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12/26/12

Canine Lyme disease still raises debate on definitive diagnosis

Canine Borreliosis or Canine Lyme disease (CLD), is a vector borne disease caused by the spirochete, Borrelia burgdorferi sensu lato. Four subgroups of the genus Borrelia have been identified with Borrelia burgdorferi sensu stricto being the most common isolate in the United States that consistently produces clinical disease in the dog. Two additional Borrelia species, Borrelia afzelii and a Borrelia species (Florida isolate) have been isolated from dogs with neurologic manifestations, uveitis, lymphadenopathy and joint disease. Surface proteins of the borrelia species are virulence factors that allow spirochetes to attach to mammalian cells. The first documented case in a dog was published in 1984 from an endemic area in Connecticut. Since that clinical description, the incidence of the reported disease in dogs has substantially increased.


Location as a primary factor Geographically, CLD is more prevalent in the three major endemic geographic regions in the United States associated with infections in man. In the year 2000, approximately 18,000 cases of human infections of Lyme disease were reported based on data from the Centers for Disease Control. Approximately 95 percent of human cases were from 12 states in the Northeastern-Mid-Atlantic and upper Midwest regions. The Western (Northern California-Oregon) is another region within the United States with a high incidence of cases in man. Studies on the seroprevalence of CLD appear to show a significant spatial pattern that is correlated with the incidence of Lyme disease in humans and with the abundance of tick vectors.



Major arthropod vectors for CLD in the United States is the Ixodes ricinus complex of ticks (Photo 1). Ixodes scapularis is found predominately in the Northeastern-MidAtlantic and upper Midwesterern region, while Ixodes pacificus is the major species in the Western United States. Ixodes species have a two-year life cycle, with borrelia organisms being maintained in infected nymphs during the winter. Nymphs and adult stages of the tick are the most effective transmitters of the spirochete to mammalian hosts. Each life stage of the tick feeds on more than one host (white footed mouse, whitetail deer, woodrat and lizards) during different phases in their life cycle. Other arthropod vectors, including fleas, flies and mosquitoes are infected in nature, but the role in transmission of disease by these vectors is unknown at this time.

Transmission and manifestation Dogs are infected through deposition of the infecting organism from the tick into the skin during attachment. Infection rates increase directly to the duration of time the tick is attached ( 48 hours) and the infection rate in the stage of the tick present in the particular geographic region. After infection of the dog, local multiplication of the organism occurs predominately in skin, fascia and muscle tissues in proximity to the tick bite. Dissemination of the organism occurs to a number of different tissues of the body. Isolation of the organism by culture, demonstration of the organism by histopathology, and polymerase chain reaction (PCR) reveal the organism in peripheral blood, skin, joint and synovial tissues, central nervous tissue and urine for extended periods. Immunologic responses by the host to infection produces the clinical manifestations in the dog, and persistence of the organism in infected dogs, with or without treatment, can be detected for years.

The major clinical manifestations in dogs with natural and experimentally induced infection include fever, malaise, joint pain and effusion, and lameness. The inflammatory arthritis may be a mono- or polyarthritis with approximately half of dogs having multiple joints affected at the time of presentation (Photo 2, p. 26). The carpus, digits, shoulder, elbow, tarsus and stifle were the most frequently affected joints in reported series of dogs. Incubation periods for naturally infected dogs are unknown, but joint disease after experimentally induced infection is delayed for approximately two to five months. Episodes of arthritis and lameness in untreated dogs may be recurrent, with the first episode of arthritis in the limb closest to the tick bite.

Several other clinical syndromes have been described in naturally infected dogs. These syndromes have not been conclusively linked to infection with the borrelia organism, but there is a sufficient body of evidence to suspect borrelia infection as a potential cause of these syndromes.

