Scapularis Ticks Research Articles

Fungal fauna of Ixodes scapularis say and Rhipicephalus Sanguineus (Latreille) (Acari: Ixodida) with special reference to species-associated internal mycoflora

Body contents of the black-legged tick, Ixodes scapularis, and brown dog (or kennel) tick, Rhipicephalus sanguineus, were screened by fungal culturing for more virulent strains to use in biological control and to investigate the propensity of ticks as endosymbiotic hosts. A low incidence of internal fungi was observed in both ticks, averaging < 20% recovery. Noteworthy in R. sanguineus was identification of Scopulariopsis carbonaria Morton and Smith (previously unknown within ticks) among the few internal isolates as well as the common internal associate S. brevicaulis (Sacc.) Bainier. Ixodes scapularis ticks were similarly devoid of fungi except for recovery of Metarhizium anisopliae (Metsch.) that is likely present as a parasite. Compositions of external mycoflora were alike (13 Deuteromycetes and three Zygomycetes) and reflect fungi prevalent in soil, leaf litter and organic debris. Despite this rich external mycoflora, internal fungi appear to be restricted to Scopulariopsis spp. and harboring this fungus is linked to the large wax glands (‘sagittiform sensilla’) that are characteristic of metastriate, but not prostriate (Ixodes), ticks. However, low levels of fungi within R. sanguineus indicate that the prevalence of tick-borne fungi is not as widespread as previously thought. The fact that not all classified entomopathogenic fungi recovered from tick surfaces were found inside the tick suggests that successful parasitism may be limited to a few select fungi (e.g., Metarhizium spp.) in nature, thus affecting potential biological control applications.

Distribution of Borreliae among Ticks Collected from Eastern States

Lyme disease is the most commonly reported vector-borne disease in the United States and is transmitted by Borrelia burgdorferi-infected Ixodes species. The disease is typically characterized by an erythema migrans (EM) rash at the site of tick feeding. EM rashes have also been associated with feeding by Amblyomma americanum ticks despite evidence suggesting that they are incompetent vectors for Lyme disease. In 1996, a Borrelia organism only recently cultivated in the laboratory was described in A. americanum ticks and designated B. lonestari. This Borrelia is believed to be the etiologic agent of a novel Lyme-like disease, southern tick associated rash illness (STARI). This study examined ticks collected from eight eastern states to evaluate the epidemiology of B. lonestari, B. burgdorferi, and their tick hosts. Three hundred individual or small pool samples were evaluated from tick genera that included Amblyomma, Ixodes, and Dermacentor. DNA was extracted following tick homogenization and the polymerase chain reaction (PCR) was performed using primers derived from the flagellin gene that amplify sequences from both B. burgdorferi and B. lonestari. Species specific digoxigenin labeled probes were designed and used to differentiate between B. burgdorferi and B. lonestari. Borrelia DNA was detected in approximately 10% of the A. americanum and I. scapularis tick samples, but none was present in any of the Dermacentor samples tested. Positive samples were detected in ticks collected from Kentucky, Maryland, Massachusetts, New Jersey, New York, and Virginia. This is the first known report of B. lonestari from Massachusetts and New York and the first detection in I. scapularis. This suggests that B. lonestari and its putative association with STARI may be more widespread than previously thought.

Seroprevalence of antibodies against Borrelia burgdorferi and Anaplasma phagocytophilum in cats

