No evidence of African swine fever virus replication in hard ticks

Abstract

African swine fever (ASF) is caused by African swine fever virus (ASFV), a tick-borne DNA virus. Soft ticks of the genus Ornithodoros are the only biological vectors of ASFV recognized so far. Although other hard ticks have been tested for vector competence, two commonly found tick species in Europe, Ixodes ricinus and Dermacentor reticulatus, have not been assessed for their vector competence for ASFV. In this study, we aimed to determine whether virus replication can occur in any of these two hard tick species (I. ricinus and/or D. reticulatus), in comparison with O. moubata (the confirmed vector), after feeding them blood containing different ASFV isolates using an improved in vitro system. DNA quantities of ASFV in these infected hard ticks were measured systematically, for 6 weeks in I. ricinus, and up to 8 weeks in D. reticulatus, and the results were compared to those obtained from O. moubata. There was evidence of virus replication in the O. moubata ticks. However, there was no evidence of virus replication in I. ricinus or D. reticulatus, even though viral DNA could be detected for up to 8 weeks after feeding in some cases. This study presents the first results on the possible vector competence of European hard (ixodid) ticks for ASFV, in a validated in vitro feeding setup. In conclusion, given the lack of evidence for virus replication under in vitro conditions, D. reticulatus and I. ricinus are unlikely to be relevant biological vectors of ASFV.

Introduction

African swine fever (ASF) is a highly contagious haemorrhagic disease of swine, caused by African swine fever virus (ASFV), an enveloped double-stranded DNA virus from the family Asfarviridae, genus Asfivirus. Infection usually results in high morbidity and mortality (Costard et al., 2012). Since it was first described (1921), African swine fever has been present in most of sub-Saharan Africa, with most of the incidental introductions into Europe and the Americas eventually resulting in eradication (with the exception of Sardinia). However, after the 2007 ASFV outbreak in Georgia (Rowlands et al., 2008), the disease continued to spread, reaching European neighbouring countries. At the time of writing, ASFV is still circulating in Russia (OIE, 2013), mostly between wild boar and free-ranging domestic pigs (Gogin et al., 2013).

ASFV is a tick-borne virus (Labuda and Nuttall, 2004), and soft ticks (Ornithodoros spp.) have been identified as vectors of ASFV. In Africa, an intricate cycle between warthogs (Phacochoerus africanus), domestic swine, and Ornithodoros ticks (particularly O. moubata), is relevant in the maintenance of an endemic infection (Plowright et al., 1994, Thomson, 1985). On the Iberian Peninsula, Ornithodoros erraticus has also been associated with disease reoccurrence in a sporadic ASF outbreak in Portugal in 1999 (Boinas et al., 2011). Upon ingestion of blood containing ASFV, ticks may develop a persistent infection, with high virus titres in a number of tissues and organs, both in the O. moubata/porcinus complex (Greig, 1972, Kleiboeker et al., 1998, Plowright et al., 1970a, Plowright et al., 1970b) and in O. erraticus ticks (Basto et al., 2006, Endris and Hess, 1992, Endris et al., 1992).

Moreover, ASFV-infected Ornithodoros sonrai ticks (Vial et al., 2007) have been found in the field, and in vitro studies have suggested that several other Ornithodoros species such as O. savignyi (Mellor and Wilkinson, 1985), O. puertoricensis (Hess et al., 1987), O. turicata (Hess et al., 1987) and O. coriaceus (Groocock et al., 1980) can also act as vectors of ASFV. However, none of the latter ticks has yet been confirmed as vector for the transmission of ASFV in the field.

ASFV vector competence of hard ticks (Ixodidae) has been assessed on Rhipicephalus spp. (Sanchez Botija, 1963), Rhipicephalus simus (Plowright, 1977, Plowright et al., 1994), Rhipicephalus bursa (Kovalenko et al., 1967, Plowright et al., 1994), Amblyomma variegatum (Plowright, 1977), Hyalomma spp. (Plowright et al., 1994), Amblyomma americanum and Amblyomma cajennense (Groocock et al., 1980) either by experimental infection or by field collection to determine the presence of ASFV. Field specimens were negative for ASFV, and although ASFV could be detected in R. simus nymphs (Plowright et al., 1994) for up to 5–6 weeks, and in both A. americanum and A. cajennense for 4–7 days after a viraemic blood meal, no hard ticks transmitted ASFV to susceptible pigs after experimental infection. Given that some hard ticks may carry ASFV for some time, it is possible that they may act as mechanical vectors; similar to the European stable fly, Stomoxys calcitrans, which has been shown to mechanically transmit ASFV to pigs up to 24 h post-infective meal (Mellor et al., 1987).

Of the hard ticks studied so far, only Hyalomma spp. and Rhipicephalus bursa are present in Europe (Estrada-Peña et al., 2012). However, no studies have been published addressing replication or maintenance of ASFV in other ticks commonly reported in Europe, such as Ixodes ricinus (Medlock et al., 2013) and Dermacentor reticulatus (Estrada-Peña et al., 2012). Both tick species are known to be involved in the epidemiology of other tick-borne viruses; i.e. with I. ricinus being a vector of tick-borne encephalitis virus (TBEV) and D. reticulatus being a vector of Omsk haemorrhagic fever virus (OHFV) (Jongejan and Uilenberg, 2004, Labuda and Nuttall, 2004).

However, feeding habits differ between the two tick species. Ixodes ricinus ticks feed on a wide range of hosts, including domestic and wild pigs (Farkas et al., 2013). In contrast, D. reticulatus host preference strongly depends on life stage, with adults feeding on larger mammals and nymphs mostly on small mammals (Farkas et al., 2013). Both tick species could be involved in ASFV transmission either via mechanical transmission by interrupted feeding (by adult males) or via biological transmission, either transovarially (via ASFV-infected females) or transstadially (via ASFV-infected I. ricinus nymphs).

In this study, I. ricinus, D. reticulatus, and O. moubata (one of the confirmed vector species) were fed in vitro with blood containing different ASFV isolates. DNA quantities of ASFV in these hard ticks were measured systematically, for 6 weeks in I. ricinus and up to 8 weeks in D. reticulatus, and the results were compared to those found in O. moubata. The purpose of this comparison was to examine if replication can occur in both species of hard ticks. Such knowledge is relevant to better understand the possible role these hard ticks could play as biological or mechanical vectors of ASFV.