RESEARCH lated with the Ar-248 strain of WNV, but transmission from infected ticks to mice was not observed (22,23). Vermeil et al. (1959) infected O. maritimus and O. errati- cus ticks by feeding on inoculated (Uganda 28B strain) chickens, guinea-pigs, mice, or gerbils. Infected ticks transmitted the virus to uninfected mice (24). More recent- ly, an artificial membrane system was used to infect Argas arboreus ticks, which were then able to transmit the virus to uninfected hosts, although transstadial transmission of WNV was not observed (25,26). Our study demonstrated that both I. ricinus and O. moubata ticks become infected with WNV (NY99 strain) through feeding on virus-infected rodent hosts, but only when these hosts were viremic (i.e., systemic transmis- sion). Thirty days after engorgement, we no longer found any evidence of WNV infection in the I. ricinus ticks. This finding suggests that nymphs of this tick species do not support replication of the virus, and therefore are not com- petent vectors for WNV. By extrapolation, the closely related tick species, I. scapularis (the main U.S. Lyme dis- ease vector) is also unlikely to be a competent vector of WNV, although this hypothesis will need to be confirmed experimentally. In contrast, infected O. moubata ticks maintained infec- tious virus for at least 132 days (length of experiment), and WNV persisted transstadially through at least two develop- mental stages. Evidence for tick-to-host transmission of WNV was found in our study, although the level of infec- tion observed (subclinical) makes assessing its importance without further investigation difficult. Whitman and Aitken (1960) observed much higher levels of transmis- sion from WNV-infected (Eg101 strain) O. moubata ticks to day-old chicks but only when very high feeding densi- ties were used (an average of 49 ticks per chick) (16). Although ticks often feed in large numbers on individual hosts (27), tick-to-host transmission appears to be very inefficient when compared to mosquito transmission of WNV (23). Consequently, this mode of transmission is unlikely to be important in the natural transmission cycle of WNV. Perhaps higher levels of infection (and therefore transmission) would be found with ticks that feed on birds, the natural reservoir hosts of WNV. Some avian species exhibit much higher (>1010 PFU/mL serum) and more pro- longed viremia when infected with WNV than the mice used for this investigation (28,29). Although neither of the tick species that we tested are obligate bird feeders, I. rici- nus ticks often feed on pheasants in the United Kingdom (30), and several species of Ornithodoros ticks feed almost exclusively on birds, for example, the O. capensis group of ticks that are established along the southern coast of the United States (31). As members of this group have been shown to be competent vectors for WNV (24), these ticks could represent a reservoir of the virus in the United States. The transmission of flaviviruses such as TBEV and LIV from infected to noninfected ixodid ticks through co- feeding on nonviremic hosts (nonsystemic transmission) is a well-established phenomenon (32). Indeed, this mode of transmission is believed to play a substantial role in the epidemiology of these diseases (27). We tested for co-feed- ing transmission of WNV between infected and uninfected O. moubata ticks. More than 22% of the uninfected ticks were positive for WNV RNA 5 days after co-feeding. A similar percentage of ticks were positive 40 days later, after having molted to the next developmental stage. As co-fed ticks were in contact with the mice for <24 hours, this finding strongly suggests that WNV was nonsystemi- cally transmitted between infected and uninfected ticks, since viremia had insufficient time to develop. Our study represents the first unequivocal report of co-feeding trans- mission by an argasid tick species. Argasid ticks, unlike ixodid ticks, typically feed for <2 hours. Vesicular stomati- tis virus has been transmitted between infected and nonin- fected co-feeding black flies (Simulium vittatum), insects that typically feed for 4–5 min (33). Langerhans cells are believed to be the agents of viral transmission between feeding sites of infected and noninfected co-feeding hard ticks (32,34). Langerhans cells, which are susceptible to WNV infection (35), have been shown to migrate rapidly (within 2 hours) from localized antigen-stimulated epider- mal sites (36). Therefore, these cells could possibly play a similar role in the co-feeding transmission of WNV by soft tick species. Although this study is not exhaustive, it does demon- strate that tick species can become infected with the U.S. strain of WNV through feeding upon infected hosts and through co-feeding with infected ticks on noninfected hosts. In some tick species, WNV can be maintained through the transstadial stages of the tick lifecycle, and infected ticks may be capable of infecting hosts through further feeding. When compared to experimental studies with mosquito species (37–39), ticks are clearly not effi- cient vectors of WNV and therefore are unlikely to be important vectors for WNV in the current U.S. epidemic. However, our results demonstrate that WNV can persist for a comparatively long time in infected ticks and be transmitted between vertebrate hosts; this finding suggests a reservoir potential of ticks for WNV that justifies further investigation. Dr. Lawrie is a postdoctoral researcher in the Nuffield Department of Clinical Laboratory Sciences, University of Oxford. His research interests include identifying and character- izing cancer-associated antigens that are recognized by autolo- gous antibody responses, molecular aspects of the tick-host interface, and transmission of flaviviruses in tick species. 656 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 4, April 2004