All four hosts inoculated with blood from dog A72 developed severe acute CME, which was demonstrated by hematologic and clinical signs. Pyrexia (i.e., rectal temperature >39 °C) was observed in all four inoculated dogs by 10 – 14 dpi. Body temperatures reached maximum values of 39.8 – 41.0 °C at 14 – 18 dpi, before gradually decreasing back to the normal range prior to administration of treatment regimens. Platelet counts indicated severe thrombocytopenia in all dogs, typically reaching a nadir 14 – 25 dpi. E. canis infections were demonstrated by 200 bp amplicons consistent with the PCR assay. These dogs all tested PCR-negative prior to inoculation with carrier blood, and they all tested PCR-positive for E. canis by 18 dpi.
Results for dogs treated with doxycycline are represented in . For dog AKE, pyrexia was observed 10 – 20 dpi with a peak of 40.6 °C at 14 dpi. Precipitous drops in peripheral leukocytes and platelets were observed 7–14 dpi, from average baseline values of 10.1 × 103 WBC/µl and 444.2 × 103 platelets/µl, reaching nadirs of 1.93 × 103 platelets/µl at 18 dpi and 1.29 × 103 WBC/µl at 49 dpi. Hematocrit values dropped from the baseline average of 44.9% to 16.9% by 32 dpi. Although signs of CME were observed within 10 dpi, peripheral blood samples did not test PCR-positive for E. canis until 18 – 24 dpi. Rectal temperatures for this dog returned to the normal range by 21 dpi, and remained so through the remainder of the study; however, treatment was required for this dog at 32 dpi. Hematological values rose rapidly after treatment began, and were close to pre-infection levels at the end of the study. CBC values rose from 6.03 to 6.29 × 103 WBC/µl, 3.54 to 304 × 103 platelets/µl and hemotocrits of 16.9 to 34% during the 28 day course of doxycline treatment. These parameters eventually reached maximum values of 9.49 × 103 WBC/µl, 403 × 103 platelets/µl and 42.6% after completion of therapy. With the exception of a single sample from 42 dpi, all of the buffy coats tested PCR-negative after treatment began.
Dog AHL was treated with the same doxycycline regimen at 64 dpi. Pyrexia was first observed 14 – 19 dpi with a peak of 40.4 °C (18 dpi), and spikes > 39.0 °C at 28, 35, 45, 52, 57, 59 and 61 dpi. Precipitous drops in peripheral leukocytes and platelets were observed 7 to 14 dpi, dropping from average baseline values of 371.3 × 103 platelets/µl and 6.15 × 103 WBC/µl to nadirs of 17.8 × 103 platelets/µl and 1.43 × 103 WBC/µl at 14 dpi and 32 dpi, respectively. Hematocrit values dropped a baseline average of 41.9% to a nadir of 19.9% at 42 dpi. PCR-positive buffy coats were observed 14 – 28 dpi and again at 42 dpi, but E. canis was not detected again even though CBC values remained low through 63 dpi, just prior to treatment, and mild fevers repeatedly rose >39 °C. However, rectal temperature remained below 39 °C from initiation of treatment at 64 dpi through the remainder of the study, and CBC values rebounded shortly after treatment began from 2.85 × 103 to 4.27 × 103 WBC/µl, 71 × 103 to 273 × 103 platelets/µl and hematocrits of 27.9 to 38.6% during the 28 day course of doxycline treatment. These values eventually reached 5.01 × 103 WBC/µl, 291 × 103 platelets/µl and a hematocrit of 40.4% after therapy. There was no further PCR evidence of infection after 42 dpi.
