Robert S. Lane

Infection with a Babesia-Like Organism in Northern California

BACKGROUND Human babesiosis is a tick-transmitted zoonosis associated with two protozoa of the family Piroplasmorida: Babesia microti (in the United States) and B. divergens (in Europe). Recently, infection with an unusual babesia-like piroplasm (designated WA1) was described in a patient from Washington State. We studied four patients in California who were identified as being infected with a similar protozoal parasite. All four patients had undergone splenectomy, three because of trauma and one because of Hodgkins disease. Two of the patients had complicated courses, and one died. METHODS Piroplasm-specific nuclear small-subunit ribosomal DNA was recovered from the blood of the four patients by amplification with the polymerase chain reaction. The genetic sequences were compared with those of other known piroplasm species. Indirect immunofluorescent-antibody testing of serum from the four patients and from other potentially exposed persons was performed with WA1 and babesia antigens. RESULTS Genetic sequence analysis showed that the organisms from all four patients were nearly identical. Phylogenic analysis showed that this strain is more closely related to a known canine pathogen (B. gibsoni) and to theileria species than to some members of the genus babesia. Serum from three of the patients was reactive to WA1 but not to B. microti antigen. Serologic testing showed WA1-antibody seroprevalence rates of 16 percent (8 of 51 persons at risk) and 3.5 percent (4 of 115) in two geographically distinct areas of northern California. CONCLUSIONS A newly identified babesia-like organism causes infections in humans in the western United States. The clinical spectrum associated with infection with this protozoan ranges from asymptomatic infection or influenza-like illness to fulminant, fatal disease.

A COMPARATIVE STUDY OF MAMMALIAN AND REPTILIAN ALTERNATIVE PATHWAY OF COMPLEMENT-MEDIATED KILLING OF THE LYME DISEASE SPIROCHETE (BORRELIA BURGDORFERI)

The potential bactericidal activity of the alternative complement pathway of mammalian and reptilian sera to Borrelia burgdorferi sensu stricto (s.s.) was evaluated in vitro. Complement-mediated killing was observed when cultured spirochetes were inoculated into sera from the western fence lizard (Sceloporus occidentalis) and from the southern alligator lizard (Elgaria multicarinata), but not when they were inoculated into serum from either the deer mouse (Peromyscus maniculatus) or from humans. Spirochetes were still alive after 4 hr in lizard serum that had been preheated at 56 C for 30 min to inactivate complement. Furthermore, when lizard serum was chelated with 10 mM ethylenediaminetetraacetic acid to block all complement activation, borreliacidal activity was arrested. When lizard serum was chelated with 10 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid plus 4 mM MgCl2 to block only classical complement pathway activation, >85% of spirochetes were immobilized within 1 hr. Differences in B. burgdorferi s.s. mortality were not observed when chelators with or without MgCl2 were added to serum from either deer mice or humans. Proteins comprising the alternative complement pathway are responsible for the borreliacidal activity observed in the blood of S. occidentalis and E. multicarinata.

Transmission of Borrelia Burgdorferi by Ixodes Pacificus Nymphs and Reservoir Competence of Deer Mice (Peromyscus maniculatus) Infected by Tick-Bite

The transmission of Borrelia burgdorferi to deer mice (Peromyscus maniculatus) by Ixodes pacificus nymphs was investigated experimentally. Deer mice were exposed to infected nymphs for 24, 48, or 72 hr, or until ticks had fed to repletion (> or = 96 hr). Infection status of hosts was assessed 4 wk later by culture of ear-punch biopsies in BSK II medium and by indirect immunofluorescence. Eight mice exposed to ticks for 24 hr did not become Infected. In contrast, infection was acquired by 1 of 9 (11%), 2 of 8 (25%), and 8 of 10 (80%) mice exposed for 48, 72, and > or = 96 hr, respectively. Eight weeks after exposure to infected nymphs, the infectivity of 5 deer mice for I. pacificus larvae was assessed. Overall, 33% of I. pacificus larvae fed on these mice acquired and transstadially passed spirochetes. We conclude that most I. pacificus nymphs require 4 days or longer to transmit spirochetes to deer mice, and that larvae efficiently acquire and maintain spirochetes from mice that have been infected by tick-bite.