To determine whether cats in the northeastern United States develop serum antibodies against antigens of Borrelia burgdorferi and Anaplasma phagocytophilum and whether coinfection with the 2 organisms occurs. Serum samples from 84 healthy cats and 9 cats with lameness, fever, anorexia, or fatigue. Serum antibodies against B. burgdorferi and A. phagocytophilum were measured with an ELISA incorporating a whole-cell preparation or purified recombinant antigens, by means of Western blot analysis, or indirect fluorescent antibody (IFA) staining. ELISA results indicated that 44 of 93 (47%) sera contained antibodies against > or = 3 B. burgdorferi antigens, whereas 43 (46%) were reactive to whole-cell B. burgdorferi. Serum reactivity to protein 35, VlsE, and outer surface proteins A and F was most common. Seropositivity to > or = 3 antigens occurred at the same rate (5/9) in the 9 ill cats as in the 84 healthy cats (46% [39/84]). Of 13 sera reactive to recombinant antigens, 9 were seropositive as measured by Western blot testing with whole-cell antigen. Seropositivity rates of 30% and 38% were detected for antibodies against A phagocytophilum via IFA and ELISA testing, respectively. Fifteen (16%) sera had antibodies against both pathogens. Cats living in areas infested by Ixodes scapularis ticks are exposed to B. burgdorferi and A. phagocytophilum and, in some instances, may be coinfected. Most cats appeared healthy. An ELISA incorporating specific recombinant antigens may be used adjunctively with Western blot and other assays to confirm B. burgdorferi and A. phagocytophilum infection in cats.

Prevention of Lyme borreliosis

Lyme borreliosis, the most common tick-borne disease in both North America and Europe, is acquired through the bite of certain tick species in the genus Ixodes. The number of Ixodes ticks in the environment can be reduced by relatively simple interventions such as removing leaf litter and brush, which increases exposure of the tick to sun and air and takes advantage of the tick's vulnerability to desiccation, or by application of acaricides to property. Deer elimination or exclusion, application of topical acaricides to mice or deer, and application of systemic acaricides to deer are more complex approaches. However, none of these methods for reducing tick numbers, nor any of the recommended personal prevention measures, such as reducing the amount of exposed skin, use of tick repellents on exposed skin or clothing, and frequent tick checks to remove attached ticks expeditiously, has been demonstrated to decrease significantly the incidence of Lyme borreliosis in humans. Only two strategies have been shown to do so. A recombinant outer surface protein A (OspA) vaccine was approximately 80% effective in clinical trials in the United States, and a single 200 mg dose of doxycycline given within 72 hours of an I. scapularis tick bite, was shown to be 87% effective. The OspA vaccine is no longer manufactured due to poor sales. Consequently, single-dose doxycycline prophylaxis is rapidly gaining acceptance in the United States. Limiting single-dose doxycycline to just the highest risk tick bites can be accomplished if the health care provider has learned to differentiate engorged from unengorged I. scapularis ticks. Limitations of single-dose doxycycline prophylaxis are that the majority of patients with Lyme borreliosis do not recall a tick bite, and that there is no evidence that other Ixodes transmitted infections, such as human granulocytic ehrlichiosis, would be prevented. A safe, effective, inexpensive and well-accepted vaccine would be welcome.

Presence ofBorrelia burgdorferi(Spirochaetales: Spirochaetaceae) in Southern Kettle Moraine State Forest, Wisconsin, and Characterization of Strain W97F51

