To infect larval D. variabilis with F. tularensis, mice were infested with larvae either the day before (type B) or the day of infection (A1b and A2) with F. tularensis, and ticks were allowed to feed to repletion. In all cases, replete larvae dropped off the infected mice 3–5 days post-infestation. Groups of four to six mice were each infected with A1b, A2, or type B, and the larvae were pooled per each individual mouse. Analysis of bacterial burdens in the blood of infected mice indicated that replete larvae were obtained on the day before and the day of peak bacteremia (). All larvae efficiently acquired F. tularensis infections (), and no significant differences were observed in bacterial acquisition rates between A1b [mean = 0.640, standard deviation (SD) = 0.261, N = 5 pools of 5 larvae], A2 (mean = 0.800, SD = 0.179, N = 6 pools of 5 larvae), and type B (mean = 0.933, SD = 0.103, N = 6 pools of 5 larvae; ANOVA: F = 3.38, degrees of freedom (df) = 2 and 14, P = 0.0632) infections.
Acquisition, transstadial transmission, and maintenance of F. tularensis types A1b, A2, and type B in D. variabilis
To determine if F. tularensis was transmitted from the larval to the nymphal life stage, transstadial transmission rates (number of infected nymphs divided by total number nymphs tested) were determined just after the larval molt (21 dpi) (). A1b, A2, and type B infections were all found to be passed efficiently from infected D. variabilis larvae to nymphs, with no difference in transstadial transmission rates observed between A1b- (mean = 0.933, SD = 0.115, N = 5 pools of 6 larvae), A2- (mean = 0.967, SD = 0.082, N = 6 pools of 5 larvae), and type B- (mean = 1.00, SD = 0, N = 6 pools of 5 larvae) infected ticks.
To confirm that infections were maintained in nymphs at 44 days post-larval molt (the day of the nymphal blood meal for A1b and type B), the infection rate was measured in A1b- and type B-infected nymphs. Because of high mortality rates and the need to use 50 nymphs for transmission studies, insufficient numbers of A2-infected nymphs survived for these comparisons. No difference was seen in infection rates between A1b- (mean = 0.971, SD = 0.064, N = 5 pools of 7 nymphs) and type B-infected nymphs (mean = 0.933, SD = 0.163, N = 6 pools of 5 nymphs; ANOVA: F = 0.2376, df = 1 and 9, P = 0.6376), with the majority of all ticks maintaining F. tularensis infections throughout the nymphal stage.
Survivorship of uninfected and A1b-, A2-, and type B-infected D. variabilis.
The survival rate was measured 65 dpi (44 days post-larval molt) for A1b-, A2-, and type B- infected nymphs (the number of live nymphs divided by the total number of nymphs analyzed) () and compared with survival rates of uninfected nymphs to determine if fitness costs were associated with F. tularensis infection in ticks. Significant differences in survivorship were detected compared with uninfected, A1b-, A2-, and type B-infected nymphs (ANOVA: F = 11.14, df = 3, 21, P = 0.0002). Overall, survivorship was significantly higher for uninfected nymphs (mean = 0.585, SD = 0.127, N = 368 ticks derived from four larval pools) compared with A2- (mean = 0.116, SD = 0.072, N = 764 ticks derived from six larval pools) and type B-infected nymphs (mean = 0.298, SD = 0.190, N = 917 ticks derived from six larval pools). The highest mortality (88.0%) was observed among A2-infected nymphs. Of note, survivorship of A1b-infected nymphs (mean = 0.353, SD = 0.157, N = 912 ticks derived from five larval pools) did not differ significantly from uninfected groups. Survivorship of the A2-infected nymphs was significantly lower than the A1b- and type B-infected groups. The daily mortality rate for uninfected, A1b-, A2-, and type B-infected nymphs was 0.9%, 1.5%, 2.0%, and 1.6%, respectively.
Figure 1. Mean survival-rate comparison on 65 dpi for uninfected, A1b-, A2-, and type B-infected D. variabilis. Error bars are the standard deviation in survivorship of group replicates (N = 4–6 groups). Sample size represents the number of ticks that were (more ...)
Overall feeding success of uninfected, A1b-, A2-, and type B-infected nymphs.