Environmentally Related Variability in Risk of Exposure to Lyme Disease Spirochetes in Northern California: Effect of Climatic Conditions and Habitat Type

Abstract Risk of exposure to Borrelia burgdorferi sensu lato (s.l.) spirochetes, which include the causative agents of Lyme disease, is, in part, determined by the density of questing infected vector ticks. We sought to clarify the temporal patterns of nymphal activity, and the extent of variation in peak and cumulative densities of B. burgdorferi s.l.-infected Ixodes pacificus Cooley & Kohls nymphs, at 12 sites within the ecologically diverse Mendocino County in northwestern California. Also, we assessed the impact of various environmental characteristics (e.g., climatologic variables, habitat type, deer usage) on the aforementioned tick-related traits. The average durations of total and peak (nymphal density > 75% of absolute peak) questing activity were 31% and 82% longer, respectively, in areas with conifers present than in oak woodlands, which represented the warmest and driest habitat type examined. Peak and cumulative densities of infected nymphs varied > 400-fold between sites. Both traits were positively associated with the presence of Quercus spp. oaks or deer, and lower in redwood/tanoak versus oak and oak/Douglas fir habitats. However, a prolonged duration of nymphal activity in redwood habitats, relative to oak woodlands, resulted in a shift from peak nymphal densities occurring in oak woodlands in spring to redwood/tanoak habitats in summer. In conclusion, our data clearly show significant variability in seasonal as well as spatial risk of exposure to Lyme disease spirochetes within a small but ecologically, diverse geographic area. Hence, temporally dynamic and spatially explicit models are needed to assess the risk of exposure to tick-borne pathogens at spatial scales encompassing diverse climatologic or ecological conditions.

Research on the ecology of ticks and tick-borne pathogens—methodological principles and caveats

Interest in tick-transmitted pathogens has experienced an upsurge in the past few decades. Routine application of tools for the detection of fragments of foreign DNA in ticks, together with a high degree of interest in the quantification of disease risk for humans, has led to a marked increase in the number of reports on the eco-epidemiology of tick-borne diseases. However, procedural errors continue to accumulate in the scientific literature, resulting in misleading information. For example, unreliable identification of ticks and pathogens, erroneous interpretations of short-term field studies, and the hasty acceptance of some tick species as vectors have led to ambiguities regarding the vector role of these arthropods. In this review, we focus on the ecological features driving the life cycle of ticks and the resulting effects on the eco-epidemiology of tick-transmitted pathogens. We review the factors affecting field collections of ticks, and we describe the biologically and ecologically appropriate procedures for describing tick host-seeking activity and its correlation with environmental traits. We detail the climatic variables that have biological importance on ticks and explain how they should be properly measured and analyzed. We also provide evidence to critically reject the use of some environmental traits that are being increasingly reported as the drivers of the behavior of ticks. With the aim of standardization, we propose unambiguous definitions of the status of hosts and ticks regarding their ability to maintain and spread a given pathogen. We also describe laboratory procedures and standards for evaluating the vectorial capacity of a tick or the reservoir role of a host. This approach should provide a coherent framework for the reporting of research findings concerning ticks and tick-borne diseases.

Detection of a Borrelia miyamotoi Sensu Lato Relapsing-Fever Group Spirochete from Ixodes pacificus in California

Abstract We investigated whether host-seeking nymphs and adults of the western blacklegged tick, Ixodes pacificus Cooley & Kohls, the primary vector of Lyme disease spirochetes in far-western North America, are infected naturally with relapsing-fever group spirochetes in Mendocino County, California. Relapsing-fever group borreliae were detected in four (1.7%) of 234 nymphal and two (0.7%) of 282 adult host-seeking I. pacificus ticks by polymerase chain reaction and sequence analysis of the 16S rRNA and flagellin genes, respectively, exhibiting 99 and 98.5% sequence homology to Borrelia miyamotoi Fukunaga. Phylogenetic analysis based on these two genes revealed that the borreliae detected in these ticks belong to the relapsing-fever group and that these are closely related to, if not identical with, B. miyamotoi.

Life Cycle of Ixodes pacificus (Acari: Ixodidae): Timing of Developmental Processes Under Field and Laboratory Conditions

Abstract The developmental timing of Ixodes pacificus Cooley & Kohls, the primary vector of the Lyme disease spirochete and the agent of human granulocytic ehrlichiosis in the far-western United States, was determined under field and laboratory conditions. During their seasonal peaks of abundance, each of the three parasitic stages of I. pacificus, both fed and unfed, was placed inside silk-screen packets. These packets were apportioned between four topographic exposures of two hilltop sites in northwestern California. The sites differed in vegetational composition and elevation: the first (elevation 390 m) was dominated by woodland-grass, the second (elevation 914 m) by chaparral. The timing of oviposition, larval eclosion, molting, and mortality were recorded in the field every 2–3 wk for 2.5 yr. Microenvironmental temperatures were measured on all four exposures at both sites. Accelerated developmental rates of all three stages were correlated with warmer soil temperatures and the time of placement in the field. In the laboratory, replete female I. pacificus maintained under uniform environmental conditions sustained constant preovipositional and prehatch periods independent of date-of-feeding. In the field, all unfed stages survived through one active feeding season with most larvae and nymphs remaining in behavioral diapause between late summer and early spring. No life stage survived through two active feeding periods which suggests that cohorts do not overlap. We concluded that I. pacificus takes a minimum of 3 yr to complete its life cycle in northwestern California. Ixodes pacificus, life history, microhabitat, development, Lyme disease

