Our findings demonstrate that C. sonorensis is a potential competent vector for EHDV-7, as evidenced by the ability of the midge to become infected following ingestion of a blood meal from an experimentally infected WTD and subsequent bite transmission of EHDV-7 from C. sonorensis to a naive WTD. The observed viral dynamics, clinical abnormalities, and postmortem findings in the challenge animal were consistent with previous reports of HD in WTD, as reviewed by Howerth et al.. Four days after feeding infected midges on the naïve WTD, the animal had a high-titer viremia and the exhibited clinical signs and the incubation period were consistent with HD in this species[28, 29]. Lymphopenia, erythrocytosis, hypoproteinemia, thrombocytopenia, and prolongation of PT and APTT were observed, all of which are common abnormalities in WTD infected with EHDV and/or BTV[25, 29]. The coagulopathy in this animal was severe, based on both the clinical pathology findings, as well as clinical observation of bleeding tendencies at the time of euthanasia on 9 dpf.
Only 27 of the 44 midges that took a blood meal from the naïve deer were found to be infected at the time of feeding, thus it may be assumed that these 27 midges were responsible for the EHDV transmission. However, based on the titration data, only a portion of infected midges were likely to be competent (12/27, 44%). Thus, it is suspected that one or more of these 12 midges were responsible for transmission. While only 44 of 100 midges had a visible blood meal after the transmission attempt, we cannot rule out the possibility of probing without blood feeding by any of the 14 other potentially competent midges, which could have potentially contributed to virus transmission. The efficiency of EHDV transmission to WTD by C. sonorensis has not been investigated, nor is a minimum infectious dose known. The bite from a single infected C. sonorensis has been shown to transmit BTV-1 to domestic sheep[30, 31] and similar studies are necessary to better understand virus transmission to WTD.
Our results suggest that, for EHDV-7, infection of blood fed midges is less efficient (4.6%, 6/130) when viremia was < 104.0 TCID50/ml. This lower infection rate is not surprising, considering the poor virus recovery rate on 0 dpf (33%; 5/15) when midges were provided a lower-titer blood meal (Table1). A previous experimental infection of C. sonorensis with EHDV encountered a similar low infection rate when the blood meal titer ranged from 102.1 – 103.0 TCID50/ml. Our study did not examine an infection threshold, but the finding is interesting and highlights our lack of understanding of some very fundamental factors regarding the transmission of EHDV. Additionally, we do not know for how long a viremic WTD is infectious to Culicoides, specifically when viremia is of low titer (<102.3 TCID50/ml). It is known that WTD experimentally infected with EHDV can be viremic for as long as 56 days post-inoculation; however, only transiently is the viremia comparable to the peak titers in our study (>107.0 TCID50/ml) which resulted in high infection rates in the midges. Previous studies show that peak viremia in WTD generally occurs 5–7 days post inoculation and blood virus titers of 105.0–6.0 TCID50/ml may be present for as long as seven days, whereas titers > 106.0 TCID50/ml are generally only present for 2–3 days early in the infection[25, 32]. After the appearance of neutralizing antibodies the blood virus titer generally declines sharply 8 to 12 dpi. Future studies should examine the epidemiological significance of the prolonged low-titer viremia in WTD. Although the efficiency of transmission from these animals to Culicoides has not been explored, it is important to consider additional factors regarding transmission efficiency. For instance, the attack rate on WTD by Culicoides midges can be remarkably high. In a study by Smith and others, >10,000 C. debilipalpis (formerly lahillei) were aspirated from a WTD on five of six autumn mornings, with a maximum single collection of 20,840 midges.
The World Health Organization (WHO) has established criteria for the designation of an arthropod as a confirmed vector of an arbovirus: 1) recovery of virus from non-blood-fed wild-caught specimens, 2) demonstration of the ability to become infected experimentally, 3) demonstration of biological transmission, and 4) an accumulation of field evidence confirming an association of the infected arthropods and the appropriate vertebrate host. This study has fulfilled the second and third WHO criteria, thus, we conclude that C. sonorensis should be considered a potential vector of EHDV-7. Since EHDV-7 is exotic to North America, criteria 1 cannot be fulfilled. Although field research is scarce, previous studies have provided some evidence for both criteria 1[18, 35, 36] and 4, as they relate to C. sonorensis serving as a vector of EHDV serotypes endemic to the US for WTD.
Although there is significant field evidence to show that C. sonorensis is the primary vector of BTV for sheep and cattle throughout most of the US, the evidence is not as strong for EHDV and BTV transmission to WTD in wild habitat. With the exception of a previous study in Kentucky, where C. sonorensis was the predominate species trapped in captive WTD pens during an HD outbreak, C. sonorensis has been absent or present in very low numbers in subsequent field studies that have been performed in WTD habitat or on captive WTD facilities, using WTD as bait[33, 37–39]. In fact, most field studies directly related to WTD suggest that species other than C. sonorensis are likely to also be involved in EHDV and BTV transmission to WTD, specifically in the southeastern US. Based primarily on midge abundance, seasonal occurrence, and host preference, it has been suggested that C. debilipalpis, C. stellifer, C. obsoletus, C. paraensis, and C. spinosus warrant further investigation as potential vectors of EHDV and BTV to WTD in the southeastern US[33, 37, 39] and C. debilipalpis has been shown experimentally to support replication of EHDV-2. Furthermore, multiple Culicoides spp. other than C. sonorensis and C. insignis, both confirmed BTV vectors, have been listed as suspect or potential vectors of BTV to cattle and sheep in the southeastern US. The above mentioned studies highlight the gaps in our knowledge of the transmission of EHDV and BTV among free-ranging WTD in certain parts of the US where EHDV and BTV are enzootic.
Despite gaps in our knowledge regarding EHDV and BTV transmission among wild ruminants in some portions of the US, C. sonorensis was chosen in this study for multiple reasons: 1) it represents the only confirmed vector of EHDV in North America, 2) this species is widely distributed throughout much of the US and commonly feeds on domestic ruminants, thus its competence represents a large geographic risk for outbreaks and establishment following an introduction, and 3) C. sonorensis has been colonized, making it possible to test large numbers of insects that consistently feed in captivity.