Acquisition of Borrelia burgdorferi Infection by Larval Ixodes scapularis (Acari: Ixodidae) Associated With Engorgement Measures.

Measuring rates of acquisition of the Lyme disease pathogen, Borrelia burgdorferi sensu lato Johnson, Schmid, Hyde, Steigerwalt & Brenner, by the larval stage of Ixodes scapularis Say is a useful tool for xenodiagnoses of B. burgdorferi in vertebrate hosts. In the nymphal and adult stages of I. scapularis, the duration of attachment to hosts has been shown to predict both body engorgement during blood feeding and the timing of infection with B. burgdorferi. However, these relationships have not been established for the larval stage of I. scapularis. We sought to establish the relationship between body size during engorgement of larval I. scapularis placed on B. burgdorferi-infected, white-footed mice (Peromyscus leucopus Rafinesque) and the presence or absence of infection in larvae sampled from hosts over time. Body size, time, and their interaction were the best predictors of larval infection with B. burgdorferi. We found that infected larvae showed significantly greater engorgement than uninfected larvae as early as 24 h after placement on a host. These findings may suggest that infection with B. burgdorferi affects the larval feeding process. Alternatively, larvae that engorge more rapidly on hosts may acquire infections faster. Knowledge of these relationships can be applied to improve effective xenodiagnosis of B. burgdorferi in white-footed mice. Further, these findings shed light on vector-pathogen-host interactions during an understudied part of the Lyme disease transmission cycle.

Relative humidity and activity patterns of Ixodes scapularis (Acari: Ixodidae).

Laboratory studies have shown clear relationships between relative humidity (RH) and the activity and survival of Ixodes scapularis Say (blacklegged tick). However, field studies have produced conflicting results. We examined this relationship using weekly tick count totals and hourly RH observations at three field sites, stratified by latitude, within the state of Rhode Island. Records of nymphal tick abundance were compared with several RH-related variables (e.g., RH at time of sampling and mean weekly daytime RH). In total, 825 nymphs were sampled in 2009, a year of greater precipitation, with a weighted average leaf litter RH recorded at time of sampling of 85.22%. Alternatively, 649 nymphs were collected in 2010, a year of relatively low precipitation, and a weighted average RH recorded at time of sampling was 75.51%. Negative binomial regression analysis of tick count totals identified cumulative hours < 82% RH threshold as a significant factor observed in both years (2009: P = 0.0037; 2010: P < 0.0001). Mean weekly daytime RH did not significantly predict tick activity in either year. However, mean weekly daytime RH recorded with 1-wk lag before sample date was a significant variable (P = 0.0016) in 2010. These results suggest a lag effect between moisture availability and patterns of tick activity and abundance. Differences in the relative importance of each RH variable between years may have been due to abnormally wet summer conditions in 2009.

Identification of Borrelia burgdorferi ospC genotypes in canine tissue following tick infestation: implications for Lyme disease vaccine and diagnostic assay design.

In endemic regions, Lyme disease is a potential health threat to dogs. Canine Lyme disease manifests with arthritis-induced lameness, anorexia, fever, lethargy, lymphadenopathy and, in some cases, fatal glomerulonephritis. A recent study revealed that the regional mean for the percentage of seropositive dogs in the north-east of the USA is 11.6%. The outer surface protein C (OspC) of Lyme disease spirochetes is an important virulence factor required for the establishment of infection in mammals. It is a leading candidate in human and canine Lyme disease vaccine development efforts. Over 30 distinct ospC phyletic types have been defined. It has been hypothesized that ospC genotype may influence mammalian host range. In this study, Ixodes scapularis ticks collected from the field in Rhode Island were assessed for infection with B. burgdorferi. Ticks were fed on purpose bred beagles to repletion and infection of the dogs was assessed through serology and PCR. Tissue biopsies (n=2) were collected from each dog 49 days post-tick infestation (dpi) and the ospC genotype of the infecting strains determined by direct PCR of DNA extracted from tissue or by PCR after cultivation of spirochetes from biopsy samples. The dominant ospC types associated with B. burgdorferi canine infections differed from those associated with human infection, indicating a relationship between ospC sequence and preferred host range. Knowledge of the most common ospC genotypes associated specifically with infection of dogs will facilitate the rational design of OspC-based canine Lyme disease vaccines and diagnostic assays.