Phylogenetic Analysis of the Spirochetes Borrelia parkeri and Borrelia turicatae and the Potential for Tick-Borne Relapsing Fever in Florida

ABSTRACT Isolates of Borrelia turicatae, Borrelia parkeri, and the Florida canine borrelia (FCB) were examined to further phylogenetically characterize the identities of these spirochetes in the United States. DNA sequences of four chromosomal loci (the 16S rRNA gene, flaB, gyrB, and glpQ) were determined for eight isolates of B. turicatae and six isolates of B. parkeri, which grouped the spirochetes into two distinct but closely related taxa (>98% sequence identity) separate from Borrelia hermsii. The FCB was clearly separated with the group identified as B. turicatae, confirming this bacterium as a relapsing fever spirochete. Therefore, the potential for tick-borne relapsing fever in humans and other animals exists in Florida and future efforts are needed to determine the enzootic hosts and distribution of this spirochete in the southeastern United States. Analysis of plasmids demonstrated both linear and circular forms in B. turicatae but only linear plasmids in B. parkeri, which should be of interest to investigators concerned with plasmid diversity and evolution within this group of spirochetes.

Seasonal activity patterns of Ixodes pacificus nymphs in relation to climatic conditions.

Abstract In western North America, the tick Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) is the primary vector to humans and domestic animals of the disease agents causing Lyme disease and granulocytic ehrlichiosis. We examined the seasonal activity patterns of I. pacificus nymphs over a 4‐year period, including the wet and cold El Niño winter/spring of 1998, in a dry oak/madrone woodland, and for one year in a cooler and moister redwood/tanoak woodland in Mendocino County, California. Linear regressions were used to estimate when nymphal densities first exceeded and then fell below 25, 50 and 75% of the recorded yearly peak densities. In oak/madrone woodland, nymphs typically were active by mid‐March, reached 50% of their yearly peak densities in early to mid‐April, peaked by early May, fell below 50% of their peak densities by early to mid‐June, and were absent by late July to mid‐August. The lengths of the periods with nymphal densities exceeding 50 and 75% of the recorded yearly peaks in oak/madrone woodland were associated positively with rainfall and negatively with maximum air temperatures during April–May. Moreover, nymphal numbers typically reached 50% of their peak 10–15 days later, remained at levels above 50% of the peak 1.3–1.5 times longer, and started declining 4–6 weeks later under cooler, moister climatic conditions (oak/madrone woodland in 1998 and redwood/tanoak woodland in 2000) relative to warmer, drier conditions (oak/madrone woodland in 2000–2001). In oak/madrone woodland, nymphal densities typically started to decline when mean maximum daily air temperatures exceeded 23°C. Nymphal densities were higher in dry oak/madrone relative to moist redwood/tanoak woodland from mid‐March to late May 2000, similar in both habitat types in early June, but higher in redwood/tanoak woodland from late June onwards. We conclude that large‐scale studies of the density of I. pacificus nymphs in California need to consider spatial variation in the length of nymphal activity periods and select temporal sampling regimens that yield representative data for all included habitat types.

Relative importance of lizards and mammals as hosts for ixodid ticks in northern California

Lizards and mammals were trapped and examined for ticks from August 1992 to June 1993 in two habitat types, chaparral and woodland-grass, in northern California. Five tick species were collected from mammals (Dermacentor occidentalis, Haemaphysalis leporispalustris, Ixodes pacificus, I. spinipalpis, I. woodi), but only I. pacificus was found on lizards. Dermacentor occidentalis, I. pacificus, and I. woodi occurred in both habitats, whereas H. leporispalustris and I. spinipalpis were found only on animals trapped in chaparral. The tick species most commonly encountered on mammals was D. occidentalis in chaparral and I. pacificus in woodland-grass. Peak infestation of mammals occurred in spring for I. pacificus immatures and H. leporispalustris, summer for D. occidentalis immatures, fall through spring for I. woodi immatures, and fall through winter for I. spinipalpis. The primary aim of the study was to quantify the relative importance of the western fence lizard (Sceloporus occidentalis), which is reservoir-incompetent for Borrelia burgdorferi sensu lato (s.l.), and mammalian B.burgdorferi s.l.-reservoirs as hosts for the immature stages of I. pacificus in spring. The estimated relative utilization by I. pacificus of the western fence lizard versus mammals was 88% for larvae and 99% for nymphs in chaparral in May. When tick infestation data were corrected for a two-fold lower efficiency of field examinations for rodents than for lizards, the western fence lizard still accounted for 78% of larval and 98% of nymphal feedings. In woodland-grass, 46% of 100 I. pacificus larvae and 100% of 52 nymphs recovered from mammals or western fence lizards during May-June were collected from the lizards. However, this may represent an underestimate of the importance of the western fence lizard as a larval host in this habitat because inclement weather during the late May sampling period doubtless resulted in significantly decreased lizard activity. In conclusion, the western fence lizard was more heavily utilized by I. pacificus immatures, especially nymphs, than were rodents.