In the United States, tularemia in humans is caused by Francisella tularensis subspecies tularensis (Type A) or F. tularensis subspecies holarctica (Type B). Type A strains can be further subdivided into two distinct clades called A1 (A-east), and A2 (A-west). 1,2 Human infections caused by A1, A2, and Type B strains differ with respect to geographic location 1,2 and clinical severity.3 Infections with A2 seem to be less severe in humans than infections caused by A1 and possibly Type B.1
Ixodid ticks are considered to play a prominent role in enzootic maintenance and as bridging vectors of F. tularensis to humans.4–7 Experimental laboratory studies of tick vector competence for F. tularensis were conducted mostly from 1924 to 1956 and lacked standardization in methodology for infecting feeding ticks.8–14 This precludes meaningful comparisons among tick species or bacterial strains examined. Furthermore, all studies were conducted before the recognition of F. t. tularensis clades A1 and A2. Despite the recent interest in F. tularensis as a potential bioterrorism threat, 15 little is known about how A1, A2, and Type B are maintained in enzoonotic cycles or which tick species most commonly transmit these bacteria to humans. Understanding such dynamics is important for providing evidence-based recommendations on prevention and control of tularemia.
To set the stage to evaluate efficiency of F. tularensis (A1, A2, and Type B) transmission by common human-biting North American ticks, we sought to develop a mouse model to standardize infection of feeding larval and nymphal ticks with F. tularensis. Previous studies of tick-borne transmission of F. tularensis conducted before 1980 typically used guinea pigs or rabbits, rather than mice, to infect feeding ticks.8–14 Immature ticks (Dermacentor spp. and Ixodes spp.) will readily feed on mice under laboratory conditions. 16 Typically larval and nymphal ixodid ticks feed for 3–4 and 4–5 days, respectively, before dropping off of the host. 17 Because laboratory animals become moribund shortly after acquiring tularemia infection,18–24 it is important to have a description of temporal changes in bacterial concentration so that the period of rapid engorgement in ticks can be timed to coincide with peak bacteremia in the host.
Quantitative descriptions of the kinetics of hematogenous dissemination of F. tularensis are lacking; however, numerous studies report the time course of bacterial dissemination to liver, lung, and spleen or report single time point estimates of bacteremia. 18,19,22,23,25,26 In this study, we describe the course of infection in the blood of Swiss-Webster mice inoculated subcutaneously with three strains representing F. tularensis A1, A2, and Type B. This is the first study to directly compare these types in laboratory mice and to report daily changes in bacteremia.
Strains previously characterized as F. t. tularensisA1 (MA00-2987) and A2 (WY96-3418) and F. t. holarctica (KY99-3387) were used. These isolates were derived from human samples collected in Massachusetts in 2000, Wyoming in 1996, and Kentucky in 1999, respectively. Isolates were subcultured from −70°C stocks onto cysteine heart agar with 9% chocolatized sheep blood (CHAB) and incubated at 35°C for 48 hours. After an additional subculture for 24 hours, bacteria were resuspended in 0.85% NaCl and diluted to a final concentration of ~1.25 × 103 cfu/mL. A target inoculating dose of 1.0 × 102 cfu was selected as a low dose that could be reliably reproduced among inocula without variability that typically arises when small numbers of bacteria are used. Inoculum concentrations were confirmed by plating bacteria in duplicate on CHAB and counting colony forming units after 48- to 72-hour incubation at 35°C.
For each of the three F. tularensis strains, 25 female Swiss-Webster mice (age, 6 weeks) were inoculated subcutaneously on Day 0 with 124–126 cfu in 100 μL. Each mouse was randomly assigned to a blood collection day and was housed in an individual microisolator cage at 21°C and 50% RH. Each day post-infection (dpi) for up to 5 days, five mice per strain were euthanized, and blood was collected by sterile cardiac puncture. Bacterial concentrations in the blood were quantified by plating 10−2–10−7 serial dilutions in duplicate on CHAB and counting colony forming units after 48- to 72-hour incubation at 35°C. Mice were monitored twice daily for evidence of infection (e.g., slow response to stimuli, ruffled fur). If clinical signs became severe before the assigned blood collection date, the animal was euthanized, and blood was collected as described above. All animal methods were approved by the Division of Vector-Borne Infectious Diseases Institutional Animal Care and Use Committee (Protocol 08-003) and were conducted in ABL3 facilities.
The relationships between bacterial concentration and number of dpi were determined for each strain by fitting a linear regression model of log10 cfu/mL on dpi. Mice that were euthanized before their randomly assigned blood collection date were pooled with those euthanized on their randomly assigned date to not artificially deflate naturally occurring bacterial concentrations.