Lyme disease, caused by Borrelia burgdorferi Johnson, Schmidt, Hyde, Steigerwalt & Brenner; babesiosis, caused by Babesia microti Franca; and human granulocytic ehrlichiosis, caused by Anaplasma phagocytophilum Bakken & Dumler have been reported in Wisconsin, mainly in the endemic areas of the northwestern part of the state. People exposed to blacklegged ticks, Ixodes scapularis Say, from this region can potentially contract one or all of these diseases concurrently. Within the past several years, there have been cases of Lyme disease reported from southeastern Wisconsin, an area that contains deer, mice, and similar vegetation found at regions with high endemicity. However, past white-tailed deer surveys suggested no existence of I. scapularis in southeastern Wisconsin. However, in 1996, we identified questing adult I. scapularis in the southernmost part of the Southern Kettle Moraine State Forest, Walworth County. To determine whether an established population of I. scapularis exists in this region, we performed a detailed survey of the abundance of host-seeking I. scapularis adults and the presence and abundance of subadults feeding on white-footed mice, Peromyscus leucopus Rafinesque. We also tested for possible infections of B. burgdorferi, Ba. microti, and A. phagocytophilum in ticks and B. burgdorferi harbored by mice. In 1997 and 1998, a total of 249 P. leucopus mice and 118 questing adult I. scapularis ticks, in addition to 157 larvae and seven nymphs feeding on mice, were collected and their locations were recorded from the Nordic trails of the Southern Kettle Moraine State Forest. Only one P. leucopus and its attached engorged I. scapularis nymph were infected with B. burgdorferi, whereas none of the engorged larvae attached to mice were infected. However, 4.2% of questing adult I. scapularis were infected with B. burgdorferi. The abundance of questing adult I. scapularis was 1.6 ticks per hour. The prevalence of subadult ticks on mice was 27%, with a mean intensity on infested mice of 2.0. I. scapularis adults were not infected with either Ba. microti or A. phagocytophilum. A unique strain of B. burgdorferi s.l. (W97F51) was discovered, showing 33 nucleotide substitutions and one codon insertion in a 567-bp fragment of the OspB gene, compared with Borrelia bissettii (strain Ca389). The sequences of ospA, ospB, ospC,fla, and rrs genes and the rrf-rrl intergenic spacer region were compared between W97F51 and other B. burgdorferi s.l. species. Although W97F51 was most genetically related to B. bissettii, the genetic identity of W97F51 was less than that of B. bissettii conspecifics. This study documents the existence of an established population of I. scapularis and the presence of B. burgdorferi with a novel strain in southeastern Wisconsin.

BptA (bbe16) is essential for the persistence of the Lyme disease spirochete, Borrelia burgdorferi, in its natural tick vector.

Borrelia burgdorferi (Bb), the agent of Lyme disease, is a zoonotic spirochetal bacterium that depends on arthropod (Ixodes ticks) and mammalian (rodent) hosts for its persistence in nature. The quest to identify borrelial genes responsible for Bb's parasitic dependence on these two diverse hosts has been hampered by limitations in the ability to genetically manipulate virulent strains of Bb. Despite this constraint, we report herein the inactivation and genetic complementation of a linear plasmid-25-encoded gene (bbe16) to assess its role in the virulence, pathogenesis, and survival of Bb during its natural life cycle. bbe16 was found to potentiate the virulence of Bb in the murine model of Lyme borreliosis and was essential for the persistence of Bb in Ixodes scapularis ticks. As such, we have renamed bbe16 a gene encoding borrelial persistence in ticks (bpt)A. Although protease accessibility experiments suggested that BptA as a putative lipoprotein is surface-exposed on the outer membrane of Bb, the molecular mechanism(s) by which BptA promotes Bb persistence within its tick vector remains to be elucidated. BptA also was shown to be highly conserved (>88% similarity and >74% identity at the deduced amino acid levels) in all Bb sensu lato strains tested, suggesting that BptA may be widely used by Lyme borreliosis spirochetes for persistence in nature. Given Bb's absolute dependence on and intimate association with its arthropod and mammalian hosts, BptA should be considered a virulence factor critical for Bb's overall parasitic strategy.

Identification of novel tick salivary gland proteins for vaccine development

Methods currently used to control Ixodes scapularis ticks rely principally on acaricidal applications which suffer from a number of limitations. Recently, host vaccination against ticks has been shown to be a promising alternative tick control method. In tick salivary glands, numerous genes are induced during the feeding process. Many of these newly expressed proteins are secreted in tick saliva and may play a role in modulating host immune responses and pathogen transmission. We have performed suppression subtraction hybridization to identify unique I. scapularis salary gland proteins specifically expressed during engorgement. We have cloned and sequenced ten unique salivary gland-associated cDNAs that are up-regulated during feeding. The protein products of these genes represent potential vaccine candidates for use in the control of ticks and to prevent transmission of tick-borne diseases.

Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis ticks collected in Northern New Jersey.

