OVERVIEW A D EVOLUTIO ARY SUMMARY OF IXODES SPP. A D BORRELIA BURGDORFERI

MEREDITH GILMA MORROW

ABSTRACT The ecology of ticks and the effect of their interactions with the environment and host(s) are fundamental to the spatial and temporal variation in the risk of infection by tick-borne pathogens. What follows is a systematic review of the order Acari with a particular focus on Ixodes scapularis, the main vector of the Lyme disease spirochete bacterium, Borrelia burgdoferi (Bb) in the northeastern United States. I will cover the medical importance of this genus and the evolutionary history of ticks in general. Both traditional and molecular phylogenetic classifications will be discussed, however, the main focus of this paper will follow the traditional phylogenetic scheme. In addition, I will examine the biology and ecology of Ixodids vs. Argasids, and how it relates to Bb infection and transmission. Lastly, I will provide some specifics about current research being completed on Bb and information on the evolutionary history of Bb.

I TRODUCTIO Acari is an order of arachnids that consists of mites and ticks. More than 30,000 species have been described as of 2005, and it is estimated that there are half a million more still undescribed (Oliver a, 1996). General morphology of Acari consists of two segments- the prosoma (cephalothorax) and opisthosoma (abdomen) (Krantz, 1970). Mites and ticks may be distinguished from

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spiders by noting the placement of the mouthparts (ticks: anterior gnathosoma, spiders: cephalothorax), and portion of the body on which the legs are inserted (ticks: the podosoma, spiders: cephalothorax) (Krantz, 1970). Most adults have four pairs of legs, similar to other arachnids. However, larvae generally hatch with only three pairs of legs – the fourth set is usually acquired at the first molt (Krantz, 1970). Some Acari have less than three pairs of legs, like the gall mite Phyllocoptes variabilis, which only has two pairs of legs (Oliver b, 1989). Acari are extremely diverse and live in a variety of habitats, including freshwater and marine environments (Oliver b, 1989). Many are parasitic and affect both vertebrates and invertebrates. Most parasitic forms are external parasites, while the free living forms are mainly predaceous. Others are detritivores that help to break down forest litter or dead skin debris. In addition, others may be plant feeders and can cause considerable damage to agricultural crops (Oliver b, 1989). Acarine parasites of animals occur in all but one suborder (Oribatida), and many are of major importance to man and domestic animals. The scope of this paper will focus on parasitic Acari within the family Ixodidae and genus Ixodes, however, I will also discuss the relatedness between the three families of ticks. MEDICAL IMPORTA CE Viruses, rickettsias, bacteria, spirochetes, protozoans, and helminthes have all been isolated from various acari ectoparasites (Krantz, 1970). Many of these organisms cause virulent or debilitating diseases in man and animals throughout the world (Gubler, 1998). The best way to decrease your chances of infection once a tick has attached to you is to remove the potential vector as quickly as possible. The risk of infection increases

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between 24 to 72 hours after the tick attaches to the skin (Sood et al., 1997). It is important to note that one should not twist, crush, burn or smother the tick with any type of product since this will agitate the tick and cause it to salivate or regurgitate more infected fluids. A person should note any changes in their health and/or the bite location for the next three to four weeks (Lang, 1997). If redness or swelling develops, one should immediately see a physician. It is important to remember that the "common bulls-eye rash" that accompanies Lyme disease, only occurs 50% of the time (Piesman, 2002).

LYME DISEASE The varied symptoms of Lyme disease are caused by the Borrelia burgdorferi (Bb) spirochete bacterium which is characterized by a thin, corkscrew structure. The spirochetes are a small, cohesive group of bacteria currently divided into 9 genera, which can be readily distinguishable from other bacteria on the basis of a few important characteristics (Saier, 2000). First, spirochete cells are generally long, narrow, and helical in form. They also exhibit unusual prokaryotic characteristics such as linear chromosomes and a cytoskeleton (Saier, 2000). In addition, they are able to swim active in liquid media which allows them to respond to chemical and physical stimuli in their own environments. Bb is transferred to a host through the bite of an infected carrier, most commonly various species of ticks (including Ixodes scapularis in the northeastern United States). The major threat to humans is the tick nymph, which feeds and infects in late spring to summer (usually May to July). The nymph is tiny and hard to see when it is attached, therefore, it may never be noticed. The main reservoirs of Bb are the white-footed mouse

