The major histocompatibility complex-restricted response of recombinant inbred strains of mice to natural tick transmission of Borrelia burgdorferi.

The causative agent of Lyme disease, Borrelia burgdorferi, is transmitted by ticks of the Ixodes ricinus complex. In this study, we report the antibody response of recombinant inbred strains of mice of the H-2, b, d, and k haplotypes, infected with B. burgdorferi as a result of exposure to infected I. dammini. The patterns of antibody response assayed by Western blot analysis indicate significant major histocompatibility complex (MHC) restriction to bacterial antigens within the first 2 mo of infection in mice. Other bacterial antigens induce a significant response across the MHC haplotypes tested when assayed on the same bacterial strain used to transmit the infection, but do not crossreact with the same proteins derived from heterologous strains of B. burgdorferi. No response to outer surface protein A was detected at any time during the 60-d period we analyzed this infection. A third group of bacterial antigens appear to generate a MHC-nonrestricted response, and this lack of restriction is maintained when assaying the crossreactivity of the response with other strains of B. burgdorferi. These proteins may provide more accurate diagnostic probes than those currently in use. Finally, there appears to be a significant difference in the expression of most bacterial antigens when the spirochete is cultured for many passages since the same strain of bacterium isolated from low-passage and high-passage preparations exhibit different banding patterns in Western blots when assayed with the same sera.

Canine Babesia new to North America.

A domestic dog residing in New England suffered a fatal febrile illness caused by a Babesia infection. The morphology of these intraerythrocytic protozoa and the range of hosts that could be infected experimentally suggested that the parasite was B. gibsoni. Although this tick-bourne disease is enzootic in wild and domestic Canidae in Africa and Asia, it appears to be new to the Americas.

Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species.

The hard tick Ixodes ricinus (Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been named Midichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks for Midichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening for Midichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related to M. mitochondrii. Samples of the tick species Rhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny of Midichloria symbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

Kinetics of Borrelia burgdorferi infection in larvae of refractory and competent tick vectors.

The acquisition of Borrelia burgdorferi by the larvae of competent and refractory ixodid ticks was assessed by quantitative polymerase chain reaction (PCR). Larvae were fed on infected mice, and the spirochete loads were determined during feeding and up to 93 d postfeeding. Amblyomma americanum (L.) was refractory to B. burgdorferi infection, with almost no detection of spirochete DNA during or postfeeding. In contrast, Ixodes scapularis Say supported high loads of spirochetes (10(3)-10(4) per larva). In Dermacentor variabilis (Say), B. burgdorferi uptake was reduced, with an average of 16 spirochetes per larvae acquired after 4 d of feeding, representing 1/195 of the counts in I. scapularis. However, during the first day postfeeding, the spirochete growth rate in D. variabilis reached 0.076 generations per hour, 7.7 times greater than the highest growth rate detected in I. scapularis. D. variabilis supported intense spirochete growth up to the fourth day postinfection, when the counts increased to an average of 282 spirochetes per larvae or 1/8.5 of the I. scapularis counts 4 d postfeeding. The kinetics of spirochete growth was unstable in D. variabilis compared with I. scapularis, and transmission of B. burgdorferi by D. variabilis could not be demonstrated. A cofeeding experiment indicated that I. scapularis feeding increased A. americanum spirochete uptake. These collective results indicate suboptimal conditions for B. burgdorferi uptake and colonization within A. americanum or the presence of anti-Borrelia factor(s) in this nonpermissive tick species.

Spatiotemporal patterns of host-seeking Ixodes scapularis nymphs (Acari: Ixodidae) in the United States.

The risk of Lyme disease for humans in the eastern United States is dependent on the density of host-seeking Ixodes scapularis Say nymphal stage ticks infected with Borrelia burgdorferi. Although many local and regional studies have estimated Lyme disease risk using these parameters, this is the first large-scale study using a standardized methodology. Density of host-seeking I. scapularis nymphs was measured by drag sampling of closed canopy deciduous forest habitats in 95 locations spaced among 2 degrees quadrants covering the entire United States east of the 100th meridian. Sampling was done in five standardized transects at each site and repeated three to six times during the summer of 2004. The total number of adults and nymphs of the seven tick species collected was 17,972, with 1,405 nymphal I. scapularis collected in 31 of the 95 sites. Peak global spatial autocorrelation values were found at the smallest lag distance (300 km) and decreased significantly after 1,000 km. Local auto-correlation statistics identified two significant high-density clusters around endemic areas in the northeast and upper Midwest and a low-density cluster in sites south of the 39th parallel, where only 21 nymphs were collected. Peak nymphal host-seeking density occurred earlier in the southern than in the most northern sites. Spatiotemporal density patterns will be combined with Borrelia prevalence data as part of a 4-yr survey to generate a nationwide spatial risk model for I. scapularis-borne Borrelia, which will improve targeting of disease prevention efforts.