How much pilocarpine contaminates pilocarpine-induced tick saliva?

Pilocarpine is often applied or injected into ticks to induce salivation, and the resulting saliva used to test for various pharmacological, biochemical and immunological activities. To measure the amount of pilocarpine in pilocarpine-induced tick saliva, an HPLC-MS/MS method, based on capillary strong cation exchange chromatography online with an ion trap mass spectrometer, was used to measure pilocarpine in the pg to ng range. Results indicate large concentrations of pilocarpine in Ixodes scapularis Say and Amblyomma americanum (Linnaeus) (Acari: Ixodidae) saliva, ranging from 3 to 50 mm. Due to the known effects of pilocarpine on smooth muscle and immune cells, appropriate controls are proposed and discussed for proper interpretation of results using this saliva preparation.

A reverse transcriptase-polymerase chain reaction assay for detecting Highlands J virus.

Highlands J (HJ) virus is an arbovirus frequently recovered at high rates in mosquitoes collected in the eastern United States. HJ virus is primarily a veterinary pathogen causing disease in domestic birds including turkeys, chickens, and partridges. It has an enzootic cycle similar to eastern equine encephalitis (EEE) virus and is often used as an indicator species in EEE surveillance programs. Current immunologic techniques to identify HJ virus are often inefficient and can involve cross-reactivity of antibodies. Therefore, we developed a molecular-based assay by a reverse transcriptase (RT)-polymerase chain reaction (PCR) technique. Primers were constructed from conserved sequences of the E1 coding region from 19 strains of HJ virus. PCR amplifications from serial dilutions of HJ virus-infected Vero cell culture supernatants indicated that this assay could detect viral RNA at concentrations of 10 plaque-forming units per reaction. Extracted RNAs from western equine encephalitis, EEE, LaCrosse, and Jamestown Canyon viruses were not detected with this assay. RNA extracted directly from the brain tissue of a dead house sparrow and from a pool of Culiseta mosquitoes yielded a PCR product of the expected size. The RT-PCR technique developed was both sensitive and specific for detecting HJ virus from infected cell culture supernatants, bird brain tissues, and mosquitoes. This new assay will permit rapid and accurate diagnosis of HJ virus, both enhancing surveillance activities for EEE transmission risk and monitoring infections in domestic poultry and wild birds.

Assessing the association between the geographic distribution of deer ticks and seropositivity rates to various tick-transmitted disease organisms in dogs.

OBJECTIVE: To determine whether the geographic distribution of deer ticks (Ixodes scapularis) was associated with the distribution of dogs seropositive for various tick-transmitted disease organisms (ie, Borrelia burgdorferi, Rickettsia rickettsii, the human granulocytic ehrlichiosis [HGE] agent, Ehrlichia canis, and Bartonella vinsonii subsp berkhoffii). DESIGN: Serologic survey. SAMPLE POPULATION: Serum samples from 277 dogs in animal shelters and veterinary hospitals in Rhode Island. RESULTS: Overall, 143 (52%) dogs were seropositive for B burgdorferi, 59 (21.3%) were seropositive for R rickettsii, 40 (14.4%) were seropositive for the HGE agent, 8 (2.9%) were seropositive for E canis, and 6 (2.2%) were seropositive for B vinsonii. Regression analysis indicated that the natural logarithm of nymphal deer tick abundance was correlated with rate of seropositivity to the HGE agent and to B burgdorferi but not to rate of seropositivity to R rickettsii, E canis, or B vinsonii. Percentages of samples seropositive for B burgdorferi, R rickettsii, the HGE agent, and E canis were significantly higher for samples from the southwestern part of the state where ticks in general and deer ticks in particular are abundant than for samples from the northern and eastern portions of the state, where ticks are relatively rare. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that all 5 disease agents are in Rhode Island and pose a risk to dogs and humans. Knowledge concerning tick distributions may be useful in predicting the pattern of disease associated with particular tick species and may aid diagnostic, prevention, and control efforts.