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and deer (Lang, 1997). However, studies completed at the University of North Florida suggest that anoles may serve as additional reservoirs in the Southeastern United States (Clark, 2004). Three hundred different genospecies of Bb have been found worldwide so far; with 100 located in the United States alone (Vanderhoof-Forschner, 1997). While some of these genospecies are quite similar, others are extremely heterogeneous (all of the strains are collectively called Bb sensu stricto). These differences drastically affect how transmission occurs and also the apparent virulence of the disease (Carrol et al., 2000). Therefore, treatment of patients depends on which particular strain they are infected with. However, at this time, there is still no definitive diagnostic test to confirm or deny a Lyme disease infection, let alone a test to inform healthcare providers with which strain(s) patients may be infected (Guttman et al., 1996). Because of this lack of technology, and the fact that Bb is an intracellular parasite (thus explaining why blood tests for antibodies to Bb may be negative even when bacteria are present inside of cells) a vast amount of Lyme disease cases are unreported. While some patients only exhibit the typical arthritic symptoms usually associated with Lyme disease, others may experience severe neurological and/or cardiological complications (Oliver a, 1990). These variations are thought to indicate the ways in which different genospecies of Bb can manifest themselves, how different immune systems react to invasion of Bb, as well as how long the infection has persisted (Oliver a, 1990).

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Bb (SPIROCHETE) EVOLUTIO ARY THEORIES There are two main hypotheses as to how modern spirochetes evolved: the Protospirochete Hypothesis and the Convergent Evolution hypothesis (Canale-Parola, 1977).

Proto-spirochete Hypothesis Presumably, the first primitive cells evolved over 3 billion years ago. During the Pre-Cambrian era, ancestral prokaryotic cells likely arose and survived through various selection pressures (Canale-Parola, 1977). An obligately anaerobic, free living protospirochete likely evolved through mutations leading to morphological differentiation. These mutations have persisted and were retained by the ancestral spirochetes because it offered them selective advantages (helical shape, motility, protective outer sheath etc.). Eventually, new habitats became available to bacteria after the appearance of animals on Earth. Spirochetes likely persisted and thrived on or within the body of these organisms, developing complex physiological interactions with host cells and with other resident microorganisms (Canale-Parola, 1977). It is possible that modern spirochetes evolved from ancient symbiotic or commensal primitive spirochetes that gained the ability to overcome the natural defenses of the host (Canale-Parola, 1977). Primitive animals were likely infected via direct contact with a host, through contact with the host's secretions, or through primitive arthropod vectors (Canale-Parola, 1977).

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Convergent Evolution Hypothesis The convergent evolution hypothesis states that over the course of evolution, different prokaryotes developed the characteristics of modern day spirochetes (CanaleParola, 1977). Therefore, the modern spirochete did not evolve from a common, single proto-spirochete, but instead is derived from prokaryotes that had already become diversified (both physiologically and ecologically). These prokaryotes acquired spirochete characteristics through independent, but convergent evolution (Canale-Parola, 1977). It is likely that selective advantages were gained by prokaryotes that attained spirochete characteristics (Canale-Parola, 1977). One such important advantage that at least some acquired was the development of motility (Canale-Parola, 1977). It has even been suggested that spirochetes may have been the precursors to eukaryotic flagella and cilia (Canale-Parola, 1977). In addition, the possession of an outer sheath may also confer selective advantage for spirochetes as it acts as a permeable barrier which protects the organism from pH extremes and host immune response (Canale-Parola, 1977).

Molecular Evolutionary Studies Comparisons of the 16S rRNA sequences demonstrate that the spirochetes represent a monophyletic phylum within the bacteria and diverged from other bacteria during early evolution (Saier, 2000; Paster and Dewhirst, 2000). The spirochetes are presently classified in the Class Spirochaetes in the order Spirochetales and are divided into three major phylogenetic families (Fig. 1 - Paster and Dewhirst, 2000). The family Spirochaetaceae is separated into 6 genera, and contains the genus Borrelia (Paster and Dewhirst, 2000).