A nephritic syndrome characterized by membranoproliferative glomerulonephropathy, cortical tubular necrosis and progressive renal failure was described in several dogs in three different publications. Borrelia organisms were present in renal tissue in two dogs by use of the indirect fluorescent antibody technique, and in one dog the organism was isolated from a urine sample. This dog had a high serum antibody titer to Borrelia burgdorferi as well. In another series of dogs with glomerulonephropathy, azotemia, proteinuria, and edema and effusions were frequent clinical and laboratory abnormalities. In that series, 18/49 dogs were seropositive to Borrelia burgdorferi. Six dogs had rare spirochetes observed in renal tissue on histopathologic examination. Whether or not one can associate borrelia infection and glomerulonephropathy is not universally accepted as a frequently identified clinical syndrome of CLD. Experimentally, isolation of the organism by culture and by use of PCR from renal tissues is an infrequent event. Uveitis, hepatosplenomegaly, neurologic signs (seizures), cardiac arrhythmias and myocarditis are other clinical signs described in a few dogs in a number of different publications.


Photo 3: A cytological preparation made from a joint tap from a dog with CLD showing a neutrophilic response. (Magnification X1000)

Diagnostic findings Clinical laboratory findings are non-specific for CLD. Abnormalities present on the hemogram, biochemical profiles and urinalysis may include an inflammatory leukogram, renal azotemia, hypoalbuminemia, proteinuria and an inflammatory urine sediment. Joint fluid changes include an increase in cell counts that consist primarily of a neutrophilic response (Photo 3). Organisms have been identified in peripheral blood, urine, joint and CSF fluids by use of dark field or phase contrast microscopy in natural or experimentally infected dogs but these techniques are not useful in clinical practice.



What constitutes a definitive diagnosis of CLD still generates a significant debate within the veterinary community because of the subjectivity of criteria used in defining the naturally occurring disease. A history of tick exposure or bites, compatible clinical signs (fever, lameness, joint effusion), a positive serologic test or rising titer for antibodies (IFA, ELISA, C6 ELISA), or characteristic serum proteins analysis (Western blot technique) to the borrelia organism, and response of the dog to appropriate antibacterial agents are the most useful clinical criteria.
Photo 4: A peripheral blood smear showing multiple Borrelia organisms from a dog with an acute episode of fever, lameness and associated acute renal failure. (Magnification X1250)

Serologic surveys reveal up to 50 percent of dogs may be positive for antibodies to borrelia in endemic areas. These serologic surveys have used the indirect fluorescence antibody and ELISA methods for detection of antibodies. Antibody titers (IgM and IgG) in infected dogs increased one to two months after infection and reach their highest concentration by three months post-infection. Antibodies to borrelia in natural and experimentally infected dogs persist for extended periods ( one to two years). Cross-reactivity of antibodies to borrelia detected with IFA and ELISA methods are reported to other bacterial agents and with certain inflammatory disease processes. Leptospirosis, other non-pathogenic spirochetes, or different borrelia agents may cause false-positive results on serologic tests. In addition, the lack of consistent standards for antigen preparation, laboratory technique and cut-off points lead to a divergent view about what a positive antibody test means. Immunoblot techniques for demonstrating certain proteins may be used to discriminate between natural infected and vaccinated dogs.

Serologic reactivity cannot be equated with a diagnosis of CLD but only suggests the dog was exposed to the infecting agent. Acute and convalescent antibody titers provide a better idea of potential active infection, while isolation of the organism is the most definitive procedure to document infection (Photo 4). This procedure is time-consuming and limited because the number of infecting agents is low in most tissues. Skin biopsies appear to be the best sample to culture from in suspected CLD cases. Barbour-Stoenner-Kelly medium is an acceptable transport solution for isolation studies.

