Transmission dynamics of Borrelia turicatae from the arthropod vector

Abstract

Background: With the global distribution, morbidity, and mortality associated with tick and louse-borne relapsing fever spirochetes, it is important to understand the dynamics of vector colonization by the bacteria and transmission to the host. Tick-borne relapsing fever spirochetes are blood-borne pathogens transmitted through the saliva of soft ticks, yet little is known about the transmission capability of these pathogens during the relatively short bloodmeal. This study was therefore initiated to understand the transmission dynamics of the relapsing fever spirochete Borrelia turicatae from the vector Ornithodoros turicata, and the subsequent dissemination of the bacteria upon entry into murine blood.

Methodology/principal findings: To determine the minimum number of ticks required to transmit spirochetes, one to three infected O. turicata were allowed to feed to repletion on individual mice. Murine infection and dissemination of the spirochetes was evaluated by dark field microscopy of blood, quantitative PCR, and immunoblotting against B. turicatae protein lysates and a recombinant antigen, the Borrelia immunogenic protein A. Transmission frequencies were also determined by interrupting the bloodmeal 15 seconds after tick attachment. Scanning electron microscopy (SEM) was performed on infected salivary glands to detect spirochetes within acini lumen and excretory ducts. Furthermore, spirochete colonization and dissemination from the bite site was investigated by feeding infected O. turicata on the ears of mice, removing the attachment site after engorment, and evaluating murine infection.

Conclusion/significance: Our findings demonstrated that three ticks provided a sufficient infectious dose to infect nearly all animals, and B. turicatae was transmitted within seconds of tick attachment. Spirochetes were also detected in acini lumen of salivary glands by SEM. Upon host entry, B. turicatae did not require colonization of the bite site to establish murine infection. These results suggest that once B. turicatae colonizes the salivary glands the spirochetes are preadapted for rapid entry into the mammal.

Figure 1. Immunoblots from four mice demonstrating seroconversion in animals when spirochetes were detected by microscopy or qPCR after ticks fed to repletion (A), or were interrupted after attaching for 15 seconds (B).

Animals that were fed on by infected ticks yet spirochetes were undetectable in murine blood (C and D). B. turicatae protein lysates and rBipA were electrophoresed in lane 1 and 2, respectively. Serum samples were collected from animals four weeks after tick feeding. Molecular masses (kilodaltons) are indicated to the left of each immunoblot.

Figure 2. Evaluation of salivary gland colonization by B. turicatae.

IFA of salivary glands considered infected (A), in vitro cultivated spirochetes as a positive control (B), and uninfected tick salivary glands (C). These findings from a single tick are representative of the remaining infected ticks.

Figure 3. Cumulative frequency of infection detected in mice after transmission.

Three infected ticks were fed on mice (n = 22) and spirochetemia was detected by microscopy and qPCR. Successful transmission frequencies after full repletion (squares) and interrupted feeding (diamonds) are separated for comparison.

Figure 4. Spirochete densities in murine blood after allowing three infected ticks to engorge (black triangle), or interrupting the bloodmeal after 15 seconds (white triangle).

Each triangle represents spirochete densities in a given animal. Solid horizontal lines are the average number of spirochetes per ml of blood among the spirochetemic mice on a given day. The limit of detection is indicated by the horizontal dotted line.

Figure 5. SEM of infected in vitro grown B. turicatae and salivary glands from infected O. turicata (A–F).

Images of in vitro cultured spirochetes (A) were used as reference for spirochete size and shape. A dorsal view of a tick in which the cuticle and midgut were removed, where white ([ ]) indicate the location of the salivary glands (B). An intact salivary gland was removed and the white and black (→) indicates an individual acinus and excretory duct, respectively (C). A cluster of cryofractured acini (D) and a magnified single acinus (E). The lumen of an ancinus was analyzed for the presence of spirochetes (F), as represented by white (→). The black boxes represent progressively magnified (D–F). The scale in nm and µm are represented by black bars.