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In 2008, Qiu et al. studied the DNA of Bb sensu stricto in both the United States and Europe. They found uniform high virulence clones in both continents which suggest a recent trans-oceanic migration to the United States, which may explain the recent spread of Lyme disease in North America (beginning in Lyme, Connecticut in 1975). In another study, Margos et al. researched the evolutionary history of Bb (from strains in both the United States and Europe) by looking at sequences of 8 "housekeeping genes" which tend to evolve slowly over time. The study's findings appear to show that Bb originated in Europe before the Ice Age, but that the species has been present in North America long enough to cause some adaptive selection and diversification between the strains found on both continents (Margos et al., 2008). They suggest that the large reemergence that took place in Lyme, Connecticut in the 1970s occurred after the geographic territory of the vector tick expanded, for example through the restoration of woodland habitat (Margos et al., 2008).

PHYLOGE ETIC RELATIO SHIPS (Suborder Parasitiformes) Ticks comprise the suborder Parasitiformes. The three families- Argasidae, Nuttalliellidae, and Ixodidae—are presently included in the superfamily Ixodoidea (Hoogstraal et al., 1983).

UTTALLIELLIDAE Very little is known about the tick family uttalliellidae due to a lack of specimens. Only one species, uttalliella namaqua, has been described (Oliver b, 1989; Hoogstaal et al., 1983). . namaqua is found in semiarid areas in Cape Province South Africa, and in

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higher rainfall areas of Tanzania. The external surface of . namaqua is similar to an argasid tick with some generalized ixodid-like characteristics (Oliver b, 1989). uttalliella namaqua can be distinguished from ixodid ticks and argasid ticks by noting the position of the spiracles, lack of setae, and strongly ridged integument (Hoogstraal et al., 1985). Only nymphs and females of . namaqua have been recorded and their life cycle is currently unknown (Keirans et al., 1976). In addition, the host preference of . namaqua is also uncertain (Oliver b, 1989).

ARGASIDAE A great deal is known about the other two tick families. The Argasidae (soft ticks) is smaller, containing approximately 170 species assigned to five subfamilies (Oliver a, 1996). Most soft ticks belong to two genera, Ornithodoros (~100 species) and Argas (~56 species). The general argasid life cycle is comprised of egg, larval, and several nymphal, male and female stages. Generally, nymphs and adults of most species of Argasidae feed rapidly on several hosts, while the immatures usually feed only once in each developmental stage (Oliver a, 1996). If the first feeding has been interrupted, then a single nymphal instar may feed twice before ecdysing to the next stage (Oliver a, 1996). Adults feed several times, each meal followed by production of a group of eggs in females and additional sperm in males. The number of nymphal instars varies (from 2 up to 8) among different species and even varies intraspecifically (Oliver a, 1996). Although the number of nymphal instars of a species is usually genetically determined, it may be altered slightly by nutritional and other factors.

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Argasids are, for the most part, nidicolous (nest dwellers), and their rapid feeding and inability to migrate quickly result in their remaining at sheltered sites that are usually attractive to a variety of vertebrates, including reptiles, birds, and mammals (Oliver a, 1996).

IXODIDAE The Ixodidae (hard ticks) is the dominant tick family, with regard to number of species and their medical and veterinary importance. Fifty species are arranged in two major groups: Prostriata and Metastriata (Oliver b, 1989; Black et al., 1997). These two groups consist of 5 subfamilies and 13 genera (Oliver b, 1989). Ixodids undergo four stages: eggs, larvae, nymphs, and adults. They have only one nymphal instar, in contrast to the several nymphal instars of argrasids (Oliver b, 1989). They also differ from argasids in that each stage requires several days or longer to fully engorge with blood (Oliver b, 1989). Females feed only once, produce one large egg mass, and then die. Most Ixodids require three different individual hosts, however, a few Ixodids no longer require multiple hosts and have evolved a two-host or one-host feeding behavior (Oliver b, 1989). In the two-host pattern, larvae attach to a host, and upon completion of feeding (not dropping from the host as occurs in three-host species), they ecdyse on the host and the resulting nymphs reattach and complete feeding. After feeding the nymph then drops from the host. After several days, it ecdyses and, as an adult, seeks a second host. Upon finding a second host, the adult feeds and the female drops from the host. Several days later, she deposits one large egg mass before dying. The one-host tick species complete the larval, nymphal, and adult periods on one individual animal by

