Milan Labuda

Incidence from coincidence: patterns of tick infestations on rodents facilitate transmission of tick-borne encephalitis virus

Tick-borne encephalitis (TBE) virus has a highly focal distribution through Eurasia. Endemic cycles appear to depend on the transmission of non-systemic infections between ticks co-feeding on the same rodent hosts. The particular features of seasonal dynamics and infestation patterns of larval and nymphal Ixodes ricinus, but not Dermacentor reticulatus, from 4 regions within TBE foci in Slovakia, are such as to promote TBE virus transmission. The distributions of larvae and nymphs on their principal rodent hosts are highly aggregated and, rather than being independent, the distributions of each stage are coincident so that the same ca. 20% of hosts feed about three-quarters of both larvae and nymphs. This results in twice the number of infectible larvae feeding alongside potentially infected nymphs compared with the null hypothesis of independent distributions. Overall, co-feeding transmission under these circumstances brings the reproductive number (R0) for TBE virus to a level that accounts quantitatively for maintained endemic cycles. Essential for coincident aggregated distributions of larvae and nymphs is their synchronous seasonal activity. Preliminary comparisons support the prediction of a greater degree of coincident seasonality within recorded TBE foci than outside. This identifies the particular climatic factors that permit such patterns of tick seasonal dynamics as the primary predictors for the focal distribution of TBE.

Association of Borrelia afzelii with rodents in Europe

Borrelia burgdorferi sensu lato (s.l.) is maintained in nature by complex zoonotic transmission cycles, involving a large variety of vertebrates as hosts and hard ticks of the genus Ixodes as vectors. Recent studies suggest that the genospecies of B. burgdorferi s.l. and sometimes their subtypes are propagated by different spectra of hosts, mainly birds and rodents. In order to test the concept of host-association, we analysed the relationships between Borrelia genospecies, rodent hosts and I. ricinus ticks in an endemic focus of Lyme borreliosis in western Slovakia. Rodents and questing ticks were collected at a forested low land locality near Bratislava. Tick infestation levels on rodents were determined, and spirochaete infections in ticks and in ear punch biopsies were analysed by PCR followed by genotyping. Mice were more heavily infested with ticks than bank voles, and a higher proportion of mice was infected with spirochactes than voles. However, the infectivity of soles was much higher than that of mice. The vast majority of infections detected in the skin and in ticks feeding on the rodents represented B. afzelii. In contrast, more than half of all infections in questing ticks collected in the same region of Slovakia were identified as B. valaisiana and B. garinii. In conclusion, whilst the study reveals that mice and voles play different quantitative roles in the ecology of Lyme borreliosis, it demonstrates that B. afzelii is specifically maintained by European rodents, validating the concept of host-association of B. burgdorferi s.l.

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

ABSTRACT In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

Tick-borne viruses

At least 38 viral species are transmitted by ticks. Virus-tick-vertebrate host relationships are highly specific and less than 10% of all tick species (Argasidae and Ixodidae) are known to play a role as vectors of arboviruses. However, a few tick species transmit several (e.g. Ixodes ricinus, Amblyomma variegatum) or many (I. uriae) tick-borne viruses. Tick-borne viruses are found in six different virus families (Asfarviridae, Reoviridae, Rhabdoviridae, Orthomyxoviridae, Bunyaviridae, Flaviviridae) and at least 9 genera. Some as yet unassigned tick-borne viruses may belong to a seventh family, the Arenaviridae. With only one exception (African swine fever virus, family Asfarviridae) all tick-borne viruses (as well as all other arboviruses) are RNA viruses. Tick-borne viruses are found in all the RNA virus families in which insect-borne members are found, with the exception of the family Togaviridae. Some tick-borne viruses pose a significant threat to the health of humans (Tick-borne encephalitis virus, Crimean-Congo haemorrhagic fever virus) or livestock (African swine fever virus, Nairobi sheep disease virus). Key challenges are to determine the molecular adaptations that allow tick-borne viruses to infect and replicate in both tick and vertebrate cells, and to identify the principal ecological determinants of tick-borne virus survival.

Exposed and concealed antigens as vaccine targets for controlling ticks and tick‐borne diseases

Tick vaccines derived from Bm86, a midgut membrane‐bound protein of the cattle tick, Boophilus microplus, are currently the only commercially available ectoparasite vaccines. Despite its introduction to the market in 1994, and the recognized need for alternatives to chemical pesticides, progress in developing effective antitick vaccines (and ectoparasite vaccines in general) is slow. The primary rate‐limiting step is the identification of suitable antigenic targets for vaccine development. Two sources of candidate vaccine antigens have been identified: ‘exposed’ antigens that are secreted in tick saliva during attachment and feeding on a host and ‘concealed’ antigens that are normally hidden from the host. Recently, a third group of antigens has been distinguished that combines the properties of both exposed and concealed antigens. This latter group offers the prospect of a broad‐spectrum vaccine effective against both adults and immature stages of a wide variety of tick species. It also shows transmission‐blocking and protective activity against a tick‐borne pathogen. With the proliferation of molecular techniques and their application to vaccine development, there are high hopes for new and effective antitick vaccines that also control tick‐borne diseases.