Results for dogs subjected to the rifampin regimen are represented in . Both dogs in this group received treatment 64 – 71 dpi. Pyrexia was first observed in ABH at 11 – 19 dpi with a peak of 39.8 °C at 17 dpi, and a single day post-acute phase spike 104 above 39.0 °C at 65 dpi. Precipitous drops in peripheral leukocytes and platelets were observed 7 – 14 dpi, from mean baseline values of 8.2 × 103 WBC/µl and 363.5 × 103 platelets/µl to nadirs of 1.24 × 103 WBC/µl and 21.7 × 103 platelets/µl (each at 14 dpi). Hematocrit values dropped from the baseline average of 52.4% to a nadir of 30.5% at 12 dpi. ABH buffy coats initially tested PCR-positive 7 –10 dpi, and negative 14 dpi, followed by robust positive results 18 – 35 dpi and again 59 – 63 dpi. CBC’s rebounded shortly after initiation of therapy from 3.1 × 103 to 7.08 × 103 WBC/µl, 74.4 × 103 to 305 × 103 platelets/µl and hemotocrits of 36.6 to 39.1%, all during the 7 day course of rifampin treatment. Although leukocytes again dropped to 4.5 × 103 112 WBC/µl after therapy, the remaining CBC values eventually reached up to 305 × 103 platelets/µl and 50.7% hemotocrit by the end of the study. Body temperature again rose above 39 °C at 80 dpi, but there was no further PCR evidence of infection after 63 dpi.
For dog ACJ, pyrexia was first observed 13 – 19 dpi with a peak of 41 °C at 15 dpi, followed by a single spike >39.0 °C (21 dpi). CBC’s dropped from average baseline values of 8.2 × 103 WBC/µl and 416.8 × 103 platelets/µl to nadirs of 0.81 × 103 WBC/µl (49 dpi) and platelets/µl below detectable levels (25 dpi). Hematocrit values dropped from the baseline average of 42.5% to a nadir of 28.4% (28 dpi). ACJ buffy coats were PCR-positive 7 – 35 dpi and again at 63 dpi. CBC values rebounded shortly after treatment began from 2.2 × 103 to 6.32 × 103 WBC/µl, 69.4 × 103 to 237 × 103 platelets/µl and 34.1 to 36.7% hematocrits during the 7 day course of rifampin treatment, eventually reaching values up to 9.9 × 103 WBC/µl, 351 × 103 123 platelets/µl and 47.3% hematocrit after therapy. Body temperature remained below 39 °C after 21 dpi, with no further PCR evidence of infection after 63 dpi.
Previous work indicated that a 14-day doxycycline regimen did not prevent acquisition of E. canis
by R. sanguineus
nymphs fed on dogs that were infected through tick transmission.2
However, only R. sanguineus
larvae were commercially available for xenodiagnosis in the current study, so these larvae were placed on dogs AHL, ABH and ACJ prior to antibiotic treatment at 53 dpi, and post-treatment tick feeding began 78 dpi for ABH and ACJ, and 67 or 108 dpi for AKE and AHL, respectively. Cohorts of the ticks used to feed on infected dogs were fed on an uninfected dog (BCI) as a negative control. Nymphs fed as larvae on these dogs at 53 dpi were allowed to transmission feed to repletion on naïve dogs. Buffy coat samples from dogs used for transmission feeding of nymphs acquisition fed as larvae prior to antibiotic treatment did not test PCR-positive (), and these dogs did not display clinical signs of CME. Therefore ticks that fed on dogs after antibiotic treatment were not tested for E. canis
Attempted transmission of E. canis to dogs by R. sanguineus nymphs fed as larvae during post-acute phase CME
Resolution of both disease and infection with either doxycycline or rifampin suggested that rifampin might be used as an alternative treatment for CME. These results confirmed other studies where hosts were infected with E. canis
by needle-inoculation, but not investigations where dogs were infected via tick feeding.2
Although the same PCR assay was used in the previous investigation, continued efforts to enhance PCR assay sensitivity is warranted to help address this issue. Discrepancies between experimental designs and results of this and other studies together suggested that transmission mode or disease phase might influence efficacy of antibiotics to clear of E. canis
infections. Timing of antibiotic administration was among these discrepancies, but disease status might be more relevant with regard to treatment efficacy. For example, severe pancytopenia was still present in every case of the current study while only mild clinical signs were observed among dogs in an earlier report.2, 3
Route of host infection was another difference between the present and the former study; the present study involved needle-inoculation (iv.) with E. canis
carrier blood while the earlier study utilized male R. sanguineus
ticks to infect dogs. Interestingly, there is at least one report of persistence of natural E. canis
infections, most likely acquired through tick transmission, despite doxycycline therapy.6
Further work is needed to compare similar treatments of hosts during different phases of CME as well as hosts infected through tick feeding versus needle-inoculation.