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feeding and ecdysing during each stage before the engorged female leaves the host and subsequently deposits eggs. Most of the two- or one- host species are obligate in this behavior, but a few are flexible under certain conditions and can utilize one or two hosts depending upon the given hosts (Oliver b, 1989). Larval and nymphal ixodids feeding on warm-blooded animals usually require 3-7 and 4-8 days, respectively, while feeding on cold-blooded organisms requires a few days longer (Oliver b, 1989). Adult females parasitizing birds and mammals usually require 712 days, while those parasitizing reptiles take longer. The gorging behavior and long feeding periods of immatures and females might seem disadvantageous, making them easy targets for host grooming and predation, however, studies have shown that feeding is gradual throughout most of the period until the last day of attachment, when the gut rapidly fills with blood. Therefore, the feeding tick is most vulnerable for a relatively short period (Oliver b, 1989). While lack of comprehensive fossil evidence does not permit a phylogenetic evaluation of the Arachnida in general, indications are that the arachnids were already well represented on land by the mid-Palaeozoic era (Krantz, 1970). Exchange of carbon dioxide and oxygen in the Acari is accomplished in ways which are extremely diverse within the group as to rule out any theory of single line evolution of respiratory systems (Krantz, 1970). It has generally been accepted that there are three superorders within Acari: Opilioacariformes, Acariformes, and Parasitiformes. The Opilioacariformes consists of a single order and family (Opilioacaridae) with about 20 known species (Hoogstraal et al., 1983). The Acariformes contain over 300 families and over 30,000 described species.

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Two major lineages are recognized, the Sarcoptiformes (Oribatida and Astigmata) and Trombidiformes (Prostigmata) (Hoogstraal et al., 1983). The Parasitiformes consists of three orders: Ixodida, Holothyrida, and Mesostigmata. The Mesostigmata contains in excess of 65 families and 10,000 described species, the other two parasitiform orders each comprise three families (Hoogstraal et al., 1983). About 850 species of ticks are known, but only about 30 species of holothyrans are noted (Hoogstraal et al., 1983). The phylogenetic relationships of the three families of ticks, the Argasidae, Ixodidae and Nuttalliellidae, are still somewhat unresolved due to lack of specimens. The monospecific Nuttalliellidae has morphological features of both the Argasidae and Ixodidae, so its relationship to them phylogenetically is unclear (Oliver b, 1989). Although molecular analyses of the phylogenetic relationships of the Argasidae and Ixodidae have been done, these studies did not include the Nuttalliellidae because few individuals of . namaqua have ever been found, and they have not been available for molecular applications (Hoogstraal et al., 1983; Black, 1997). The evolution of ticks was originally believed to have involved a high degree of morphological adaptation to hosts (Hoogstraal and Aeschlimann, 1982). It has been hypothesized that high degrees of host adaptation could lead to restricted host range and cospeciation with specific host lineages, thus serving as a major mechanism in tick speciation. However, in 1996, Klompen et al. argued that many ticks spend a large proportion of their life cycle off the host and that habitat adaptation and ecological specificity should also be considered as potential mechanisms in tick evolution. In most respects, the phylogeny of argasids and ixodids, based on the 18S rDNA gene, resembles the morphologically based phylogeny. However, in a study utilizing the

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16S mitochondiral rDNA gene, Black et al. found five general differences from the traditional classification originally set forth by Hoogstraal:

"First, members of Amblyomminae were not monophyletic. Second, members of Haemaphysalinae arose within Amblyomminae. Third, Hyalomminae arose within Rhipicephalinae. Fourth, there was only weak support for the Ornithodorinae and branch lengths among members of Ornithodorinae were longer than among all other subfamilies. The fifth conclusion was that Argasidae is not monophyletic and that Argasinae is a sister group to the hard ticks."

