Potential significance of transovarial transmission in the circulation of tick-borne encephalitis virus.

Transovarial transmission of tick-borne encephalitis (TBE) virus in Ixodes ricinus (L., 1758), its vector tick species, has been shown both experimentally and epidemiologically to be one of the important modes of long-term virus circulation in a natural focus (Benda R. 1958: J. Hyg. Epidemiol. Microbiol. Immunol. (Prague) 2: 314-330; Řeháček J. 1962: Acta Virol. 6: 220-226; Danielová V., Holubová J. 1991: In: F. Dusbábek, V. Bukva (Eds.): Modern Acarology. Academia, Prague and SPB Academic Publishing bv, The Hague, Vol. 2, pp. 7-10). From experimental data (maternal transmission around 20%; filial infection 0.23-0.75%), it is possible to imagine the quantitative ratios of such transmission from one generation of ticks to another. The results cited were obtained from experiments in animals as well as in cells in vitro, i.e., only values having exceeded the infection threshold could be detected. Sub-threshold values that occur in nature appear negative, although under natural conditions they may contribute to virus circulation. The discovery of saliva-activated transmission (Nuttall P.A., Jones L.D. 1991: In: F. Dusbábek, V. Bukva (Eds.): Modern Acarology. Academia, Prague and SPB Academic Publishing bv, The Hague, Vol. 2, pp. 3-6), and non-viraemic transmission of TBE virus from infected to noninfected I. ricinus ticks during co-feeding on a host (Labuda M., Jones L.D., Williams T., Danielová V., Nuttall P.A. 1993a: J. Med. Entomol. 30: 295-299), provided novel alternative views on circulation of this virus in nature that call for further observation of what degree larvae participate in circulation of the virus. In identifying conditions for the existence of natural foci (Randolph S.E., Miklisová D., Lysý J., Rogers D.J., Labuda M. 1999: Parasitology 118: 177-186; Labuda M., Randolph S.E. 1999: Zentralbl. Bakteriol. 289: 513-524), the authors propose that a fundamental condition for the existence of a natural focus is the synchronisation of aggregation in the co-feeding of larvae and nymphs. Thus under the precondition that virus is transmitted from nymphs to uninfected larvae, only the spring season is considered to be important. Feeding in the autumn season is considered unimportant, as is transovarial transmission. These hypotheses are the basis for consideration in further papers (Randolph S.E. 2000: Adv. Parasitol. 47: 217-243; Randolph S.E., Green R.M., Peacey M.F., Rogers D.J. 2000: Parasitology 121: 1523; Randolph S.E. 2001: Philos. Trans. R. Soc. Lond. B 356: 1045-1056), hence we wished to test through experiments in nature, the degree and manner in which larvae participate in virus circulation. In localities of the South Bohemian natural focus of TBE (Czech Republic) where, in the preceding year, TBE virus was repeatedly isolated from tick nymphs and imagoes, we trapped small terrestrial mammals for serological investigations and for ticks that were investigated for the presence of TBE virus. Most of the ticks were I. ricinus larvae, the other developmental stages being represented in negligible numbers. Small terrestrial mammals were trapped in two periods, May 22-25 and October 2-5, 2001, in selected localities and habitats where ticks had been collected by flagging and investigated the year before (Danielová V., Holubová J., Daniel M. 2002: Exp. Appl. Acarol. 26, in press). Small mammals were live-trapped in bridge metal traps, in each collecting site, for two consecutive nights. All parasites were collected from each animal individually; ticks were selected from the material, identified, counted and cooled. Trapping occurred in the area within 48°44’-48°54’N and 14°29’-14°35’E in the localities of Borovany (border zone between tall mixed woods [oak, pine, spruce] and stand of young oak trees), Kaplice (small isolated deciduous wood stands [oak, birch, lime trees and shrubs of Corylus avellana] surrounded by cornfields), and Římov (ravine in mixed wood, bottom covered by abundant herb layer). Ixodes ricinus ticks (mostly larvae), after removal from hosts were cooled thus they were live before processing. The ticks were investigated in virus isolation experiments using the porcine cell culture, PS, and examined by indirect immunofluorescence (IIF). Larvae removed from each host were processed separately in numbers of 1 to 61. In cases where they were also present, nymphs or a female were processed individually. Irrespective of their numbers, each batch of ticks was triturated in 1 ml Leibovitz medium L-15 supplemented with 3% bovine serum, 200 μg gentamycin and 2.5 μg amphotericin B. After incubation for 1.5 h at 4°C the suspension was centrifuged 15 min at 5000 rpm, and 0.1 ml supernate inoculated onto two incubation chamber slides for the IIF test and into two wells of a 96-well plate for further passaging; the third passage was carried out on cover slips in Leighton tubes for another IIF test. Additionally, in the first passage a plaque assay was carried out (0.2 ml in a 24-well plate) as previously described (Danielová et al. 2002, op. cit.). In the spring season, 27 small mammals were trapped, ticks being found on 18 of them; in the autumn season, 76 small mammals were trapped, 29 of which were parasitised by ticks. Species composition was the following: Apodemus flavicollis (Melchior), A. sylvaticus (Linnaeus), Clethrionomys glareolus FOLIA PARASITOLOGICA 49: 323-325, 2002