Transstadial and intrastadial experimental transmission of Ehrlichia canis by male Rhipicephalus sanguineus

Abstract

The acquisition and transmission of rickettsial pathogens by different tick developmental stages has important epidemiological implications. The purpose of this study was to determine if male Rhipicephalus sanguineus can experimentally acquire and transmit Ehrlichia canis in the absence of female ticks. Two trials were performed where nymphal and male R. sanguineus were simultaneously acquisition fed on the same infected donor hosts, and transstadially or intrastadially exposed male ticks were fed on separate pathogen-free dogs as a test for transmission. A single-step p30-based PCR assay was used to test canine and tick hosts for E. canis infections before and after tick feeding. E. canis was detected after either intrastadial or transstadial passage in male ticks, the organism remained detectable in both tick groups after transmission feeding, and both tick groups transmitted the rickettsia to susceptible dogs. Infection of dogs via tick feeding resulted in milder clinical signs and lower antibody titers than intravenous inoculation of carrier blood, but further investigation is needed to understand the mechanisms responsible for this observation. These results demonstrate that male R. sanguineus can take multiple feedings, and that they can both acquire and transmit E. canis in the absence of female ticks. This tick development stage could be important in transmission of E. canis, and perhaps related pathogens, between vertebrate hosts under natural and experimental conditions.

Fig. 1

Detection of E. canis in acquisition hosts. PCR assays were performed on buffy coat samples of dogs used to acquisition feed ticks as described in the text. Lanes containing the 200 bp target amplicon (arrowhead) were considered PCR-positive. Dogs ATK (panel A) and BAA (panel B) were inoculated with whole blood from an E. canis carrier on day 0, and used to acquisition feed ticks on days 21-31. Dog AXM (panel C) was not exposed to E. canis, and was used to feed cohorts of ticks fed on the infected dogs as a negative control. For each panel, template-free reactions served as negative controls (-), template DNA (1 ng) collected from E. canis-infected DH82 cells served as positive control (+) and 0-42 indicate days post-inoculation (panels A and B) or days post-tick attachment (panel C). The molecular size standard (m) is a 100 bp ladder.

Fig. 2

Detection of E. canis in transstadially exposed male R. sanguineus. R. sanguineus nymphs were fed to repletion on E. canis-infected dogs, allowed to molt into adults, and male ticks were assayed before and after transmission feeding. Panels A and B represent ticks acquisition fed on dogs ATK and BAA, respectively, which were assayed after acquisition (AF) and transmission (TF) feeding on dogs AHK and AIP, respectively. Panel C represents male R. sanguineus that molted from nymphs allowed to feed on the negative control, dog AXM. Lanes containing the 200 bp target amplicon were considered PCR-positive. For each panel, template-free reactions served as negative controls (-), template DNA (1 ng) collected from E. canis-infected DH82 cells served as positive control (+) and lanes labeled 1-10 represent individual ticks. The molecular size standard (m) is a 100 bp ladder.

Fig. 3

Transstadial transmission of E. canis to dogs by male R. sanguineus. PCR assays were performed on peripheral blood from dogs AHK (panel A) and AIP (panel B). Lanes containing the 200 bp target amplicon (arrowhead) were considered PCR-positive. For each panel, template-free reactions served as negative controls (-), template DNA (1 ng) collected from E. canis-infected DH82 cells served as positive control (+) and 0-42 indicate days post-tick attachment. The molecular size standard (m) is a 100 bp ladder.

Fig. 4

Detection of E. canis in intrastadially exposed male R. sanguineus. Panels A and B represent male R. sanguineus exposed to E. canis on dogs ATK and BAA, respectively, which were assayed after acquisition (AF) and transmission (TF) feeding on dogs AHG and AUF, respectively. Panel C represents male R. sanguineus allowed feed on the negative control, dog AXM. Samples resulting in a 200 bp amplicon (arrowhead) were considered PCR-positive. For each panel, template-free reactions served as negative controls (-), template DNA (1 ng) collected from E. canis-infected DH82 cells served as positive control (+), and lanes labeled 1-10 represent individual ticks. The molecular size standard (m) is a 100 bp ladder.

Fig. 5

Intrastadial transmission of E. canis to dogs by male R. sanguineus. PCR assays were performed on peripheral blood from dogs AHG (panel A) and AUF (panel B). Lanes containing the 200 bp target amplicon (arrowhead) were considered PCR-positive. For each panel, template-free reactions served as negative controls (-), template DNA (1 ng) collected from E. canis-infected DH82 cells served as positive control (+), and 0-42 indicate days post-tick attachment. The molecular size standard (m) is a 100 bp ladder.

Fig. 6

Clinical signs of ehrlichiosis among dogs infected with E. canis by different measures. Panels A-D represent body temperature, packed cell volumes, platelets and total leukocyte counts, respectively. For each panel, days -21 through 0 represent values observed prior to exposure to carrier blood or infected ticks. Gray lines represent average values for acquisition hosts inoculated with carrier blood, dark solid lines represent average values for hosts exposed to intrastadially infected male R. sanguineus, and dashed lines represent average values for hosts exposed to transstadially infected male R. sanguineus.