Purification, cloning, and expression of a novel salivary anticomplement protein from the tick, Ixodes scapularis.

The alternative pathway of complement is an important defense against pathogens and in tick rejection reactions. The tick Ixodes scapularis is able to feed repeatedly on its natural host and has a salivary anticomplement activity that presumably facilitates feeding. In this study, we purified and then obtained the amino-terminal sequence of the I. scapularis salivary anticomplement (Isac). We found a full-length clone coding for Isac by random screening of a salivary gland cDNA library. Expressing Isac cDNA in COS cells reproduced the activity found in tick saliva, namely, inhibition of rabbit erythrocyte lysis by human serum in the presence of Mg(2+) and EGTA, inhibition of C3b binding to agarose in the presence of Mg(2+) and EGTA, and acceleration of factor Bb uncoupling from the C3 convertase generated by the alternative pathway. Recombinant Isac had no effect on the recalcification time of human platelet-poor plasma or in the classical complement pathway, indicating that it is a specific inhibitor similar to the regulators of complement activation of the alternative pathway such as factor H. Isac, however, has no similarity to any protein in the GenBank(TM) data base, indicating that it is a novel and relatively small (18.5 kDa) anticomplement molecule.

Complement-mediated killing of Borrelia burgdorferi by nonimmune sera from sika deer.

Various species of cervid deer are the preferred hosts for adult, black-legged ticks (Ixodes scapularis and Ixodes pacificus) in the United States. Although frequently exposed to the agent of Lyme disease (Borrelia burgdorferi), these animals, for the most part, are incompetent as transmission reservoirs. We examined the borreliacidal activity of normal and B. burgdorferi-immune sera from sika deer (Cervus nippon) maintained in a laboratory setting and compared it to that of similar sera from reservoir-competent mice and rabbits. All normal deer sera (NDS) tested killed > 90% of B. burgdorferi cells. In contrast, normal mouse and rabbit sera killed < or = 22% of the Borrelia. Anti-B. burgdorferi antibodies could not be detected in any normal sera by indirect fluorescent antibody assay (IFA). Sera collected from deer 6 wk after exposure to B. burgdorferi by tick feeding exhibited IFA titers of 1:256, whereas sera from mice and rabbits similarly exposed had titers of > 1:1,024. Heat treatment (56 C, 30 min) of NDS reduced borreliacidal activity, with < 20% of the B. burgdorferi cells killed, suggesting complement-mediated killing. The chelators EGTA and EDTA were used to block the classical or both the classical and alternative complement pathways, respectively. Addition of 10 mM EGTA to NDS had a negligible effect on borreliacidal activity, with > 90% of the cells killed. Addition of 10 mM EDTA reduced the killing to approximately 30%, whereas the addition of Mg2+ (10 mM) restored borreliacidal activity to NDS. The addition of zymosan A, an activator of the alternative pathway, increased the survival of B. burgdorferi cells to approximately 80% in NDS. These data suggest that the alternative complement activation pathway plays a major role in the borreliacidal activity of NDS. Additionally, 10 mM EGTA had almost no effect on the killing activity of B. burgdorferi-exposed deer sera, suggesting that the classical pathway is not involved in Borrelia killing, even in sera from B. burgdorferi-exposed deer.

Efficacy of a nonadjuvanted, outer surface protein A, recombinant vaccine in dogs after challenge by ticks naturally infected with Borrelia burgdorferi.