Three multiplex assays for detection of Borrelia burgdorferi sensu lato and Borrelia miyamotoi sensu lato in field-collected Ixodes nymphs in North America.

Two hundred fifty New Jersey field-collected Ixodes scapularis Say ticks and 17 Colorado Ixodes spinipalpis Hadwen & Nuttall ticks were tested using three separate multiplex real-time polymerase chain reaction (PCR) assays. One assay targets the rrs-rrlA IGS region of Borrelia spp. to detect Borrelia burgdorferi sensu lato (s.l.) and Borrelia miyamotoi s.l. The second assay targets the ospA region of B. burgdorferi s.l. to detect B. burgdorferi sensu stricto (s.s.), Borrelia bissettii, and Borrelia andersonii. The final assay targets the glpQ region of B. miyamotoi s.l. to differentiate B. miyamotoi LB-2001 and Borrelia lonestari. A testing scheme combining these tests yielded 18% of tested I. scapularis ticks surveyed from New Jersey positive for B. burgdorferi s.s., 3.2% I. scapularis ticks positive for B. miyamotoi LB-2001, and 41.2% I. spinipalpis ticks positive for B. bissettii surveyed from Colorado.

Differential infectivity of the Lyme disease spirochete Borrelia burgdorferi derived from Ixodes scapularis salivary glands and midgut.

Blood fed nymphal Ixodes scapularis Say infected with Borrelia burgdorferi were dissected to obtain salivary gland and midgut extracts. Extracts were inoculated into C3H/HeJ mice, and ear, heart, and bladder were cultured to determine comparative infectivity. Aliquots of extracts were then analyzed by quantitative polymerase chain reaction to determine the number of spirochetes inoculated into mice. A comparative median infectious dose (ID50) was determined for both salivary gland and midgut extract inoculations. Our data demonstrated a statistically significant difference (P < 0.002) in the ID50 derived from salivary gland (average = 18) versus midgut (average = 251) extracts needed to infect susceptible mice. A rationale for the differential infectivity of salivary and midgut derived spirochetes is discussed.

Vector competence of the Australian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochete Borrelia burgdorferi.

Clinical and serologic evidence of Lyme disease in Australia, including the typical rash, erythema migrans, has been reported. The vector tick transmitting Borrelia burgdorferi in Australia, however, has not been determined. The Australian paralysis tick, Ixodes holocyclus, is a logical candidate vector of the Lyme disease spirochete in Australia; therefore, we tested the ability of I. holocyclus to acquire and maintain a North American isolate of B. burgdorferi. Larval I. holocyclus ingested spirochetes, but none of 84 derived nymphs were infected. These experiments should be repeated with Australian strains of spirochetes.

An evaluation of media for transport of tissues infected with Borrelia burgdorferi.

Currently, the best medium for culture of Borrelia burgdorferi, the etiologic agent of Lyme disease, is Barbour-Stoenner-Kelly (BSK), or its modifications. This medium is complex, expensive, and laborious to prepare. A recent report suggested that a less expensive and simpler medium, hypertonic Columbia broth, might be useful as a transport medium for human tissues infected with B burgdorferi. To test this observation, hypertonic Columbia broth, Amies broth, distilled water, physiologic saline, phosphate-buffered saline (PBS), and modified Stuart medium were compared with BSK II as transport media, using ear and tail tissue samples from B burgdorferi-infected laboratory mice and using holding times and temperatures simulating actual transport conditions. The results showed BSK II to be markedly superior to the other media tested, although B burgdorferi remained viable in a few tissue samples held at room temperature in hypertonic Columbia broth, physiologic saline, or PBS for up to 2 days. Barbour-Stoenner-Kelly II continues to be the best medium for transport of tissues infected with B burgdorferi.

Human babesiosis on Nantucket Island: prevalence of Babesia microti in ticks.

