Horizontal transmission of Rickettsia felis between cat fleas, Ctenocephalides felis

Abstract

Rickettsia felis is a rickettsial pathogen primarily associated with the cat flea, Ctenocephalides felis. Although laboratory studies have confirmed that R. felis is maintained by transstadial and transovarial transmission in C. felis, distinct mechanisms of horizontal transmission of R. felis among cat fleas are undefined. Based on the inefficient vertical transmission of R. felis by cat fleas and the detection of R. felis in a variety of haematophagous arthropods, we hypothesize that R. felis is horizontally transmitted between cat fleas. Towards testing this hypothesis, flea transmission of R. felis via a bloodmeal was assessed weekly for 4 weeks. Rhodamine B was used to distinguish uninfected recipient and R. felis-infected donor fleas in a rickettsial horizontal transmission bioassay, and quantitative real-time PCR assay was used to measure transmission frequency; immunofluorescence assay also confirmed transmission. Female fleas acquired R. felis infection more readily than male fleas after feeding on a R. felis-infected bloodmeal for 24 h (69.3% and 43.3%, respectively) and both Rickettsia-uninfected recipient male and female fleas became infected with R. felis after cofeeding with R. felis-infected donor fleas (3.3-40.0%). Distinct bioassays were developed to further determine that R. felis was transmitted from R. felis-infected to uninfected fleas during cofeeding and copulation. Vertical transmission of R. felis by infected fleas was not demonstrated in this study. The demonstration of horizontal transmission of R. felis between cat fleas has broad implications for the ecology of R. felis rickettsiosis.

Fig. 1

Diagrammatic representation of three rickettsial transmission bioassays. (A) The experimental design for rickettsial horizontal transmission bioassay consisted of four groups (I–IV); rickettsial transmission between fleas from Rickettsia felis-infected donor fleas, and Rhodamine B-labelled uninfected recipient or uninfected recipient fleas was assessed. (B) The experimental design for the co-feeding bioassay via the bloodmeal from R. felis-infected donor to uninfected recipient fleas. (C) Experimental design for the mating bioassay from R. felis-infected donor to uninfected recipient fleas.

Fig. 2

Fluorescence microscopy images of adult fleas after feeding on Rhodamine B (RB). A representative control flea, pictured in the top left column, fed on normal bovine blood. Other columns show RB-labelled fleas collected at days 1, 7, 14, 21 and 30 post-feeding on 0.1% Rhodamine B-labelled bloodmeal.

Fig. 3

Immunofluorescence detection of Rickettsia felis in donor and recipient fleas. Fleas were exposed to 5 × 10⁹ rickettsiae and infection visualized at day 28 post-cofeeding at (A) 10×, (B) 40× and (C) 100×. Recipient fleas cofed with R. felis-infected donor fleas also became infected as seen at (C) 40× and (D) 100×. The inset rectangle in (A) is a control uninfected flea fed on normal bovine blood.

Fig. 4

Overall mean Rickettsia felis infection density of male and female R. felis-infected donor and recipient fleas. Rickettsial densities in male and female R. felis-infected donor and recipient fleas in Groups I-III (combined) were assessed by qPCR. Donor female fleas had the greatest density of R. felis, compared with donor male fleas and both sexes of recipient fleas. Bars represent means (±SEM) of Rf17kDa/Cf18S ratio; means with different letters are significantly different.

Fig. 5

Mean Rickettsia felis infection density of R. felis-infected donor and recipient fleas at different time points. Rickettsial densities in R. felis-infected donor and recipient fleas collected at days 1, 7, 14, 21 and 28 post-cofeeding were assessed by qPCR. In a temporal fashion, donor fleas typically had greater densities, compared with recipient fleas. Bars represent means (±SEM) of Rf17kDa/Cf18S ratio; means with different letters are significantly different.