In a blinded, controlled study, thirty purpose-bred, Borrelia burgdorferi negative, mixed-breed dogs 10 to 12 weeks of age were randomly divided into three groups of ten animals each for the purpose of evaluating a recombinant nonadjuvanted B. burgdorferi OspA vaccine (Recombitek Lyme [Merial Limited]) for efficacy and safety. Two groups received two doses of two different lots ofa nonadjuvanted, OspA, recombinant vaccine; the third group served as nonvaccinated controls. All dogs were challenged 3 weeks after the second vaccination with blacklegged deer ticks (Ixodes scapularis) harvested from a B. burgdorferi endemic area in Rhode Island. Clinical signs, antibody titers by ELISA, Western blot assays, and isolation and polymerase chain reaction analyses of spirochetes from biopsy specimens were used to evaluate vaccine efficacy. Direct fluorescent antibody assay was used to evaluate the infection rate in the challenge ticks and in naïve ticks allowed to feed on study dogs after the dogs were infected (xenodiagnosis). Vaccinates responded with high levels of antibodies (determined by ELISA and measured by optical density [OD]), which did not rise after challenge. Vaccinates demonstrated no clinical signs, negative isolation of spirochetes on biopsy, only an OspA antibody pattern on Western blot assay, and negative isolation of spirochetes on biopsy, confirming that the vaccine blocked infection with B. burgdorferi in all vaccinated dogs (20/20). Control dogs demonstrated clinical signs (2/10), antibodies characteristic of infection with B. burgdoferi (10/10), isolation of spirochetes (10/10), and polymerase chain reaction (PCR) detection of spirochetes (9/10). The recombinant, nonadjuvanted B. burgdoferi vaccine protected 100% of vaccinates against infection after a severe challenge that infected 100% of control dogs. The OspA vaccine proved to be highly safe and effective in this study.

Polymerase chain reaction detection efficiency of the human granulocytic ehrlichiosis agent (Rickettsiaceae: Ehrlichieae) in ticks (Acari: Ixodidae) is dependent on the DNA extraction method.

Several methods of extracting DNA from ticks were examined to improve the efficiency of polymerase chain reaction (PCR) detection of the human granulocytic ehrlichiosis (HGE) agent. DNA was extracted from laboratory-reared uninfected and HGE-infected ticks using 3 separate methods. In one treatment, unfed nymphs and engorged larvae of Ixodes scapularis Say, either individually or in pools of 3, were homogenized in 40 microliters of 1x PCR buffer and boiled for 30 min. A 2nd group of ticks was extracted using the QiaAmp Tissue kit, a silica column separation method. A 3rd group was extracted with DNA-STAT, a guanidinium thiocynate method. Five microliters of each extract was used for PCR amplification. Pathogen-free tick DNA samples did not amplify a product. Laboratory-infected ticks extracted either with the QiaAmp kit or those homogenized and boiled in PCR buffer amplified product in 37.5% and 87.5% of the samples, respectively. Infected ticks extracted with DNA STAT-60 amplified a product in 100% of samples. No differences were observed in detection efficiency between ticks tested singly or in pools.

Ixodes scapularis: salivary kininase activity is a metallo dipeptidyl carboxypeptidase.

Saliva and salivary gland homogenates of Ixodes scapularis contain a dipeptidyl carboxypeptidase activity that accounts for the previously described salivary kininase activity of this tick. Reversed phase HPLC and laser desorption mass spectrography of the reaction products identified bradykinin fragment 1-7 and 1-5 as being produced subsequent to incubation of purified salivary kininase with bradykinin. The activity was inhibited by captopril and EDTA and was activated by cobalt and manganese, a behavior similar to that displayed by angiotensin-converting enzymes of vertebrate and invertebrate origins.

Nested PCR assay for detection of granulocytic ehrlichiae.

