Wood ticks originating from the western (BL) and eastern (SK) slopes of the Bitterroot Valley were experimentally infected with six separate rickettsial strains (Tables and ). For comparison, uninfected sibling, uninfected F-0, and naturally infected F-0 ticks were also studied. For each group of experimental ticks, rickettsial infections were monitored by a PCR assay immediately prior to feeding and after molting, feeding, and oviposition. The infection status was confirmed by IFA tests for all but 17 samples, namely, fed larvae and nymphs. PCR-RFLP analysis confirmed the presence of R. rickettsii in the 17 samples negative by the IFA test. Furthermore, PCR-RFLP analysis confirmed the presence of R. rickettsii in experimentally, naturally, and vertically infected ticks. Direct counts and plaque titer determinations detected fewer than 103 rickettsiae per larva or nymph (20 larvae or nymphs tested) immediately after they fed on rickettsemic guinea pigs (infected with the Como-96 or Wachsmuth strain). Both eastern (SK) and western (BL) slope tick groups were susceptible to and transmitted R. rickettsii (Table ).
TABLE 2 Experimentally infected D. andersoni tick groups used to study the effect of R. rickettsii on tick survival andfecundity
A notable difference in infectivity for guinea pigs and ticks was observed among several rickettsial strains. The strains (Como-96 and Wachsmuth) grown in chicken embryos or animal tissues were highly virulent. Nonimmune guinea pigs fed upon by ticks infected with these strains developed classic RMSF with high fever, scrotal reaction, and presence of rickettsiae in tissues and blood. The Swan strain also induced lethal RMSF in guinea pigs, while adult ticks acquiring it oviposited nonviable eggs. Rickettsial strains (R and Sawtooth) cultured in Vero cells were of low virulence and induced mild RMSF when inoculated into guinea pigs. Ticks acquiring these strains failed to maintain rickettsiae (Table ) and remained apparently healthy. Concurrently, no rickettsiae were detected in any uninfected control ticks by the PCR assay, IFA tests, plaque titer determination, or direct counts.
Significant mortality was induced in wood ticks experimentally infected as larvae or nymphs with highly virulent R. rickettsii (Como-96 or Wachsmuth), while the majority of uninfected siblings developed into feeding adults (Fig. ). Nymphal molting and adult-female feeding success were significantly reduced (P < 0.001) for infected ticks compared with uninfected controls (Fig. ). No significant difference (P > 0.45) in molting or feeding success occurred between infected ticks held at 21 and 27°C, between two tick strains (BL and SK), and with either rickettsial strain.
FIG. 1 The nymphal molting and adult-female feeding success of D. andersoni ticks experimentally infected (I) with R. rickettsii were both significantly less (P < 0.001) than those of uninfected controls (C) as determined by chi-square tests of observed (more ...)
For nymphs infected during larval feeding, 94.1% of ticks infected with Como-96 (825 of 877) and 97.7% of those infected with Wachsmuth (252 of 258) died after an uninfected blood meal but prior to molting. In contrast, only 1.4% of uninfected nymphs (14 of 1021) failed to develop into adults. Overall, 96.9% of the dead nymphs from the infected groups (31 of 32) tested positive for R. rickettsii. Each of the surviving ticks (a total of 10 were tested) contained approximately 6.5 × 106 rickettsiae 4 weeks after the nymphs fed. Nearly equal numbers of male and female ticks (27 and 30, respectively) survived the molt, and each contained approximately 1.0 × 107 rickettsiae (a total of 5 were tested). Only 30.0% of infected females (9 of 30) successfully fed, which was significantly lower (P < 0.001) than the 90.7% of uninfected control females (108 of 119) that successfully fed (Fig. ). Of these, 66.6% of infected (6 of 9) and 81.5% of uninfected (88 of 108) fed ticks oviposited.
High mortality was also observed for ticks infected during nymphal feeding. For these ticks, 34.9% (176 of 504) died during the molt and 88.3% of adult females (189 of 214) failed to feed. For uninfected siblings, 2.1% (12 of 572) died during the molt and 19.2% of adult females (59 of 307) failed to feed. Overall, 100% of the dead ticks from infected groups (40 of 40) tested positive for R. rickettsii.
Ticks infected with Como-96 that were held at 4°C had a significantly greater (P < 0.01) survival and feeding success than those held at 21°C (Table ), but the success rate was still significantly lower (P < 0.005) than that of uninfected controls. No significant difference (P > 0.45) in survival and feeding success occurred between uninfected ticks at the two temperatures. Only 36.2% of infected ticks (25 of 69) attached (Table ), and, of these, all the salivary secretions tested were positive for R. rickettsii (a total of 10 ticks were tested). Of the 25 feeding ticks, 8 remained attached only for 3 to 5 days, ingested only a small volume of blood if any, and remained viable for an additional 3 months.
TABLE 3 Summary of developmental and feeding success of D. andersoni female ticks experimentally infected with R. rickettsii (Como-96) and uninfected controls incubated at 4 or21°C
Adult ticks experimentally infected as larvae or nymphs with highly virulent R. rickettsii
vertically transmitted rickettsiae to 39.0% of offspring (23 of 59 adults) (Table ). Although only a portion of the progeny inherited rickettsiae, 100% of the 20 adults tested were infected after feeding as nymphs, an observation consistent with transmission between cofeeding ticks (42
). Subsequently, 97.5% of infected progeny (235 of 241) died prior to adulthood. Ticks experimentally infected as adults with R. rickettsii
and R. montana
had ovarial tissues with as many as 2.5 × 107
rickettsiae following oviposition, but they failed to transmit infection vertically to offspring (Table ), a result consistent with some (38
) but not all (6
) past studies.
TABLE 4 Transovarial transmission and fecundity of D. andersoni ticks infected with rickettsiae and uninfectedcontrols
The number of progeny developing from ticks (F-1 BL and F-2 SK) infected with the highly virulent Como-96 and Wachsmuth strains as larvae or nymphs but not as adults was significantly smaller (P < 0.001) than that developing from uninfected siblings or those naturally infected with symbiotic R. peacockii (Table ). Further, four separate ticks (F-0 CL) naturally infected with R. bellii, R. montana, or R. rhipicephali exhibited reduced fecundity, with an average of 134 offspring (range, 91 to 185), compared to 2,919 offspring (range, 2,456 to 3,190) per uninfected F-0 tick. In rearing of ticks, either all or no eggs hatched and ovarial tissues dissected from ticks after oviposition retained fewer than 15 unlaid eggs.