaccination, and this study demon- strated that the OspA vaccine blocked pro- liferation of B. burgdorferi in all vaccinated dogs (20/20). Control dogs seroconverted following challenge, and 2 of the dogs expe- rienced clinical signs associated with Lyme disease (2/10). Spirochetes were reisolated from all of the controls (10/10). Results from this short-term study demonstrated that 100% of vaccinates were protected against infection after challenge,13 results similar to those reported in the present study.
CONCLUSION
The recombinant OspA vaccine adminis- tered subcutaneously to puppies 9 weeks of age and older is shown to be both safe and efficacious. The vaccine induced an
immune response that protected vaccinates against spirochete infection and clinical signs for a full 12 months after vaccination.
ACKNOWLEDGEMENTS
We gratefully acknowledge the technical expertise of Gordon Gilreath, Brian Gantly, Mark King, and Dr. Andy Eschner. Funding for this study was provided by Merial Limited.
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