An unprecedented degree of concurrent infection with multiple tick-borne pathogens was documented by serologic and molecular methods in this Walker Hound kennel. Approximately 40% of the 27 dogs had serologic evidence of infection with organisms from four genera, and an additional 52% had serologic evidence of infection with organisms from a combination of three genera. Seroprevalence studies in stray or hospitalized dogs from North Carolina have estimated exposure rates at 2.5% for E. canis
, 1.2% for E. equi
), 5% for R. rickettsii
), 3.6% for B. vinsonii
), and 3.8% for B. canis
). Almost half of the dogs had evidence of infection with four or more species or genera by PCR.
The relationship between the detection of E. canis
DNA and clinical illness indicates that infection with this organism, although not found to be a risk factor for death in this study, likely contributed to illness and may have had a contributory effect on death as an outcome. The detection of E. canis
DNA was also associated with the detection of B. canis
and E. platys
DNA, all of which may share R. sanguineus
as a tick vector, presumptively in the case of E. platys
). Signs of illness associated with E. canis
infection, as observed in the study dogs, are consistent with signs reported with E. canis
, E. platys
, or B. canis
. The natural or experimental infection of dogs with any of these organisms causes a wide range of clinical and pathological abnormalities, ranging in severity from asymptomatic infection to death (7
). Experimentally, concurrent infection with E. canis
and B. canis
can result in the potentiation of disease manifestations, which may account for some of the variability in clinical presentation attributed to these organisms, particularly when coinfections are overlooked (18
The detection of E. equi
DNA in three dogs and E. chaffeensis
DNA in nine dogs provides additional molecular evidence that E. equi
, the presumptive cause of HGE, and E. chaffeensis
, the cause of human monocytic ehrlichiosis, may be found in dogs in North Carolina (9
). The DNA sequence obtained from the blood of dog 8 was 100% identical to the typical HGE strain (12
). Similarly, the DNA sequence for the E. chaffeensis
amplicon derived from dog 6 was identical to the sequence derived from a human isolate from Arkansas (17
In this study, seroconversion to R. rickettsii antigen did not correlate with reported disease manifestations or death. Because this observation is inconsistent with the expected disease manifestations associated with clinical and experimental studies of canine Rocky Mountain spotted fever, we were suspicious that antibody reactivity was not related to exposure to R. rickettsii. However, Rickettsia species DNA was detected in the blood samples of most of the seroconverting dogs. Because our primers amplify spotted fever or typhus group rickettsiae, the PCR results were not specific for R. rickettsii. Ongoing studies in our laboratory indicate that these dogs may have been infected with an as yet unidentified rickettsial organism. Persistent detection of rickettsial DNA, as well as the relatively small number of uninfected dogs at either sample date, was not expected, due to our failure to find D. variabilis (American dog tick), the recognized vector for R. rickettsii in the southeastern United States. Infection with R. rickettsii causes acute disease manifestations followed by rapid immunologic clearance of the organisms. Collectively, this information provides evidence that another rickettsia was responsible for the seroreactivity to R. rickettsii detected in these dogs.
The association between seroreactivity to B. canis antigens and residence in the dirt lot would seem to support the hypothesis that dogs kept in the open lot had a greater likelihood of infestation with R. sanguineus. Although R. sanguineus is a vector for both E. canis and B. canis, residence in the dirt lot did not correlate with PCR detection of E. canis, potentially due to prior treatment with doxycycline. Interventions to control ticks and fleas occurred before and during the study period. As a consequence, we were unable to quantify the tick numbers found on the two groups of dogs.
By sampling on two occasions, approximately 3 months apart, our PCR data provide limited insights regarding the persistence of infection, the potential for acute infection or reexposure, and the influence of therapeutic interventions accompanied by enhanced efforts to eliminate tick exposure. The chronicity of documented infection with E. canis
in this group of dogs, along with the rate of newly acquired infections, is not unexpected given the lack of protective immunity following E. canis
) and the persistent R. sanguineus
infestation. As documented, we would expect a similar pattern of infection with B. canis
, reflecting the common tick vector. Although apparently new infections with B. canis
occurred during the study period, treatment with imidocarb diproprionate appeared to clear the B. canis
infection or, alternatively, decreased the number of organisms below the level of detection of our PCR assay. Imidocarb diproprionate also appeared efficacious for the treatment of E. chaffeensis
and E. ewingii
infections. As doxycycline did not appear to eliminate E. chaffeensis
in naturally infected dogs (9
), imidocarb diproprionate may offer an effective form of therapy. Either imidocarb diproprionate appears to be less effective in eliminating E. canis
infection or the dogs were rapidly reinfected.