Attachment rates, length of the nymphal feed, percentage of nymphs that fed to repletion, and feeding-induced mortality were compared between uninfected and infected ticks (). Attachment rates did not differ between uninfected (mean = 0.783, SD = 0.212, N = 4 pools of 50 nymphs) and A1b- (mean = 0.770, SD = 0.164, N = 5 pools of 50 nymphs), A2- (mean = 0.635, SD = 0.272, N = 6 pools of 50 nymphs), and type B-infected ticks (mean = 0.776, SD = 0.209, N = 6 pools of 50 nymphs; ANOVA: F = 0.580, df = 3, 20, P = 0.6361). By contrast, significantly prolonged feeding was observed for A1b-infected nymphs compared with uninfected or type B-infected nymphs (ANOVA: F = 73.95, df = 2, 12, P < 0.0001) (). The uninfected and type B-infected nymphs fed to repletion in approximately 5.38 days (SD = 0.334, N = 4 pools of 25 nymphs) and 5.63 days (SD = 0.277, N = 6 pools of 21 nymphs), respectively, compared with 9.83 days for A1b- (SD = 0.979, N = 4 pools of 9 nymphs) and 11 days for A2-infected nymphs (N = 1) ().
Figure 2. Overall feeding success was compared for uninfected, A1b-, A2-, and type B-infected nymphal D. variabilis by examining (A) the mean number of days to feed to repletion and (B) the mean percentages of nymphs that fed to repletion. Error bars are the standard (more ...)
Significant differences were also observed in the percentage of nymphs feeding to repletion, with more uninfected nymphs feeding to repletion (mean = 55.23%, SD = 17.10%, N = 4 pools of 40 nymphs) than A2- (mean = 3.7%, SD = 9.1%, N = 6 pools of 25 nymphs) infected nymphs (). No significant differences were observed between the percentage of uninfected nymphs compared with both type B- (mean = 56.7%, SD = 25.82%, N = 6 pools of 37 nymphs) and A1b- (mean = 23.6%, SD = 19.19%, N = 5 pools of 37 nymphs) infected nymphs. Among infected nymphs, significant differences in the percentages feeding to repletion were observed between type B-infected compared with A1b- and A2-infected nymphs. Overall, the A2-infected nymphs exhibited the lowest percentage of nymphs to feed to repletion.
Within replete nymphs, a pronounced effect of A1b infection was observed, where even after prolonged feeding, the A1b-infected replete nymphs were very small compared with the uninfected replete nymphs (). The average weight of the A1b-infected nymphs (mean = 6.0 mg, SD = 2.55 mg, N = 9) was significantly lower than that of the uninfected nymphs (mean = 11.0 mg, SD = 4.18 mg, N = 24; t = 4.22, P = 0.0002, pre-hoc one-tailed t test). Also, the scutal index was lower for the A1b-infected nymphs (mean = 4.79, SD = 1.06) compared with uninfected nymphs (mean = 6.99, SD = 0.806; t = 5.64, P < 0.0001, pre-hoc one-tailed t test). There was no visible difference in size of replete nymphs observed between the type B-infected and uninfected controls (data not shown).
Figure 3. Physical effects of A1b infection on replete D. variabilis nymphs (Top) compared with uninfected D. variabilis replete nymphs (Bottom). This figure appears in color at www.ajtmh.org.
Additionally, both A1b- and A2-infected nymphs were subject to feeding-induced mortality. A large percentage of the A1b- and A2-infected nymphs died while attached to the host (A1b-infected nymphs: 75.0%; A2-infected nymphs: 94.4%) compared with type B-infected (19.4%) and uninfected (2.5%) nymphs. Therefore, the expected mortality rate was compared with the observed mortality rate for the length of the feed (11 days). Based on the calculated daily mortality rates, we expected to lose approximately 16.2% (N = 6 nymphs) of the A1b-infected population, 22.0% (N = 5 nymphs) of the A2-infected population, 16.2% (N = 5 nymphs) of the type B-infected population, and 10.0% (N = 4 nymphs) of the uninfected population over 11 days of feeding. The observed and expected mortality were statistically similar for type B-infected and uninfected nymphs; however, observed mortality was significantly higher than expected for the A1b- and A2-infected nymphs. For the A1b- and A2-infected nymphs, 75.0% (N = 36 nymphs; χ2 = 26.38, P < 0.0001, df = 1, Fisher's exact test) and 94.4% (N = 18 nymphs; χ2 = 25.82, P < 0.0001, df = 1, Fisher's exact test) died on the host, respectively. For the type B-infected nymphs, only 19.4% (N = 31; χ2 = 0.111, P = 0.739, df = 1, Fisher's exact test) died on the host. For the uninfected population, 2.5% (N = 1 nymph; χ2 = 2.05, P = 0.153, df = 1, Fisher's exact test) mortality was observed.