In order to derive direct evidence implicating Ixodes dammini as a vector of human babesiosis, we determined the prevalence of Babesia microti infection in nymphal I. dammini collected on Nantucket Island. In experiments in the laboratory we found that nymphs remained attached to hamsters for about 3 days. Babesial infection was transmitted more often during 54 hours of attachment then during 36 or 48 hours. Since parasites were demonstrable in salivary glands solely after 48 hours, we derived an engorgement index for identifying ticks attached for 2 days or more. Of 156 nymphal I. dammini, collected from white-footed mice in 1979, 86 were engorged sufficiently to satisfy this index of attachment, and the salivary glands of four contained B. microti parasites. This demonstrates that about 5% of nymphal I. dammini are infected in nature. Risk of human infection can be reduced by prompt removal of attached ticks.

Field trial of an outer surface protein A (OspA) antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect Borrelia burgdorferi in Ixodes scapularis.

Field-collected adult male Ixodes scapularis from Westchester County, New York were bisected and Borrelia burgdorferi infection rates were ascertained by both a direct fluorescent antibody test and an outer surface protein A (OspA) antigen-capture enzyme-linked immunosorbent assay (ELISA). Both assays gave identical antigen positivity rates with 89% concordance between the two assays. Storing dried ticks before ELISA analysis had no significant effect on the ability of the ELISA to determine the presence of OspA compared with assaying live ticks. The OspA antigen positivity rate for dried ticks was 49% compared with 53% for live ticks, with mean OspA antigen spirochete equivalents of 3,388 and 2,823 for dried and live ticks, respectively.

Growth kinetics of the Lyme disease spirochete (Borrelia burgdorferi) in vector ticks (Ixodes dammini).

Lyme disease spirochetes (Borrelia burgdorferi) multiplied rapidly in larval Ixodes dammini, reaching a mean density of 2,735 spirochetes/tick on day 15 post-repletion. A 5-fold drop in spirochete levels occurred during the subsequent premolting period. Recently molted nymphs contained a mean of less than 300 spirochetes/tick. Following nymphal repletion, spirochete multiplication renewed, reaching a mean abundance of 61,275 spirochetes/nymph on day 75 post-repletion. A 10-fold drop in spirochete abundance occurred again when ticks molted to the adult stage. Tick-derived spirochetes proved to be infectious when greater than 10(4) spirochetes were inoculated ip into hamsters (4 of 4 animals infected). Inocula of 10(3-4) spirochetes were not always infectious (8 of 23 animals infected), and inocula of less than 10(3) spirochetes were insufficient for establishing infection (0 of 8 animals infected).

Host availability limits population density of Panstrongylus megistus.

In order to determine whether host availability limits triatomine population growth, 5th-stage Panstrongylus megistus were maintained in feeding chambers containing 0, 1, 2, or 3 mice. During the 5-day feeding period, triatomines exposed to two or three mice gained significantly more weight than did bugs exposed to one mouse. In addition, half of the bugs exposed to two or three mice molted, as compared to one-fifth of the P. megistus exposed to one mouse. Thus, weight gain and molting were related to host density. In contrast, bug mortality was related to the triatomine-mouse ratio, being greatest among bugs exposed to one mouse. Twenty-nine nonplastered mud-stick houses in a Chagas' disease endemic area were censused and examined for triatomines. About 70% of houses with greater than or equal to 4 persons contained dense bug populations, while only 20% of houses with 1-3 persons were densely infested. Moreover, blood-meal identifications demonstrated that two-thirds of the P. megistus collected from these houses fed on man. The density of triatomines present in infested houses is related to the number of persons available as hosts.

Reservoir competence of white-footed mice for Lyme disease spirochetes.

Using the vector tick, Ixodes dammini, we described the reservoir competence of the white-footed mouse, Peromyscus leucopus, for the Lyme disease spirochete, Borrelia burgdorferi. Nymphal I. dammini were used to infect mammals, and larval ticks were used to diagnose infection (a form of xenodiagnosis). One tick was nearly as efficient as more than 1 in transmitting the spirochete to mice. The duration of the prepatent period was about 1 week. Prevalence of infection approached 100% in ticks that fed as larvae on mice infected 2 or 3 weeks previously. Thereafter, infectivity gradually decreased, but duration exceeded 200 days. Hamsters, too, became infectious for larval I. dammini. This report formally demonstrates the life cycle of B. burgdorferi as it seems to occur in nature. We conclude that the white-footed mouse is a competent reservoir for the Lyme disease spirochete.

Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene.