Infection with E. platys
appeared to be acquired during the study period, supporting that R. sanguineus
may be a vector for this organism, as previously hypothesized (43
). The cyclic nature of ehrlichiemia associated with this organism may, however, also have been responsible for the appearance and disappearance of E. platys
DNA. The absence of newly acquired infection with E. chaffeensis
and E. ewingii
in these dogs indicates that there was not a persistent vector for transmitting infection. Our inability to find A. americanum
during follow-up visits to the kennel supports this assumption, particularly for E. chaffeensis
and possibly for E. ewingii
. As E. equi
results in an acute self-limiting infection, recent exposure to a vector-competent tick was expected. However, we were not able to find any I. scapularis
(black-legged tick) ticks, a documented vector for E. equi
, during our visits.
The only significant associations with death in this population of dogs included living in the dirt and brush lot and male gender. Because only males were kept in the dirt lot and only females were kept in the kennel, we were unable to differentiate between the effect of gender and that of location within the kennel. Males and females were exposed to identical training regimens, parasite control measures, and food rations, with their only ambient difference being type of housing. The statistical association between residence in the dirt lot with pale gums, decreased hematocrit values, and seroreactivity to babesia antigens suggests either an effect of chronic babesia infection (anemia frequently accompanies acute B. canis
infection) or the possibility of an interaction with other individual or combined, documented or undocumented organisms. As infections with Babesia
, and Bartonella
species are reported to induce immunosuppression, secondary opportunistic infections may have played a role in the disease manifestations reported in this kennel (27
). Conditions in the dirt lot were more conducive to the transmission of intestinal parasites and other urine- or fecally transmitted organisms, since the fenced kennel had a cement floor which was hosed off approximately daily. Although not expected to be a primary cause of death in adult dogs, fecally transmitted organisms may have contributed to death in dogs coinfected with multiple blood-borne pathogens. However, this would seem unlikely due to the historical use of antihelminthics and the infrequency of gastrointestinal manifestations.
Although tick collection efforts associated with this study were limited, only A. americanum and R. sanguineus ticks were found on the dogs and in the environment. Tick collection results were likely influenced by extensive tick control efforts undertaken by the owners of the kennel during the course of the study to try to halt the progression of illness in existing dogs. The only A. americanum tick collected during the study was from an early visit. It is unlikely that an A. americanum infestation was present at the site of the kennel since so few were collected. It is therefore unlikely that dogs were continually infected with organisms transmitted by this tick. Presumably, exposure to A. americanum and I. scapularis ticks occurred during exercise periods in the spring and summer months, and while deer hunting in the fall and winter months. The continued presence of R. sanguineus ticks, both on the dogs and in the kennel environment, indicates that dogs could have been continually reinfected with organisms transmitted by this tick throughout the study period.
Considering the severity of tick-borne infection in this kennel, our results indicate that these dogs posed a minimal risk of contributing to human infection, particularly with E. chaffeensis
. Although some of the people with dog contact had antibodies to tick-borne pathogens, most had antibodies only to B. henselae
, an organism for which dogs have not been implicated as a reservoir. In clinical reports, ticks have been implicated in the transmission of Bartonella
). The species of bartonella causing infection in these dogs was not determined, as current PCR techniques in our laboratory did not allow for the species characterization of the bartonella amplicons derived from EDTA-blood samples. Due to substantial cross-reactivity among Bartonella
species, serologic differentiation was not attempted. The association of B. henselae
infection with people who spent fewer hours outside may be related to increased exposure to cats and cat fleas on the premises. Although flea bites were reported frequently by the respondents, we did not attempt to collect fleas from inside the house and did not collect any specimens from the cats living around the neighborhood. The two patients with antibodies to E. chaffeensis
or R. rickettsii
antigens did not participate in hunting activities but did have regular contact with at least one dog. Although they complained of ongoing symptoms, neither had hematologic or biochemical abnormalities detected, supporting the hypothesis of prior exposure rather than active infection.
In summary, this study illustrates that kennel dogs with heavy tick exposure can be simultaneously infected with multiple tick-borne pathogens of potential clinical importance to both veterinary and human medicine. Dogs seroreactive to E. canis antigen were shown to be simultaneously infected with multiple Ehrlichia species when blood samples were assessed by PCR amplification techniques. Public and private health providers, particularly veterinarians and physicians, should be aware of the increased potential for vector-borne illnesses in both dogs and humans, especially when large numbers of dogs with extensive tick exposure are maintained for recreational purposes.