Non-viraemic transmission of tick-borne encephalitis virus : a mechanism for arbovirus survival in nature

The vectors of arthropod-borne viruses (arboviruses) become infected by feeding on the viraemic blood of an infected animal. This theory is based on transmission studies involving artificial infection of vertebrate hosts by syringe inoculation. To reproduce natural conditions of virus transmission, infected and uninfected vectors (ticks) of tick-borne encephalitis virus, the most important arbovirus in Europe, were allowed to feed together on uninfected wild vertebrate hosts. The greatest numbers of infected ticks were obtained from susceptible host species that had undetectable or very low levels of viraemia. The results suggest that ‘nonviremic transmission’ is an important mechanism for the survival of certain arboviruses in nature.

Tick-host interactions: saliva-activated transmission.

The skin site at which ticks attach to their hosts to feed is the critical interface between the tick and its host, and tick-borne pathogens. This site is highly modified by the pharmacologically active molecules secreted in tick saliva. For pathogens, it is an ecologically privileged niche that many exploit. Such exploitation is referred to as saliva-activated transmission (SAT) - the indirect promotion of tick-borne pathogen transmission via the actions of bioactive tick saliva molecules on the vertebrate host. Here we review evidence for SAT and consider what are the most likely candidates for SAT factors among the tick pharmacopoeia of anti-haemostatic, anti-inflammatory and immunomodulatory molecules identified to date. SAT factors appear to differ for different pathogens and tick vector species, and possibly even depend on the vertebrate host species. Most likely we are searching for a suite of molecules that act together to overcome the redundancy in host response mechanisms. Whatever they turn out to be, the quest to identify the tick molecules that mediate SAT is an exciting one, and offers new insights to controlling ticks and tick-borne diseases.

An Antivector Vaccine Protects against a Lethal Vector-Borne Pathogen

Vaccines that target blood-feeding disease vectors, such as mosquitoes and ticks, have the potential to protect against the many diseases caused by vector-borne pathogens. We tested the ability of an anti-tick vaccine derived from a tick cement protein (64TRP) of Rhipicephalus appendiculatus to protect mice against tick-borne encephalitis virus (TBEV) transmitted by infected Ixodes ricinus ticks. The vaccine has a “dual action” in immunized animals: when infested with ticks, the inflammatory and immune responses first disrupt the skin feeding site, resulting in impaired blood feeding, and then specific anti-64TRP antibodies cross-react with midgut antigenic epitopes, causing rupture of the tick midgut and death of engorged ticks. Three parameters were measured: “transmission,” number of uninfected nymphal ticks that became infected when cofeeding with an infected adult female tick; “support,” number of mice supporting virus transmission from the infected tick to cofeeding uninfected nymphs; and “survival,” number of mice that survived infection by tick bite and subsequent challenge by intraperitoneal inoculation of a lethal dose of TBEV. We show that one dose of the 64TRP vaccine protects mice against lethal challenge by infected ticks; control animals developed a fatal viral encephalitis. The protective effect of the 64TRP vaccine was comparable to that of a single dose of a commercial TBEV vaccine, while the transmission-blocking effect of 64TRP was better than that of the antiviral vaccine in reducing the number of animals supporting virus transmission. By contrast, the commercial antitick vaccine (TickGARD) that targets only the ticks midgut showed transmission-blocking activity but was not protective. The 64TRP vaccine demonstrates the potential to control vector-borne disease by interfering with pathogen transmission, apparently by mediating a local cutaneous inflammatory immune response at the tick-feeding site.

Enhancement of tick‐borne encephalitis virus transmission by tick salivary gland extracts

Abstract. To investigate the role of ticks in TBE virus transmission, salivary gland extract (SGE) was derived from partially fed female Ixodes ricinus, Dermacentor reticulatus and Rhipicephalus appendiculatus ticks. Guinea‐pigs were infested with uninfected R.appendiculatus nymphs and inoculated with a mixture of TBE virus and SGE or with virus alone. The number of ticks which on average acquired virus from feeding on animals inoculated with TBE virus and SGE from partially fed ticks was 4‐fold greater than the number that became infected by feeding on animals inoculated with virus alone or virus plus SGE from unfed I.ricinus. Viraemia was detected in 67% of guinea‐pigs inoculated with virus plus SGE compared to 30% of guinea‐pigs inoculated with virus alone. Virus titres in the blood were similar for both groups of animals [range 2.0‐2.8 log10 plaque‐forming units (PFU)/ml of blood]; however, the number of ticks that became infected was significantly higher on animals inoculated with virus plus SGE from partially fed ticks. No significant difference was observed with respect to the tick species used to derive SGE. The results indicate that TBE virus transmission is enhanced by factor(s) associated with the salivary glands of feeding ticks, and that these factor(s) may facilitate efficient transmission of TBE virus between infected and uninfected ticks even when they feed on hosts that have no detectable viraemia.

Blackbirds and song thrushes constitute a key reservoir of Borrelia garinii, the causative agent of borreliosis in Central Europe.

ABSTRACT Blackbirds (Turdus merula) and song thrushes (Turdus philomelos) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as Borrelia garinii and Borrelia valaisiana. We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.