In this paper I chose to follow the more traditional classification scheme, however, in Figure 2 you can see a comparison of phylogeny from both the traditional versus 16S mitochondrial rDNA analysis from Black et al. 1997.

BIOLOGY/ECOLOGY OF TICKS

For mating between ticks to occur there must be a mechanism responsible for bringing males and females together. There are at least three groups of pheromones known in ticks: 1. assembly pheromones, 2. aggregation and attachment pheromones, and 3. sex pheromones (Hamilton, 1992). Assembly pheromones serve to assemble all active life stages and often attract species even in different genera (Hamilton, 1992). Aggregation/attachment pheromones induce attachment to host sites where ticks are feeding (or have recently fed). Such pheromones are produced by feeding males (Hamilton, 1992). Attractant sex pheromones and contact

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sex pheromones are often both reported in ticks. These pheromones serve to attract males to feeding females over limited distances (Hamilton, 1992). Attractant sex pheromones have not been reported in argasids. Contact sex pheromones, however, occur in both argasids and ixodids and serve as mating stimuli (Hamilton, 1992). Reproductive strategies differ greatly between the ixodids and the argasids. Unfortunately, we know nothing about reproduction in Nuttalliellidae. No males have been found yet, therefore, uttalliella namaqua may possibly be parthenogenetic. Most female ticks require a blood meal in addition to mating in order to oviposit. However, some argasid species are able to oviposit without a blood meal as an adult (autogeny). The ability to produce one or more cycles of eggs without a blood meal as an adult is not found among ixodid species (Oliver b, 1989). Ornithodoros lahorensis is obligately autogenous for its first cycle of eggs, but must feed prior to subsequent ovipositions (Oliver b, 1989). The truly obligatory autogenous ticks are Otobius spp., Antricola spp., and othoaspis spp. (Oliver b, 1989). Mouthparts of the females of these species are poorly developed and nonfunctional. Argasids only mate off the host, while Ixodes commonly mate off and on the host. All ticks transfer spermatids via a spermatophore. Males usually do not attempt copulation until spermatids have been formed (Oliver b, 1989). Argasid males fully insert their hypostome, chelicerae, and palps into the genital opening of the female (Oliver b, 1989). In constrast, Ixodes males fully insert their hypostome and chelicerae, but the palps are not inserted and instead are rested out to the sides of the genital opening (Oliver b, 1989). Ixodid females have evolved a reproductive strategy utilizing a single gonotrophic cycle, in contrast to the multigonotrophic cycle of argasid females (Oliver b, 1989). Adult

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female ixodids feed only once and take in an enormous blood meal, in contrast to argasid females which feed repeatedly and take smaller meals (Oliver b, 1989). In addition, ixodid females do not usually become engorged unless they have been mated (Oliver b, 1989). Upon completion of engorgement, she drops from the host and seeks a protected location in which to oviposit. Attachment to the host by ixodids is accomplished by piercing of the skin with the chelicerae, and anchoring to the site by inserting the hypostome into the wound (Krantz, 1970). A cement-like substance is produced by the salivary glands of most ixodid genera during feeding. This material is deposited around the mouthparts and extends into the host tissue to secure the tick to its feeding site. Attraction and attachment responses of ticks are triggered, at least in part, by stimuli received through thin walled contact and olfactory sensory setae. Host finding and attachment are ensured by 1. orientation responses leading to a distribution in vegetation layers and favorable waiting posture, 2. direct response to an approaching host by taking up a “questing” posture, and 3. reactions associated with crawling to the host and choosing attachment sites (Krantz, 1970). Chance of contact with a host often is enhanced by the tick’s habit of climbing to the tops of grass blades where animals often pass (known as “questing”).

