Imaging of Borrelia turicatae Producing the Green Fluorescent Protein Reveals Persistent Colonization of the Ornithodoros turicata Midgut and Salivary Glands from Nymphal Acquisition through Transmission

Abstract

Relapsing fever (RF) spirochetes colonize and are transmitted to mammals primarily by Ornithodoros ticks, and little is known regarding the pathogen's life cycle in the vector. To further understand vector colonization and transmission of RF spirochetes, Borrelia turicatae expressing a green fluorescent protein (GFP) marker (B. turicatae-gfp) was generated. The transformants were evaluated during the tick-mammal infectious cycle, from the third nymphal instar to adult stage. B. turicatae-gfp remained viable for at least 18 months in starved fourth-stage nymphal ticks, and the studies indicated that spirochete populations persistently colonized the tick midgut and salivary glands. Our generation of B. turicatae-gfp also revealed that within the salivary glands, spirochetes are localized in the ducts and lumen of acini, and after tick feeding, the tissues remained populated with spirochetes. The B. turicatae-gfp generated in this study is an important tool to further understand and define the mechanisms of vector colonization and transmission.IMPORTANCE In order to interrupt the infectious cycle of tick-borne relapsing fever spirochetes, it is important to enhance our understanding of vector colonization and transmission. Toward this, we generated a strain of Borrelia turicatae that constitutively produced the green fluorescent protein, and we evaluated fluorescing spirochetes during the entire infectious cycle. We determined that the midgut and salivary glands of Ornithodoros turicata ticks maintain the pathogens throughout the vector's life cycle and remain colonized with the spirochetes for at least 18 months. We also determined that the tick's salivary glands were not depleted after a transmission blood feeding. These findings set the framework to further understand the mechanisms of midgut and salivary gland colonization.

Division of the genus Borrelia into two genera (corresponding to Lyme disease and relapsing fever groups) reflects their genetic and phenotypic distinctiveness and will lead to a better understanding of these two groups of microbes (Margos et al. (2016) There is inadequate evidence to support the division of the genus Borrelia. Int. J. Syst. Evol. Microbiol. doi: 10.1099/ijsem.0.001717)

“…This is of medical and ecological importance, because it means transmission of the pathogen to the host can begin soon after feeding begins. These include species that are transmitted by either argasid, prostriate or metastriate ticks: B. duttonii in Ornithodoros moubata [44], B. anserina in Argas species [45], B. theileri in Rhipicephalus (Boophilus) microplus [46], B. crocidurae in Ornithodoros species [47], B. hermsii in Ornithodoros hermsi [48], B. turicatae in Ornithodoros turicata [49,50] and B. miyamotoi in Ixodes scapularis (personal communication, Linda Bockenstedt, Yale University). We note that this characteristic of the RF clade holds even for Borrelia species transmitted by hard ticks, which are slow-feeding in contrast to the rapid-feeding soft ticks, such as Ornithodoros and Argas species.…”

The relapsing fever spirochete Borrelia turicatae persists in the highly oxidative environment of its soft‐bodied tick vector

“…Previously, we have shown B. turicatae colonises and persists in both the salivary glands and midguts of O. turicata ticks (Boyle et al, ; Krishnavajhala et al, ). Our data indicate B. turicatae must withstand both oxidative and nitrosative environments while it resides in the midguts and salivary glands of O. turicata ticks.…”

“…We recently reported B. turicatae is localised within the acini lumen of salivary glands from unfed O. turicata nymphs (Krishnavajhala et al, ), which likely contributes to its ability to be rapidly transmitted from its tick vector to a naïve mammalian host. To further understand the microenvironment of the O. turicata acini lumen and how it may affect B. turicatae gene expression, physiology, and overall virulence, we assessed the salivary gland transcriptome of uninfected O. turicata nymphs by RNAseq.…”

“…During blood feeding on an infected mammal, B. turicatae enters the midgut of O. turicata , and within weeks, disseminates to various tick organs including the salivary glands, synganglion, and reproductive tissues (Davis, ). O. turicata goes through at least five nymphal stages prior to becoming adults, during which the spirochetes are transstadially maintained (Krishnavajhala et al, ). B. turicatae can also survive prolonged starvation periods of at least five years within O. turicata (Davis, ).…”

“…B. turicatae can also survive prolonged starvation periods of at least five years within O. turicata (Davis, ). Moreover, prior studies indicated that B. turicatae resides within the lumen of salivary gland acini, facilitating the pathogen's rapid transmission to naïve mammalian hosts (Boyle, Wilder, Lawrence, & Lopez, ; Krishnavajhala et al, ).…”