Effect of the salivary gland and midgut extracts from Ixodes ricinus and Dermacentor reticulatus (Acari: Ixodidae) on the growth of Borrelia garinii in vitro.

The interaction between pathogen and tick vector seems to be crucial for vector-borne pathogens. For instance, Borrelia burgdorferi s.l., the Lyme disease agent, must overcome at least two main barriers in the tick vector body to be effectively transmitted: the midgut and the salivary glands. It has been found that salivary gland extract (SGE) of ixodid ticks affects the immune system of vertebrate hosts (Ribeiro J.M.C. 1989: Exp. Appl. Acarol. 7: 15–20; Kuthejlová M., Kopecký J., Štěpánová G., Macela A. 2001: Infect. Immun. 69: 575–578) and also contributes to the transmission of B. afzelii to the host (Pechová J., Štěpánová G., Kovář L., Kopecký J. 2002: Folia Parasitol. 49: 153–159). In earlier studies it was observed that borreliae did not occur in host-seeking Dermacentor reticulatus (Fabricius, 1794) in contrast to Ixodes ricinus (L., 1758) (Hubálek Z., Halouzka J., Juřicová Z. 1998: Folia Parasitol. 45: 67–72), and that B. garinii survived in I. ricinus after experimental inoculation while it rapidly disappeared from D. reticulatus (Mátlová L., Halouzka J., Juřicová Z., Hubálek Z. 1996: Folia Parasitol. 43: 159–160). The aim of this study was to examine the effect of SGE and midgut extract (MGE) from both I. ricinus and D. reticulatus on the growth, motility and morphology of the B. garinii spirochaete in vitro. This might address the question of vector competence of these two tick species for B. burgdorferi s.l. at the level of their compartments (salivary glands and midgut). Salivary glands and midgut were removed from 40 unfed female I. ricinus and 20 unfed female D. reticulatus. This study attempted to simulate the effect of SGE and MGE on borrelial growth under conditions of host-seeking ticks, and therefore unfed individuals only were used. Dissected organs were homogenized with a small glass blender in phosphatebuffered saline pH 7.0 (PBS; Oxoid), placed in microtubes, and centrifuged at 9,000 g for 10 min. Clarified extracts were sterilized by filtration through the 0.2 μm Nanosep MF centrifugal device (Pall Corporation) and stored at –20°C. Final protein content (μg/ml) of the extracts was estimated (Bradford J. 1976: Anal. Biochem. 72: 248–254) as 32.8 (SGE) and 30.9 (MGE) in I. ricinus, and 29.3 (SGE) and 39.2 (MGE) in D. reticulatus. In the experiments, 100 μl of each extract (or PBS in the control) were mixed with 100 μl of a 3day culture of B. garinii strain BR 14 (about 10 spirochaetes per ml) in BSK-H medium with 6% of rabbit serum (Sigma) in 96-well U-bottomed sterile microplates (Sarstedt), and covered with a sterile sealing film (Denville Scientific). The microplates were placed in a 33°C incubator for 11–12 days. Concentration of motile spirochaetes (the number of motile cells/ml of medium) was determined at intervals (0, 2, 4, 7, 9 and 11 days in I. ricinus, and 0, 2, 5, 7, 9 and 12 days in D. reticulatus), using darkfield microscopy. (1) Estimation of per cent motility was determined in 3 wells per variant, when 100 randomly selected spirochaetes were screened for motility per well. (2) Concentration of all spirochaetes (motile plus nonmotile) was estimated in 10-μl volumes of appropriately diluted cultures on a microscope slide with a 20 × 20 mm coverslip (Hubálek Z., Halouzka J., Heroldová M. 1998: J. Med. Microbiol. 47: 929–932); for each variant, 3 wells with 5 counts (total, 15 repetitions) were used. The data were analysed with the two sample t-test using SOLO (BMDP Statistical Software). Significant differences in the concentration of motile spirochaetes were considered at P < 0.01. The results are summarised in Tables 1 to 3. The proportion of motile spirochaetes decreased more slowly with the extracts of Ixodes ricinus than in corresponding control (C) since day 9 post inoculation (p.i.). On the other hand, the percentage of spirochaetal motility decreased more rapidly with SGE and MGE from D. reticulatus than in C since day 9 p.i. With I. ricinus, the concentration of motile spirochaetes increased significantly from days 2 to 11 (p.i.) with both SGE and MGE compared to C. In addition, the growth of spirochaetes was enhanced to a greater degree with SGE than with MGE on days 4, 7 and 9 p.i. With D. reticulatus, a significant increase in concentration of motile spirochaetes was only detected with SGE (compared to C) on day 5 p.i., while a marked decrease in concentration of motile spirochaetes was observed on day 9 p.i. with MGE, and on day 12 p.i. with both extracts compared to C. Moreover, many spirochaetes grown in the presence of D. reticulatus MGE were morphologically changed (compared to C and SGE) by 9 days p.i.; the cells were damaged (e.g., less discernible walls), shorter, and with a lower number of coils. The effect of SGE and MGE on the growth of B. garinii spirochaetes in vitro thus differed between the two tick species tested. While extracts derived from I. ricinus (a competent vector for Lyme borreliosis) stimulated growth significantly, extracts from D. reticulatus (a non-competent species) did not affect the growth of borreliae markedly, or even inhibited their growth on days 9 (MGE) and 12 p.i. (MGE and SGE). Our results therefore indicate that the tick compartment extracts surprisingly need not be inhibitory for pathogen survival in the body of even non-competent tick species like D. reticulatus in a short-term exposure. In such species, the role of a barrier in FOLIA PARASITOLOGICA 50: 159–160, 2003