CURRE T RESEARCH TRE DS

RECE T Bb RESEARCH

In 1981, Borrelia burgdorferi was first discovered in ticks; since then much interest has been focused on the possible biological roles of certain outer surface proteins

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of Bb in the alternating life cycle that includes ticks and vertebrate hosts (Schwan, 2003). Two major proteins, OspA and OspC, are regulated by the spirochete during the several days when ticks feed. The decrease in OspA and the simultaneous up-regulation of OspC by the spirochetes when ticks are feeding, suggests that OspA aids in spirochete adhesion (in the tick) while OspC assists in the dispersal of spirochetes to the vertebrate host (Grimm et al., 2004; Carrol et al., 2003; Schwan, 2003; Fingerle et al., 2000; LYMErix, 1998; Revel et al., 2002; de Silva et al., 1996). Researchers have been working with mutant spirochetes that lack these proteins in order to provide evidence to these hypotheses. However, up until 2004, researchers have been unable to generate an OspA/OspC deficient mutant from a virulent strain of Bb. Yang et al. were the first to do this with OspA and proved that this outer surface protein was not required for Bb infection of mice. They also confirmed that OspA function was essential for Bb colonization and survival within the tick midgut (Yang et al., 2004). This finding has prompted more research into the biological roles of other outer surface proteins and the possible use of Osps for a new vaccine against Lyme disease.

OUTER SURFACE PROTEI A The first Osp identified (OspA) in culture-derived Bb was approximately 31 kDa (Schwan, 2003). The OspA monomer has an elongated fold composed of 21 anti-parallel beta-strands followed by a single short alpha helix (Kumaran, 2001). Indirect immunofluorescence with a monoclonal antibody that was produced against OspA, allowed researchers to visualize spirochetes from an infected tick for the first time. This specific antibody-protein binding became the main method for identifying Bb and also for analyzing tick tissues to look for spirochete infection (Schwan, 2003).

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Simpson et al. (1991) studied antibody responses in animals that were experimentally infected with Bb and found that they differed from animals infected with Bb from a tick bite (as reviewed in Schwan, 2003). Rodents infected with Bb from a tick bite rarely stimulate an antibody response to OspA, whereas those inoculated with cultured Bb do (Schwan, 2003). In 1995, for the first time there was evidence for spirochetes altering their phenotypes in vitro. Schwan et al. demonstrated that Bb altered its Osps in ticks during feeding and with temperature changes during in vitro growth. Spirochetes in unfed ticks had OspA (and lacked OspC) yet after the ticks fed, spirochetes stained positive for OspC (Schwan, 2003). These were the first results to demonstrate that Bb spirochetes changed phenotypically during tick feeding since OspA is down-regulated while ticks feed. Most spirochetes in the midgut of unfed ticks produce OspA, but after 3-4 days of feeding, only 30-40% of the spirochetes contain this protein (de Silva et al., 1996). The fact that there are a high number of OspA positive spirochetes in the midgut of unfed ticks, and only OspA negative spirochetes are likely to persist in mammals during infection, led to the hypothesis that OspA acts as a tick midgut adhesion molecule to prevent the spirochetes from being eliminated in tick feces (Yang et al., 2004; Schwan, 2003; Fingerle et al., 2000; Pal et al., 2000; LYMErix, 1998; de Silva et al., 1996). As mentioned earlier, in February 2004, Yang et al. lent support to prove this hypothesis by creating an OspA deficient mutant of an infectious strain of Bb (strain 297).

OUTER SURFACE PROTEI C One year after identifying OspA, researchers described a 22 kDa protein found in ticks and patients in Europe; this protein was eventually named OspC (Schwan, 2003).

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The OspC monomer is almost all helical, with four long helices plus a short fifth helix (Kumaran, 2001). Originally, researchers believed that OspC was found only in sprirochetes from Eurasia, however, many studies since then have shown that the OspC (gene or protein) is present in all North American strains of Bb (Schwan, 2003; Carrol et al., 2003; Fingerle et al., 2000).

In 2004, Grimm et al. created and analyzed a Bb mutant that lacked OspC. All of the six mice that were challenged with the mutant spirochete remained sero-negative; spirochetes were not acquired by feeding ticks, and spirochetes could not be cultured from mouse tissues. Their findings showed that Bb strictly required OspC in order to infect mice and therefore is necessary to spread infection and could possibly influence the virulence of disease (Grimm et al., 2004; Schwan, 2003).