Comparison of transmission efficiency among A1b, A2, and type B.
For analysis of transmission efficiency, three possible modes of transmission were considered: incomplete feeding, tick-borne, and consumption of the tick. Incomplete feeding refers to incidents where a nymph attached to the mouse but failed to feed to repletion. Tick-borne refers to the classical concept of biological transmission whereby a nymph attaches to the mouse, feeds to repletion, and drops off of the host. In instances where the tick was not recovered because it could not be found in the water or on the mouse, it was assumed that the tick was consumed by the mouse (e.g., consumption form of transmission).
Overall, transmission of F. tularensis by nymphal D. variabilis was observed for type B and A2, whereas no transmission was observed for A1b (). Comparisons of individual rates of transmission indicated that 0% [N = 9; 95% confidence interval (CI) = 0.00–29.91] of A1b-, 0% (N = 1; 95% CI = 0.00–79.35) of A2-, and 14.3% (N = 21; 95% CI = 3.91–30.06) of type B-infected nymphs transmitted by classical biological transmission. Transmission by tick consumption occurred at a rate of 0% (N = 1, 95% CI = 0.00–79.35) for A1b-, 28.6% (N = 7, 95% CI = 5.57–65.85) for A2-, and 33.3% (N = 5, 95% CI = 6.61–72.59) for type B-infected nymphs. Transmission by incomplete feeding was observed for 0% of A1b- (N = 27, 95% CI = 0.00–12.46), 0% of A2- (N = 17, 95% CI = 0.00–18.43), and 10.0% (N = 10, 95% CI = 0.51–40.43) of type B-infected nymphs. Although we did not observe transmission for A1b, maximum likelihood estimates indicated that transmission could be as high as 30%, 80%, and 65% for classical, consumption, and incomplete feeding, respectively. In all cases, the absence of bacterial growth and seroconversion against F. tularensis in mice surviving 14–19 days post-tick attachment confirmed the lack of transmission by ticks. Similarly, all mice showing signs of infection after being fed on by F. tularensis-infected ticks were F. tularensis antigen- and culture-positive.
Comparison of transmission rates among D. variabilis nymphs infected with F. tularensis A1b, A2, and type B
Bacterial kinetics of infection—bacterial acquisition through nymphal blood meal.
To determine if bacterial loads changed in infected ticks over the entire time course of the experiment, colony-forming units were determined at various time points: replete larvae 1 day after drop-off (1 dpi), just after larval molt (21 dpi), just before nymphal feed (65 dpi), and after nymphal feed and drop-off (). A similar trend was observed in A1b-, A2-, and type B-infected ticks; replication of the bacteria occurred between initial acquisition of infection at the larval stage and the completion of the nymphal blood meal. Measuring from 1 dpi to when the nymphs were replete, we observed a 5.39-log increase for A1b- (χ2 = 15.37, df = 1, P < 0.0001, Mann–Whitney U test with χ2 approximation), a 2.35-log increase for type B- (χ2 = 32.83, df = 1, P < 0.0001, Mann–Whitney U test with χ2 approximation), and a 2.76-log increase for A2-infected nymphs (no statistical analyses performed because only one infected nymph fed to repletion). Replication of F. tularensis occurred throughout most stages (), with the most significant changes in bacterial numbers occurring during the blood meal. For A1b-infected nymphs, a 2.66-log increase was seen between 65 dpi and replete nymphs (χ2 = 18.61, df = 1, P < 0.0001, Mann–Whitney U test with χ2 approximation), and for type B-infected nymphs, a 1.67-log (χ2 = 26.09, df = 1, P < 0.0001, Mann–Whitney U test with χ2 approximation) increase was observed for the same time period.
Figure 4. Bacterial loads within A1b-, A2-, and type B-infected ticks 1 day after larval feeding (1 dpi), shortly after the larval molt (21 dpi), 1 day before the nymphal feed (65 dpi), and the day of nymphal drop-off. Each data point represents the bacterial load (more ...)
Bacterial loads in infected ticks were also compared among F. tularensis infections at 65 dpi. A1b-infected nymphs had significantly higher bacterial loads (median = 6.38 log10 cfu/mL, range = 5.78–6.80, N = 31) compared with type B-infected nymphs (median = 5.73 log10 cfu/mL, range = 4.11–6.56, N = 23; χ2 = 28.10, df = 1, P < 0.0001, Mann–Whitney U test with χ2 approximation).