A sensitive and specific nested PCR assay was developed for the detection of granulocytic ehrlichiae. The assay amplifies the 16S rRNA gene and was used to examine acute-phase EDTA-blood and serum samples obtained from seven humans with clinical presentations compatible with human granulocytic ehrlichiosis. Five of the seven suspected cases were positive by the PCR assay using DNA extracted from whole blood as the template, compared with a serologic assay that identified only one positive sample. The PCR assay using DNA extracted from the corresponding serum samples as the template identified three positive samples. The sensitivity of the assay on human samples was examined, and the limit of detection was shown to be fewer than 2 copies of the 16S rRNA gene. The application of the assay to nonhuman samples demonstrated products amplified from template DNA extracted from Ixodes scapularis ticks collected in Rhode Island and from EDTA-blood specimens obtained from white-tailed deer in Maryland. All PCR products were sequenced and identified as specific to granulocytic ehrlichiae. A putative variant granulocytic ehrlichia 16S rRNA gene sequence was detected among products amplified from both the ticks and the deer blood specimens.

Molecular cloning and sequencing of three granulocytic Ehrlichia genes encoding high-molecular-weight immunoreactive proteins.

Granulocytic Ehrlichia was isolated from canine blood obtained from animals challenged with field-collected Ixodes scapularis and propagated in HL60 cells. PCR primers specific for the 16S ribosomal DNA (rDNA) of the Ehrlichia genogroup comprising E. equi, E. phagocytophila, and the agent of human granulocytic ehrlichiosis (HGE) amplified DNA from extracts of these cells. Sequence analysis of this amplified DNA revealed that it is identical to the 16S rDNA sequence of the HGE agent. A genomic library was constructed with DNA from granulocytic Ehrlichia and screened with pooled sera from tick-challenged, granulocytic Ehrlichia-infected dogs. Several clones were isolated and sequenced. Three complete genes encoding proteins with apparent molecular masses of 100, 130, and 160 kDa were found. The recombinant proteins reacted with convalescent-phase sera from dogs and human patients recovering from HGE. This approach will be useful for identifying candidate diagnostic and vaccine antigens for granulocytic ehrlichiosis and aid in the classification of genogroup members.

Duration of immunity to reinfection with tick-transmitted Borrelia burgdorferi in naturally infected mice.

The ability of naturally infected and cured mice to resist reinfection with tick-transmitted Borrelia burgdorferi was tested over a 1-year period. All of the mice were resistant to reinfection when they were challenged at 1.5 months after cure. The majority of animals were resistant to reinfection for up to 10.5 months after cure, but this resistance was lost at 1 year after cure. Both protected and unprotected animals showed a diverse array of antibodies on Western immunoblots. Protection was not associated with the killing of spirochetes in ticks, and naturally infected mice produced no antibodies to outer surface protein A (OSP A). The titers to whole Borrelia sonicate and OSP C, however, remained high throughout the 1-year study period. The levels of borreliacidal antibodies were highest in the 1.5 month-after-cure group. Natural immunity to reinfection with B. burgdorferi is limited in time, is complex, and may involve both humoral and cellular components.

Serologic and molecular detection of granulocytic ehrlichiosis in Rhode Island.

A new indirect fluorescent-antibody (IFA) assay with antigen produced in vitro in the human promyelocytic leukemia cell line HL60 was used to identify the first recognized case of human granulocytic ehrlichiosis in Rhode Island. This IFA assay was used to detect granulocytic ehrlichiae in white-footed mice and in a dog inhabiting the area surrounding the patient's residence. Host-seeking Ixodes scapularis ticks found in the same habitat also were infected. I. scapularis ticks collected from other locations were fed on dogs and New Zealand White rabbits to assess the competency of these species as hosts of granulocytotropic Ehrlichia. Tick-induced infections of dogs were confirmed by serologic testing, tissue culture isolation, and PCR amplification, whereas several rabbits seroconverted but were PCR and culture negative. PCR amplification of the 16S rRNA gene and DNA sequencing of the PCR products or culture isolation was used to confirm granulocytic Ehrlichia infections in humans, dogs, white-footed mice, and ticks.

