Bartonella species are gram-negative bacteria that infect erythrocytes, endothelial cells, and macrophages, often leading to persistent blood-borne infections. Of the 22 identified Bartonella species and subspecies, five are known to be transmitted by lice, fleas, or sandflies. Bartonella spp. have either been cultured from patients and reservoir hosts or detected by PCR analysis from a variety of arthropod vectors, including numerous tick species. Research was initiated to further elucidate the role ticks may play in the transmission of Bartonella spp.
In the first study, a molecular epidemiology survey was performed screening Amblyomma americanum, the Lone Star tick, North Carolina (n = 98) and Virginia (n = 466) for the presence of Bartonella DNA. Amplicons were obtained from two questing A. americanum from Virginia were most closely related to B. tamiae by DNA sequencing (74.48–85.22% similarity). Bartonella tamiae has been isolated from the blood of three patients from Thailand, but has never been identified within a vector or patient in the United States. All other ticks examined did not harbor detectable bacteria (prevalence in VA: 0.43%). Potential transmission of Bartonella spp. by A. americanum should be the focus of future experimental studies.
Next, a study was performed to determine whether Bartonella spp. can infect and replicate with an A. americanum, AAE12, cell line. We demonstrated successful infection of the AAE12 cell line by 7 different Bartonella isolates and 3 Candidatus Bartonella species by either electron or light microscopy. With the exception of B. bovis, infection of AAE12 cells with all other examined Bartonella species induced cytopathic effects characterized by heavy cellular vacuolization and eventually cell lysis. Interestingly, two B. henselae isolates appeared to form morulae-like inclusion bodies, collections of bacteria present within a clearly defined vacuole, which have previously only been identified within tick cells infected with Ehrlichia/Anaplasma species and Midichloria mitochondrii . Furthermore, using quantitative real time PCR (qPCR), we demonstrated significant amplification of the two B. henselae genotype I isolates in the A. americanum cell line over a 5 day period. Ultimately, tick-cell derived Bartonella antigens may prove useful for the development of more sensitive diagnostic reagents and may assist in the development of an effective vaccine to prevent the further spread of disease caused by these organisms. The remaining studies described within this dissertation were focused on further increasing our knowledge regarding the transmission of B. vinsonii subsp. berkhoffii , a recognized cause of endocarditis in dogs. Using an in vitro model system, we demonstrated the invasion of canine erythrocytes by a B. vinsonii subsp. berkhoffii isolate. Dog erythrocytes were infected with B. vinsonii subsp. berkhoffii and then treated with gentamicin at 12, 24, and 48 hour post-infection. There was a gradual increase in the number of intra-erythrocyctic bacteria recovered at each collection time point, with the largest recovery occurring 48 hours post-infection. These results suggest that canine erythrocytes may serve in the maintenance of B. vinsonii subsp. berkhoffii within an infected host.
Circumstantial evidence suggests that Rhipicephalus sanguineus , the Brown Dog tick, may be responsible for the transmission of B. vinsonii subsp. berkhoffii. Using capillary tube feeding, we attempted to infect R. sanguineus adult females with a solution containing a B. vinsonii subsp. berkhoffii genotype II isolate. Of the 40 females that passed the housekeeping PCR, 4 (10%) harbored detectable bacteria. Furthermore, one pool of males (9 pools total) fed and mated with females on a rabbit host also produced a positive PCR amplicon using Bartonella specific primers. Though Bartonella was not detected in examined eggs or larvae, our data suggests that B. vinsonii subsp. berkhoffii may be transmitted by R. sanguineus via co-feeding or through sexual transmission.