Rhipicephalus appendiculatus (Acari: Ixodidae): dynamics of Thogoto virus infection in female ticks during feeding on guinea pigs
Introduction
Successful transmission of arboviruses from vector to host requires that imbibed virus survives in the vector’s gut for a period of time, enters the hemolymph through the gut wall and, during a subsequent meal, enters the saliva through the salivary gland epithelium. For certain vectors (most tick species) a molting period, involving extensive tissue histolysis and renewal, frequently intervenes during this whole process. Little is known about the specific mechanisms that permit survival of the pathogen in a vector undergoing molting, and about the vector’s cellular mechanisms that influence transmission to the host. These factors probably vary significantly from one vector–pathogen system to another (Nuttall et al., 1994; Schwan, 1996).
In an earlier study, THO virus was not detected in the salivary glands of trans-stadially infected Rhipicephalus appendiculatus until the ticks had fed on a host for about 7 days (Booth et al., 1989). Moreover, the latter study suggested that virus resides primarily in the synganglion by the time the adult molt is complete. This observation was in harmony with an assumption that the synganglion probably does not suffer histolysis during the molting period (Till, 1961). Nevertheless, such infected ticks apparently are able to transmit virus to a susceptible host (Syrian hamster) as early as 24 h following attachment (Davies, 1988), and accelerated transmission of THO virus was demonstrated when the feeding of infected ticks was interrupted for a period of up to 28 days (Wang and Nuttall, 2001). How ticks can transmit virus via the salivary secretions during a time that no virus can be detected in the salivary glands was an enigma for some years. Then we observed that virus can pass directly from the hemolymph to the saliva through the salivary gland epithelium independently of viral replication within the tick (Kaufman and Nuttall, 1996); the mechanism of this passage of virus through the salivary gland epithelium remains unknown.
It seems surprising that virus would persist for several weeks in a molting tick and be excluded from the very tissue (salivary gland) that will ultimately be responsible for viral transmission. One possibility is that virus is present in the salivary glands, but the detection system used by Booth et al. (1989; immunofluorescence by light-microscopy and immuno-gold labelling by electron microscopy), although eminently suitable for determining the intracellular localization of virus, may not have been sensitive enough to detect low virus titers in the tissue. Thus we re-examine here the question of trans-stadial virus persistence, and virus titers in various organs throughout a 7-day feeding cycle on guinea pigs, using a plaque assay to quantify infectious virus.
Section snippets
Source of virus and infected ticks
Rhipicephalus appendiculatus nymphs were taken from a long-established colony maintained at CEH-Oxford as described by Jones et al. (1988). The Sicilian isolate of THO virus (106–107 PFU/ml Eagle’s minimal essential medium supplemented with 6–10% fetal calf serum) was prepared as described by Davies et al. (1986). Nymphs were fed on guinea pigs (Dunkin Hartley strain) and engorged ticks were inoculated parenterally with virus as described by Davies et al. (1986). When such inoculated ticks molt
Controls for loosely associated virus
‘Wash medium control’ was titrated for THO virus in 23 samples and ‘exogenous virus control’ was titrated for virus in six samples. None of these controls contained a detectable titer of virus. On the basis of these observations, we believe that the virus titers reported in this study represent virus endogenous to the tissue.
Analysis of percent infection of tissues
The percent of tissues infected with virus from days 0 to 7 of feeding varied among tissue types (Table 1). On day 0, about 14% of the salivary gland pairs were infected.
Discussion
The time at which an arbovirus appears in the salivary gland of its vector is critically important epidemiologically. This period determines the safety margin during which an infected vector can be detached from a host without the host becoming infected. Booth et al. (1989) suggested that THO virus could not be detected in the salivary gland of unfed, infected female R. appendiculatus. Instead, they detected virus primarily in the synganglion prior to feeding. Only towards the very end of the
Acknowledgements
This study was supported by the Natural Environment Research Council (UK). Travel expenses of W.R.K. to Oxford were supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada. We are most grateful to Dr. Rich Moses, Department of Biological Sciences, University of Alberta, for performing the logistical regression analysis for us.
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