Entomologic index for human risk of Lyme disease.

An entomologic index based on density estimates of Lyme disease spirochete-infected nymphal deer ticks (lxodes scapularis) was developed to assess human risk of Lyme disease. The authors used a standardized protocol to determine tick density and infection in numerous forested sites in six Rhode Island towns. An entomologic risk index calculated for each town was compared with the number of human Lyme disease cases reported to the Rhode Island State Health Department for the same year. A strong positive relation between entomologic risk index and the Lyme disease case rate for each town suggested that the entomologic index was predictive of Lyme disease risk.

Experimental Babesia microti infection in golden hamsters: immunoglobulin G response and recovery from severe hemolytic anemia.

We described the parasitemia, hematologic changes, and immunity developed by golden hamsters during 8 wk of infection with Babesia microti following experimental inoculation. All 8 hamsters used in this study were readily infected. Animals attained peak parasitemias asynchronously but within a 2-wk period. Most of the animals reached their peak parasitemia by 4 wk postinoculation, attaining a mean +/- SD of 21.9 +/- 9.4% infected erythrocytes (range = 20-35%). Red blood cell count, packed cell volume, and hemoglobin level were used to monitor the course of the hemolytic anemia experienced by infected hamsters. All 3 measures corresponded inversely to the parasitemia; significant hematologic changes (P = 0.0001) were observed during the 8 wk of monitoring. Although all hamsters suffered from severe hemolytic anemia, they also recovered within the same period. Golden hamsters developed a detectable anti-B. microti IgG response by 2 wk postinoculation. Individual animals typically attained peak antibody levels (> or = 1:8, 192) 1 wk after the peak parasitemia. Hamsters retained a high IgG titer (> or = 1:4,096), although parasitemias fell dramatically, fluctuating thereafter at low levels (< 5%).

Methods for evaluating Lyme disease risks using geographic information systems and geospatial analysis.

Lyme disease is a tick-transmitted borreliosis of humans and domestic animals emerging as one of the most significant threats to public health in north temperate regions of the world. However, despite a myriad of studies into symptomology, causes, and treatment of the disease, few researchers have addressed the spatial aspects of Lyme disease transmission. Using statewide data collected in Rhode Island (United States) as a test case, we demonstrated that exposure to deer ticks and the risk of contracting Lyme disease occurs mostly in the peridomestic environment. A Geographic Information System model was developed indicating a strong association among Lyme disease in humans, the degree of nymphal blacklegged tick, Ixodes scapularis Say, abundance in the environment, and prevalence of Borrelia burgdorferi infection in ticks. In contrast, occurrence of plant communities suitable for sustaining I. scapularis populations (forests) was not predictive of Lyme disease risk. Instead, we observed a highly significant spatial trend for decreasing number of ticks and incident cases of Lyme disease with increasing latitude. Geostatistics were employed for modeling spatial autocorrelation of tick densities. These findings were combined to create a model that predicts Lyme disease transmission risk, thereby demonstrating the utility of incorporating geospatial modeling techniques in studying the epidemiology of Lyme disease.

Entomological correlates of Babesia microti prevalence in an area where Ixodes scapularis (Acari:Ixodidae) is endemic.

Zoonotic prevalence of Babesia microti Franca piroplasms infecting white-footed mice, Peromyscus leucopus Rafinesque, was determined at 34 sites in Rhode Island where nymphal blacklegged tick, Ixodes scapularis Say, densities ranged from low to hyperabundant (1.7-525.3 nymphs per hour of flagging). Babesia was only detected at sites where tick abundance was moderate to high (> 20 nymphs per hour of flagging) and appeared to exhibit a clumped distribution. Where B. microti was detected, the mean number of nymphal ticks collected per hour of flagging was 229.2 compared with a mean of 40.1 at sites where Babesia was not detected. By combining the spatial occurrence of Babesia with a tick density database in a geographic information system, it may be possible to predict the pattern of zoonotic and human infection with B. microti.