PCR analysis of Ixodes scapularis ticks collected in New Jersey identified infections with Borrelia burgdorferi (33.6%), Babesia microti (8.4%), Anaplasma phagocytophila (1.9%), and Bartonella spp. (34.5%). The I. scapularis tick is a potential pathogen vector that can cause coinfection and contribute to the variety of clinical responses noted in some tick-borne disease patients.

OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands

Outer surface protein C (OspC) is a differentially expressed major surface lipoprotein of Borrelia burgdorferi. ospC is swiftly upregulated when spirochetes leave the Ixodes scapularis tick gut, migrate to the salivary gland, and exit the arthropod vector. Here we show that OspC strongly binds to the tick salivary gland, suggesting a role for OspC in spirochete adherence to this tissue. In vivo studies using a murine model of Lyme borreliosis showed that while OspC F(ab)2 fragments did not influence either the viability of spirochetes or ospC gene expression, they did interfere with B. burgdorferi invasion of tick salivary glands. We then generated ospC knockout spirochetes in an infectious clone of B. burgdorferi and examined them within the vector. OspC-deficient or wild-type spirochetes persisted equally within the gut of unfed ticks and multiplied during the tick engorgement; however, unlike wild-type B. burgdorferi, the mutants were unable to invade salivary glands. Salivary gland colonization of OspC-deficient spirochetes was completely restored when this mutant was complemented in trans with a plasmid harboring the wild-type ospC gene. These studies conclusively demonstrate the importance of OspC in the invasion of tick salivary glands by B. burgdorferi, a critical step in the transmission of spirochetes from the arthropod vector to the mammalian host.

OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands.

Outer surface protein C (OspC) is a differentially expressed major surface lipoprotein of Borrelia burgdorferi. ospC is swiftly upregulated when spirochetes leave the Ixodes scapularis tick gut, migrate to the salivary gland, and exit the arthropod vector. Here we show that OspC strongly binds to the tick salivary gland, suggesting a role for OspC in spirochete adherence to this tissue. In vivo studies using a murine model of Lyme borreliosis showed that while OspC F(ab)(2) fragments did not influence either the viability of spirochetes or ospC gene expression, they did interfere with B. burgdorferi invasion of tick salivary glands. We then generated ospC knockout spirochetes in an infectious clone of B. burgdorferi and examined them within the vector. OspC-deficient or wild-type spirochetes persisted equally within the gut of unfed ticks and multiplied during the tick engorgement; however, unlike wild-type B. burgdorferi, the mutants were unable to invade salivary glands. Salivary gland colonization of OspC-deficient spirochetes was completely restored when this mutant was complemented in trans with a plasmid harboring the wild-type ospC gene. These studies conclusively demonstrate the importance of OspC in the invasion of tick salivary glands by B. burgdorferi, a critical step in the transmission of spirochetes from the arthropod vector to the mammalian host.

Identification of four genes expressed by feeding female Ixodes scapularis, including three with sequence similarity to previously recognized genes.

We created a cDNA library from feeding, female Ixodes scapularis ticks and screened the library with a subtracted probe to eliminate most genes common to feeding female and mating male I. scapularis ticks. Four unique genes were identified in this screen. One gene, Is 9, (represented by 16 cDNAs) was more highly expressed in female ticks. This gene encodes a putative glycine-rich protein, which matched a number of glycine-rich proteins including attachment cement proteins from Rhipicephalus appendiculatus. A second gene, Is 10 (represented by one cDNA) was also more highly expressed in female ticks, but did not match any other sequences in the GenBank database. The third gene, Is 11 (represented by one cDNA) was very similar to Drosophila sp. hsp68 and hsp70 genes and was expressed about equally in male and female ticks. The fourth gene, Is 12 (represented by two cDNAs) was also about equally expressed in male and female ticks, and was similar to a salivary gland gene from Ixodes ricinis. This gene also showed limited similarity to some cuticle genes from insects.

Inability of a variant strain of Anaplasma phagocytophilum to infect mice.

Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. Polymerase chain reaction and DNA sequencing were used to determine the presence and prevalence of Anaplasma phagocytophilum human agent (AP-ha) and a genetic variant not associated with human disease (AP-variant 1). The remaining ticks from each cohort were allowed to feed to repletion on either white-footed (Peromyscus leucopus) or DBA/2 (Mus musculus) mice. The engorged ticks and murine blood samples were evaluated for the presence of AP-ha and AP-variant 1. Although a high percentage of the infecting ticks harbored AP-variant 1, only AP-ha was amplified from the murine blood samples. Additional ticks were fed on immunocompromised SCID mice, and, again, only AP-ha was capable of establishing an infection, and only AP-ha could be detected by xenodiagnosis. These data suggest that AP-variant 1 cannot establish an infection in mice, and we propose that AP-variant 1 has an alternative natural reservoir, possibly white-tailed deer.

Geographic information systems and spatial analysis of adult Ixodes scapularis (Acari: Ixodidae) in the Middle Atlantic region of the U.S.A.

In the Middle Atlantic region of the U.S.A., the vector of Lyme disease, human granulocytic ehrlichiosis, babesiosis, and other human and veterinary pathogens is the black-legged tick, Ixodes scapularis Say. In 1997 and 1998, 663 adult I. scapularis ticks were collected from 320 transects spanning 66,400 km2 in five states of the Middle Atlantic region. Tick abundance patterns were clustered, with relatively high numbers along the coastal plain of the Chesapeake Bay, decreasing to the west and south. There were significant associations between tick abundance and land cover, distance to water, distance to forest edge, elevation, and soil type.

Transmission efficiency of the AP-variant 1 strain of Anaplasma phagocytophila.

Nymphal Ixodes scapularis ticks were collected from several sites in Rhode Island. DNA was extracted from a subset of these ticks, and PCR and DNA sequencing of the 16S rRNA gene were used to determine the ratio of Anaplasma phagocytophila-human agent (AP-ha) to a genetic variant not associated with human disease (AP-Variant 1). The remaining ticks were allowed to feed to repletion on either white-footed (Peromyscus leucopus) or DBA/2 (Mus musculus) mice. The engorged ticks, and blood samples drawn from each mouse at one-week intervals, were evaluated by PCR and DNA sequencing for the presence of AP-ha and Variant 1. Although a high percentage of the infecting ticks harbored AP-Variant 1, only AP-ha was amplified from the mouse blood samples. Because the A. phagocytophila variant did not establish an infection either in the natural reservoir of AP-ha, the white-footed mouse, or in a common research laboratory mouse (DBA/2), AP-Variant 1 may have an alternative natural reservoir, possibly the white-tailed deer.

Epidemiology and impact of coinfections acquired from Ixodes ticks.

Ixodes scapularis and other ticks in the Ixodes ricinus complex may transmit multiple pathogens, but research on coinfections has been limited. Coinfections occur with varying frequency in ticks, but single infections are more common than dual infections. The proportion of I. scapularis or I. ricinus ticks coinfected with both Borrelia burgdorferi sensu lato and Anaplasma phagocytophila is generally low, ranging from < 1% to 6% in six geographic areas. A higher prevalence of tick coinfection (26%) has been reported in Westchester County, New York. Genetic variants of the human disease-causing strain of A. phagocytophila are present in some tick populations, and they may affect the risk of coinfection or clinical illness. The proportion of Ixodes ticks coinfected with B. burgdorferi and Babesia microti has ranged from 2% in New Jersey to 19% on Nantucket Island, Massachusetts. In humans, cross-sectional seroprevalence studies have found markers of dual infection in 9-26% of patients with a tick-borne infection, but such studies often fail to distinguish simultaneous coinfection from sequential infections. Several studies have prospectively assessed the occurrence of acute coinfection. Among patients with a confirmed tick-borne infection, coinfection rates as high as 39% have been reported. The most commonly recognized coinfection in most of the eastern United States is Lyme borreliosis (LB) and babesiosis, accounting for approximately 80% of coinfections. LB and human granulocytic ehrlichiosis coinfections are less common, occurring in 3-15% of patients with a tick-borne infection in Connecticut or Wisconsin. Studies of clinical outcomes suggest that patients with acute Babesia coinfection have more severe symptoms and a longer duration of illness than patients with LB alone, but the risk of spirochete dissemination is similar. Coinfections can modify the immune response and alter the severity of arthritis in animal models. Future coinfection research should focus on long-term clinical outcomes, the role of genetic variants, immunologic effects, and the potential role of Bartonella species as tick-borne pathogens.