Table 1

Recently, an identified region located within the variable surface domain (VlsE) of Borrelia burgdorferi, contain immunodominant conserved regions (IR 1-6) that can be detected serologically in infected dogs. Antibody responses occur to different IR region in man, non-human primates, dogs and mice. The IR6 region of VlsE is one region that responds vigorously to infection and can be serologically detected in dogs. Use of detection of this antibody response has subsequently been made into a commercially available ELISA test. This test is confounded by vaccination of dogs for CLD and antibody titers to this region decrease after antibiotic treatment. The commercial test uses a synthetic peptide (C6), based on the IR6 sequence of the variable domain of Borrelia burgdorferi as the antigen. When the dog's serum is mixed with the kit agents, a visible blue color denotes a positive test. When evaluated in experimental infections, the antibody response to IR6 was first detected at four to six weeks post- infection and persisted for approximately 70 weeks. Sensitivity of this test was 100 percent in dogs when compared to traditional ELISA and Western Blot tests, which have been considered the "gold standard" for detecting natural infection and vaccination status of dogs. Falae positives can ocur (3.6 percent) and should be confirmed with a Western blot test.

Treatment options Treatment of CLD involves use of several different antibiotics. Doxycycline and amoxicillin are frequently used in treatment of naturally infected dogs and have advantages of low cost and few adverse effects. Duration of treatment for naturally infected dogs with acute joint and lameness presentations should be for a period of 14 to 21 days. This recommendation for duration of therapy is not supported by treatment of dogs that were experimentally infected. Two different experimental studies have revealed that a 30-day course of therapy with amoxicillin, doxycylcine, ceftriaxone and azithromycin failed to eliminate the infection but did reduce the ability of investigators to isolate the organism from tissues (Table 1, p. 29). It is also interesting to note, in one reported series consisting of 58 dogs with a tentative diagnosis of CLD that were not administered antibiotics, limb and joint disease resolved within three weeks. This response was similar to 69 dogs with CLD in the same study that was treated. However, in most instances dogs treated with antibiotics usually respond rapidly. Oral preparations are ample for treatment of most dogs but intravenous preparations may be indicated in dogs sufficiently ill and unable to take medications by this route.

Taking preventive measures Prevention of CLD depends on reduction of exposure of the dog to the vector. Environmental control is difficult but reduction of areas where tick infestations are noted can be helpful in reducing exposure. A variety of different types of acaricids for dogs are available and include the permethrins, amiatraz, fipronil and methoprene combination, and imidacloprid and permethrin combination. Tick checks and tick removal conducted by owners on a daily basis can be helpful to reduce the time that ticks are attached and the potential to transmit the infection.


Suggested Reading

Vaccination for CLD is one method that can be used to reduce infection rates and potential clinical disease. This vaccination procedure should be considered a non-core vaccine but may be indicated in a proportion of dogs in endemic areas. Currently, two vaccines are available for use in dogs. A whole cell bacterin has been available in dogs since 1992 and a non-adjuvanted recombinant product (Outer surface protein A-OspA) was licensed in 1996. Both vaccines are recommended for use in naive dogs. Vaccination would be useful in preventing another infection. One study on use of the whole cell bacterin did show a reduction in the incidence of lameness in dogs vaccinated with this product compared to non-vaccinated controls dogs. A reduction in sero-positivity determined by use of the C6 ELISA test, was also noted in vaccinated versus nonvaccinated dogs, where the whole cell bacterin was used in an endemic area of CLD in Connecticut. Potential adverse effects from use of the whole cell bacterin is suggested by some veterinarians, but immediate side-effects related to vaccination were mild and occur in less than 2 percent of vaccinates. Long-term effects from repeated use of the whole cell bacterin are unknown at this time. A non-adjuvanted recombinant OspA product induces a response to OspA in vaccinated dogs and prevents the spirochete from becoming infective when the tick feeds on the host. Dogs were protected from infection by repeated challenge of infected ticks in several studies with the OspA vaccine.

Multiple tick-borne infections with other pathogens such as Bartonella, Ehrlichia, Babesia and rickettsial organisms are commonplace today. Dogs presented with appropriate clinical manifestations of CLD may also require additional diagnostic or serologic testing to evaluate the possible of these vector borne diseases.
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