Bb spirochetes are able to change gene expression during tick feeding possibly through environmental signals such as temperature and pH. Several genes, including OspC, appear to be regulated in vitro by pH (Carrol et al., 2003). These differences in pH in culture are reflective of the pathway that the spirochetes would experience during the feeding cycle from tick (pH 8.0) to mammal (pH 7.0). As the pH is lowered, OspC is produced while OspA is down-regulated. In addition, temperature has been shown to stimulate regulation of OspC. Changing the in vitro temperature from 24ºC to 37ºC stimulates spirochetes to produce OspC, yet changing the temperature back to 24ºC caused OspC production to cease (Schwan, 2003). This was another experiment that is reflective of the alterations that the spirochetes would experience during the feeding

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cycle. It seems, to survive in both hosts, spirochetes have evolved mechanisms for sensing the different host environments and responding accordingly.

Furthermore, in an experiment done by Piesman and Burgdorfer, spirochetes in the midgut of unfed ticks that were OspC negative were not infectious when inoculated into mice, however, OspC positive spirochetes in recently fed ticks were found to successfully transmit disease. In addition, the migration of Bb from the tick midgut to the salivary glands is repressed when infected ticks feed on OspC immunized mice (as reviewed in Schwan, 2003). This finding could possibly support a role for OspC (in certain strains) to assist spirochetes in dissemination from the midgut to infect the host. Since there are 300+ strains of Bb worldwide, some researchers are looking at how OspC is regulated in different strains. Carrol et al. (2003) developed and implemented a GFP reporter system in Bb that monitored gene regulation in response to pH and temperature in vitro. They concluded that strains B31 and N40 may differ in their mechanisms of OspC regulation, such that B31 clones down-regulated OspC at a pH of 8.0, while N40 did not display any change in the amount of OspC. Since distinct strains appear to differ in their regulation of Osps, further strains should be tested and compared to other North American strains if vaccine development (using Osps) is imminent.

OspC proteins are considered to be polymorphic; this variability also extends to genospecies collected from a single geographical area. Alleles of OspC collected from a single site (Shelter Island, New York) could be clustered into 19 major groups, based on DNA sequence homology (Wang et al., 1999). Of the 19 major groups only 4 were invasive, while the others are non-human pathogens or infect only the surrounding skin

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(Wang et al., 1999). This variabilty will obviously also have consequences in the development of OspC vaccines.

Two of the invasive strains from Shelter Island, NY (HB19 and B31) were studied by Kumaran et al. (2001). They compared the electrostatic potential of the two strains with similar models of other non-invasive strains. It turned out that there was a key difference between invasive and non-invasive strains (in terms of electrostatic potential). The surface potential is highly negative for OspCs from invasive strains, while noninvasive strains are slightly more positively charged (Kumaran et al., 2001). If OspC is indeed involved in influencing the virulence of infection, perhaps the negatively charged regions interact with positively charged host molecules, (such as human fibronectin) thus increasing infection rate due to electrostatic attraction (Kumaran et al., 2001). This is a possible explanation as to why invasive strains have the negatively charged OspC while the non-invasive strains lack the negative charge. However, definitive proof of this theory is still lacking.

Variability in the pattern of OspC and OspA synthesis has been reported for different strains of Bb (Schwan, 2003; Wang et al., 1999; Kumaran et al., 2001) and a variety of OspA and OspC phenotypes can exist after infected ticks have fed (Schwan, 2003; Yang et al., 2004).

Up until now, only OspA vaccines have been researched. In the coming years, the possibility of an OspC vaccine will continue to be explored. However, some believe that experimental OspA and OspC vaccines have limited utility since they are usually only effective against challenge by the single strain (used to create the vaccine) and not by

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heterologous strains (Kumaran et al., 2001; Wang et al., 1999). Therefore, researchers are also interested in developing a mixture of different antibodies for perhaps both OspA and OspC or more Osps in one vaccine so that it may be effective against multiple strains in different countries (Kumaran et al., 2001; Vanderhoof-Forschner, 1997).

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Figure 1 – Paster and Dewhirst, 2000

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Figure 2 – Black et al., 1997

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