Borrelia burgdorferi-specific monoclonal antibodies derived from mice primed with Lyme disease spirochete-infected Ixodes scapularis ticks.

We have generated a panel of IgG monoclonal antibodies (MAbs) directed against Borrelia burgdorferi strain B31 antigens, using a method whereby mice were primed with organisms naturally inoculated by Ixodes scapularis nymphal ticks. Western blot analysis showed that these MAbs recognized several B. burgdorferi B31 antigens, including the complement inhibitor factor H-binding proteins ErpA/I/N and ErpC. Two other MAbs were specific for the RevA protein, and have enabled characterization of that previously unknown protein. The data presented here suggest that the production of MAbs from animals infected by tick-bite is a potentially useful tool for the identification of novel proteins synthesized by B. burgdorferi during mammalian infection.

Geographic uniformity of the Lyme disease spirochete (Borrelia burgdorferi) and its shared history with tick vector (Ixodes scapularis) in the Northeastern United States.

Over 80% of reported cases of Lyme disease in the United States occur in coastal regions of northeastern and mid-Atlantic states. The genetic structure of the Lyme disease spirochete (Borrelia burgdorferi) and its main tick vector (Ixodes scapularis) was studied concurrently and comparatively by sampling natural populations of I. scapularis ticks along the East Coast from 1996 to 1998. Borrelia is genetically highly diverse at the outer surface protein ospC. Since Borrelia is highly clonal, the ospC alleles can be used to define clones. A newly designed reverse line blotting (RLB) assay shows that up to 10 Borrelia clones can infect a single tick. The clone frequencies in Borrelia populations are the same across the Northeast. On the other hand, I. scapularis populations show strong regional divergence (among northeastern, mid-Atlantic, and southern states) as well as local differentiation. The high genetic diversity within Borrelia populations and the disparity in the genetic structure between Borrelia and its tick vector are likely consequences of strong balancing selection on local Borrelia clones. Demographically, both Borrelia and I. scapularis populations in the Northeast show the characteristics of a species that has recently expanded from a population bottleneck. Major geological and ecological events, such as the last glacial maximum (18,000 years ago) and the modern-day expansion of tick habitats, are likely causes of the observed "founder effects" for the two organisms in the Northeast. We therefore conclude that the genetic structure of B. burgdorferi has been intimately shaped by the natural history of its main vector, the northern lineage of I. scapularis ticks.

Prophylaxis with Single-Dose Doxycycline for the Prevention of Lyme Disease after an Ixodes scapularis Tick Bite

Prophylaxis with Single-Dose Doxycycline for the Prevention of Lyme Disease after an Ixodes scapularis Tick Bite

Single-dose doxycycline prevented Lyme disease after an Ixodes scapularis tick bite

Source Citation Nadelman RB, Nowakowski J, Fish D, et al., for the Tick Bite Study Group. Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick...

Mating, male Ixodes scapularis express several genes including those with sequence similarity to immunoglobulin-binding proteins and metalloproteases.

We created a cDNA library from mating, male Ixodes scapularis ticks and screened the library with a subtracted probe to eliminate genes common to feeding female and mating male I. scapularis ticks. A total of seven unique cDNAs were identified in this screen. One cDNA had sequence similarity to the IGBP-MC gene from Rhipicephalus appendiculatus, and another cDNA potentially encodes a protein with similarity to metalloproteases. RT-PCR, using RNA isolated from male and female I. scapularis ticks, confirmed that these genes are expressed in male, but not female ticks. The remaining five cDNAs did not match any sequences in the GenBank database.