Infection of Ixodes scapularis ticks with Rickettsia monacensis expressing green fluorescent protein: A model system

Abstract

Ticks (Acari: Ixodidae) are ubiquitous hosts of rickettsiae (Rickettsiaceae: Rickettsia), obligate intracellular bacteria that occur as a continuum from nonpathogenic arthropod endosymbionts to virulent pathogens of both arthropod vectors and vertebrates. Visualization of rickettsiae in hosts has traditionally been limited to techniques utilizing fixed tissues. We report epifluorescence microscopy observations of unfixed tick tissues infected with a spotted fever group endosymbiont, Rickettsia monacensis, transformed to express green fluorescent protein (GFP). Fluorescent rickettsiae were readily visualized in tick tissues. In adult female, but not male, Ixodes scapularis infected by capillary feeding, R. monacensis disseminated from the gut and infected the salivary glands that are crucial to the role of ticks as vectors. The rickettsiae infected the respiratory tracheal system, a potential dissemination pathway and possible infection reservoir during tick molting. R. monacensis disseminated from the gut of capillary fed I. scapularis nymphs and was transstadially transmitted to adults. Larvae, infected by immersion, transstadially transmitted the rickettsiae to nymphs. Infected female I. scapularis did not transovarially transmit R. monacensis to progeny and the rickettsiae were not horizontally transmitted to a rabbit or hamsters. Survival of infected nymphal and adult I. scapularis did not differ from that of uninfected control ticks. R. monacensis did not disseminate from the gut of capillary fed adult female Amblyomma americanum (L.), or adult Dermacentor variabilis (Say) ticks of either sex. Infection of I. scapularis with R. monacensis expressing GFP provides a model system allowing visualization and study of live rickettsiae in unfixed tissues of an arthropod host.

Introduction

Ticks (Acari: Ixodida) are unique among blood feeding arthropods in that epithelial cells lining the midgut lumen phagocytose and digest the blood meal intracellularly. That process is presumably significant to the close association of ticks with obligate intracellular bacteria within the order Rickettsiales (Balashov, 1972, Munderloh and Kurtti, 1995). The genus Rickettsia includes spotted fever group (SFG) pathogens of vertebrate and tick hosts such as Rickettsia rickettsii and Rickettsia conorii (Niebylski et al., 1999, Santos et al., 2002), as well as nonpathogenic endosymbionts such as Rickettsia peacockii that are maintained in ticks by transovarial transmission (TOT) and are believed to prevent ovarian superinfection by pathogenic rickettsiae (Burgdorfer et al., 1981, Niebylski et al., 1997, Azad and Beard, 1998, Macaluso et al., 2002, de la Fuente et al., 2003). The physiological mechanisms that mediate rickettsial interactions with arthropod hosts are poorly understood. Rickettsiae are notoriously difficult research subjects due to their small size, slow growth, instability outside host cells and resistance to genetic manipulation. Moreover, the pioneering efforts of early 20th century researchers to establish tick host models of rickettsial infection dynamics (Harden, 1990) have been only minimally improved upon (Hayes and Burgdorfer, 1989). Recent advances in genetic manipulation of rickettsiae, coupled with development of improved host model systems, should lead to a better understanding of the physiology of rickettsial host range, tissue tropisms, transmission dynamics, pathogenesis versus symbiosis and superinfection resistance in arthropods.

Rickettsiologists have relied on stain-based light microscopy, transmission electron microscopy (TEM) and immunological detection methods to observe rickettsiae within host cells and tissues. Because those techniques detect fixed rickettsiae in fixed cells, they cannot fully define the interactions of rickettsiae and hosts, a task that will ultimately require live tissue techniques. Transformation of rickettsiae to express fluorescent markers will allow study of live rickettsiae within live host tissues, analogous to the advances achieved with such techniques in study of other arthropod-borne pathogens. For example, transformation with variants of the green fluorescent protein (GFP) reporter gene from the jellyfish, Aequorea victoria (Chalfie et al., 1994) allowed characterization of tissue distributions and infection dynamics of GFP-labeled arboviruses in mosquito hosts (Brault et al., 2004, Foy et al., 2004), of Leishmania protozoans in phlebotomine sand flies (Guevara et al., 2001) and of trypanosomes in tsetse flies (Bingle et al., 2001) and triatomine bugs (Guevara et al., 2005). Imaging of the movement of GFP-labeled malarial parasites in live mosquito hosts (Frischknecht et al., 2004, Vlachou et al., 2004) demonstrated the full power of this technology.

In the first reported studies of transgenic fluorescent bacteria in ticks, Ceraul et al. (2002) described evidence for encapsulation/nodulation of GFP-labeled Escherichia coli injected into Dermacentor variabilis ticks, while Matsuo et al. (2004) described clearance from the midgut of GFP-labeled E. coli ingested by Ornithodoros moubata ticks. However, E. coli, unlike rickettsiae, is not a normal constituent of the tick microflora and does not grow intracellularly. The recent development of electroporation techniques and transposon-based transformation vectors for rickettsiae (Rachek et al., 1998, Qin et al., 2004, Baldridge et al., 2005) has made their genetic manipulation a reality.

In this report, we describe the evaluation and selection of Ixodes scapularis from three North American tick species as a model host for Rickettsia monacensis transformed to express GFP (Baldridge et al., 2005). R. monacensis is a SFG rickettsial endosymbiont isolated from the European sheep tick, Ixodes ricinus (Simser et al., 2002). Phylogenetic analyses have shown that R. monacensis and related rickettsiae detected in I. ricinus populations by molecular means (Sreter-Lancz et al., 2005 and references therein) form a separate cluster within the SFG group distinct from its known pathogenic members (Ishikura et al., 2002, Ishikura et al., 2003; Kurtti et al., unpublished). I. scapularis is a member of the I. ricinus species complex (Keirans et al., 1999) and has been shown by phylogenetic analyses to be very closely related to I. ricinus (Fukunaga et al., 2000, Xu et al., 2003). The two ticks are morphologically and behaviorally similar and, along with I. pacificus and I. persulcatus, are the principal vectors of the Lyme Disease agent Borrelia burgdorferi and related Borrelia in the northern hemisphere (Keirans et al., 1999, Fukunaga et al., 2000).

We infected ticks by capillary feeding rickettsiae to adults and nymphs and by immersion of larvae in a rickettsial suspension. Fluorescent R. monacensis were readily visualized by epifluorescence microscopy (EFM) in dissected, unfixed tick tissues but could not be observed through the strongly auto-fluorescent tick cuticle. In adult female I. scapularis, but not in Amblyomma americanum or Dermacentor variabilis, R. monacensis disseminated from the gut and infected the salivary glands that mediate transmission to vertebrate hosts. R. monacensis was transstadially transmitted (TST) in I. scapularis and was maintained in apparently healthy adult ticks for up to 6 months. We did not obtain evidence for transovarial transmission (TOT), possibly due to superinfection resistance. R. monacensis infected the tick tracheal system that surrounds and penetrates tissues within the tick hemocoel. The tracheal system could potentially serve as a dissemination pathway and infection reservoir during the tick life cycle. Infection of I. scapularis with GFP-labeled R. monacensis provides a new model system for study of the interactions of a rickettsial endosymbiont with a tick host.