We developed a polymerase chain reaction (PCR) that specifically amplifies a fragment of the flagellin gene (fla) of Borrelia burgdorferi, the causative agent of Lyme disease. This fla target, amplified with nested primers, was conserved among all 80 strains of B. burgdorferi tested. Strains examined included cultures from ticks, humans, and rodents from major B. burgdorferi-endemic regions of the United States and parts of Europe and Asia. Templates from B. hermsii, B. parkeri, B. turicatae, and B. coriaceae were not amplified, nor were eukaryotic DNAs from three tick genera. Several host DNAs potentially present in a tick blood meal also were not amplified. Approximately six B. burgdorferi per PCR reaction could be detected by ethidium bromide staining of amplified DNA. Colony-raised Ixodes dammini were used to evaluate the method. One infected nymph in a pool of 40 ticks was routinely detected. The specificity of the assay for detecting B. burgdorferi-infected ticks in pools was 94% (29 of 31). This protocol should prove useful for assessing infection rates in other putative arthropod vectors.

Short report: geographic distribution of different genetic types of Ehrlichia chaffeensis.

The 120-kD protein gene of Ehrlichia chaffeensis was used to characterize ehrlichial DNA from seven pools of adult Amblyomma americanum ticks. Ticks from Missouri, Kentucky, and North Carolina contained E. chaffeensis DNA of the Arkansas strain genotype. Ticks from North Carolina also contained ehrlichiae of the Sapulpa strain genotype, originally identified in Oklahoma.

Ability of experimentally infected chickens to infect ticks with the Lyme disease spirochete, Borrelia burgdorferi.

Chickens were used as a laboratory model to determine the conditions affecting the ability of birds to infect ticks with Lyme disease spirochetes. Chicks (Gallus gallus) were exposed to 12 nymphal Ixodes scapularis at one week or three weeks of age. Xenodiagnostic larval ticks fed these birds at weekly intervals thereafter. Chicks exposed to infected nymphs at one week of age infected 87% of larvae at three weeks of age, but only infected 3% of larvae at four weeks and 0% of larvae at five weeks. Chicks exposed to nymphs at three weeks of age infected only 12% of larvae at four weeks, and 0% thereafter. Thus, experimentally infected chicks can infect larval ticks, but only for a brief interval after exposure. Young chicks are more infectious than older chickens. The immune response of infected chicks was rapid and directed against diverse antigens.

Reservoir hosts of human babesiosis on Nantucket Island.

The host range of Babesia microti was studied on Nantucket Island in order to identify the enzootic reservoir of this human pathogen. White-footed mice (Peromyscus leucopus) were more frequently parasitized than were other indigenous animals. Infection was ubiquitous in locations where deer were abundant. Mice were most frequently parasitemic during spring and summer and adults more frequently than juveniles. Parasitemia, which was rarely intense, was sustained for as long as 4 months. Mice lived as long as 10 months, and juveniles were most abundant during early summer. Prevalence of zoonotic infection, in certain locations, appeared to be inversely correlated with abundance of mice. B. microti was present solely in regions harboring deer.

Tick transmission of Babesia microti to rhesus monkeys (Macaca mulatta).

To determine whether Ixodes dammini is capable of transmitting Babesia microti to primates, infected nymphal ticks were allowed to feed on five Macaca mulatta. The monkeys were then followed for at least 60 days with daily thick blood smears for evidence of infection. Patent B. microti parasitemia developed in four of the five animals. Prepatent periods were 13, 18, 20, and 28 days. Maximum parasitemia ranged from 83 to 7,068 organisms/mm3 blood. Splenectomy 15-17 months after exposure to ticks results in recurrences of parasitemia in three of the four infected monkeys.

Epidemiology of human babesiosis on Nantucket Island.

Between 1969 and 1977, 14 persons with parasitologically confirmed Babesia microti infections and seven persons with antibody titers to B. microti greater than or equal to 1:1,024 were identified on Nantucket Island, Massachusetts. Nineteen of these 21 persons were interviewed. About half were permanent residents of Nantucket; the others spent most of their summers on the island. There were 12 women and seven men. Patients ranged in age from 23 to 86 years; all of those with parasitologically confirmed infections were at least 49 years old. Fifteen patients had illnesses characterized by fever, chills, myalgia and fatigue. Five reported being bitten by a tick from 7 to 28 days before the onset of illness. Most cases occurred during July or August. There appeared to be no association between B. microti infection and direct contact with wild or domestic animals or specific outdoor activities. The unusual age distribution of patients with parasitologically confirmed B. microti infections may result because older persons tend to have more severe illnesses and thus are more likely to come to medical attention.