After inoculation with A1 (MA00-2987), A2 (WY96-3418), or Type B (KY99-3387) strains, all mice were found to be asymptomatic up to 2 dpi. As illness progressed, signs of disease became severe among mice inoculated with Type A strains. Six and eight mice inoculated with A1 and A2, respectively, were euthanized before their assigned blood collection dates (Table 1). As a result, all mice exposed to either A1 or A2 were euthanized by 4 dpi. In contrast, only one mouse inoculated with Type B showed overt clinical signs during the course of the experiment and was euthanized at 4 dpi.
Daily bacterial concentrations in blood after infection for A1, A2, and Type B were determined (Table 1). The maximum bacteremia observed for mice exposed to Type B (10.5 log10 cfu/mL) was higher than the maximum values recorded for Types A1 or A2 (9.7 and 9.8 log10 cfu/mL, respectively). For each strain, number of dpi was a good predictor of bacterial concentration. Based on linear regression analysis, mice were expected to reach 108 cfu/mL of blood, the concentration believed to be necessary to infect feeding ticks, by 3.4 dpi for Type A1 (log10 bacteremia = 3.18 dpi – 2.95; F = 226.45; df = 1,24; P < 0.0001; adjusted r2 = 0.90) and by 3.2 dpi for Type A2 (log10 bacteremia = 3.18 dpi – 2.05; F = 89.70; df = 1,24; P < 0.0001; adjusted r2 = 0.79). In contrast, mice infected with Type B would be expected to reach this threshold by 4.0 dpi (log10 bacteremia = 2.42 dpi – 1.70; F = 56.05; df = 1,24; P < 0.0001; adjusted r2 = 0.70).
We developed a mouse model that can be used in the future for standardized comparisons of vector competence and efficiency of ticks infected with F. tularensis A1, A2, and Type B. To maximize the likelihood of ticks acquiring infection from laboratory-infected mice before they become moribund and must be euthanized, ticks should be placed on mice so that periods of rapid engorgement occur 3–4 dpi for A1 and A2 and 4–5 dpi for Type B. Because of the small volumes of blood ingested by feeding larvae and nymphs (~2–3 μL for larvae, ~15 μL for nymphs 27), bacteremia must be very high to reliably infect feeding ticks. Indeed, previous studies indicate that transmission likely occurs primarily when the host is terminally septicemic (108–109 cfu/mL of blood 18).9
Our results are consistent with a previous study that used different strains of Type A (#33) and Type B (#108) to evaluate dissemination to liver, lung, and spleen in different strains of mice (BALB/c and C57BL/6). 18 Although that study did not report daily changes in bacteremia, similar to our study, the authors reported that 1) bacteremia was evident by 2 dpi for each of Types A and B, 2) by the day of expected death, bacteremia reached levels of > 108 cfu/mL, and 3) onset of clinical signs was delayed for mice exposed to Type B compared with Type A. Another study 28 reported daily changes in bacteremia; however, this was only for mice exposed intradermally to an attenuated strain of F. tularensis (the live vaccine strain [LVS]). Our study is the first to report daily changes in bacterial concentrations in blood from the time of infection to when the host shows severe signs of infection using virulent strains of F. tularensis and to also differentiate between A1 and A2 infections.
The ability of mice exposed to Type B to remain asymptomatic while highly bacteremic (8–10 log10 cfu/mL) supports the observation that Type B is less virulent than Type A in mice. 29 Interestingly, Type A infections are commonly associated with lagomorphs and tick-borne exposure, whereas Type B infections in the United States are commonly associated with water and water-associated rodents such as voles. 5,29 If our results are representative of naturally acquired infections in wild rodents, the ability of mice to survive very high Type B bacterial concentrations for several days suggests that it is likely that ticks could acquire infections from infected rodents. Indeed, Type B has been recorded from Dermacentor ticks during tularemia outbreaks in the United States. 30,31
Rigorous experimental studies of tick vector competence and efficiency conducted under modern standardized conditions are needed to address several significant public health issues related to preventing and controlling tularemia. These include determining 1) the relative potential for various tick species and deerflies to serve as bridging vectors to humans of F. t. tularensis (Type A1 and A2) and F. t. holarctica (Type B), 2) the duration of time a tick must be attached to its host before transmission of A1, A2, or B is likely to occur, and 3) how A1, A2, and B are maintained in enzootic cycles. Our study provides the mouse model needed as the starting point to address these questions.
Bacterial concentrations of F. tularensis A1, A2, and B in outbred Swiss-Webster mice by dpi
Address correspondence to Rebecca J. Eisen, DVBID/CDC, 3150 Rampart Road, Fort Collins, CO 80522. E-mail: dyn2@cdc.gov
Acknowledgments: The authors thank L. Eisen for comments on the manuscript and K. L. Gage for technical and logistical support.
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