Each of the 4 virus strains (BTV-2 wt, BTV-2 rg, BTV-8 wt and BTV-8 rg) used in this study efficiently infected the inoculated pregnant ewes which all became viremic and, in most cases, the presence of viral RNA in the blood was maintained for an extended period (>25 days) including the time of parturition (Figures 2 and3). In addition, all of the infected ewes seroconverted against the virus (Figure 4). A major focus of this study has been the transplacental transmission of the different viruses and this will be discussed in detail below. In addition, the unexpected horizontal transmission of BTV-2 is considered. The results show that the wt and rg strains of each serotype had very similar characteristics to each other but the BTV-2 and BTV-8 viruses show some differences. It was noted that BTV-2 wt produced more marked clinical signs (including respiratory distress and nasal discharge) than any of the other viruses and both the BTV-2 wt and the BTV-8 wt viruses produced more marked pyrexia than the rescued (rg) viruses. However, the significance of this is not clear since all animals acquired BTV RNA in their blood and seroconverted against the virus.
Transplacental transmission of BTV-2 and BTV-8 in sheep
As described previously, transplacental transmission, in both cattle and sheep, is a documented property of the North European BTV-8 strain and also of BTV modified live vaccines or laboratory adapted strains that have been extensively passaged in eggs or tissue culture[9, 17, 18]. In contrast, earlier work with other field strains of BTV (not including BTV-2) did not show this property[19, 20, 27].
In this study we now show that a field strain of BTV-2, minimally passaged in tissue culture, is able to cross the placental barrier in experimentally infected ewes. The BTV-2 wt virus had been passaged only once in KC cells and once in a sheep cell line and it is, therefore not expected to have changed its phenotype due to mutations accumulated after extensive cell culture passaging as shown for tissue culture adapted or vaccine strains. Both BTV-2 wt and BTV-2 rg were capable of transplacental transmission in approximately 50% of the infected ewes. Interestingly, transplacental transmission of BTV-2 occurred even in the negative control ewe (sheep 4) that had been infected, inadvertently, during co-housing with the experimentally infected sheep. Thus, the transplacental transmission of BTV-2 does not appear to be an artifact of the experimental inoculation procedure per se (i.e. using sub-cutaneous inoculation of high levels of virus).
There have been no previous reports on the transplacental transmission of field strains of BTV-2 but the clear detection of viremia in the lambs as well as the presence of BTV RNA in blood and tissue samples clearly demonstrate this process for both the BTV-2 wt and BTV-2 rg strains used in this study (Table 2 and Figure 5). Recently, it has been reported that an egg and cell culture-adapted strain of BTV-2 (used as a modified live vaccine) is able to cross the placenta of sheep and result in infection of foetuses. It remains to be determined whether BTV-2, which has only been passaged within animals and transmitted by transfer of blood, is also able to cross the placental barrier. The BTV-2 wt strain used in the current study was isolated from a BTV-infected sheep in Sardinia prior to the use of modified live vaccine strains (from South Africa) on this island. In addition, it has been shown, using RT-qPCR assays and genome sequencing, that this BTV-2 wt strain is distinct from the BTV-2 vaccine strain (G. Savini, unpublished results).
Transplacental transmission of the BTV-8 wt was observed at a lower frequency than expected, in only 1 of the 6 ewes, and in none of the ewes infected with the BTV-8 rg. Previous studies have indicated that transplacental transmission of BTV-8 wt has occurred in sheep[23, 24] and cattle[20, 35, 36] but the frequency can be quite variable varying from 0-69% in sheep[21, 24, 25] and 20-35% in cattle[22, 26, 36]. This may depend, in part, on the time of infection in relation to the gestation period. It should be noted that BTV infection of ewes during the early stages of gestation (5th and 6th week) can have serious neurological consequences for the lambs (see[9, 18]) when transplacental transmission (e.g. with vaccine strains) occurs. However, studies with an attenuated BTV-23 virus (passaged 20 times in Vero cells) in Merino sheep showed that vaccination with this live virus (which produced no clinical disease) during the late stage of gestation had no apparent effect on the production of lambs despite causing high losses in the first and second thirds of the pregnancy. These data are consistent with the results observed here using infection with both the BTV-2 and BTV-8 strains since no abnormalities were observed in any of the lambs.
Each of the BTVs tested clearly infected the virus inoculated sheep since all had infectious virus and BTV RNA detectable in the blood and all of the inoculated animals seroconverted against BTV. The serotype specific RT-qPCR assays confirmed that the animals had indeed been infected with the serotype of BTV used for their inoculation (Table 1). At the time of birth, only one of the lambs (number 27) had conclusively seroconverted against BTV but all lambs from BTV-infected ewes rapidly seroconverted following ingestion of colostrum. These results were consistent with previous studies indicating that the presence of anti-BTV antibodies in lambs depends on the time point during gestation at which the BTV infection occurred, when the infection is late (as in this study) then lambs can be born viremic with or without anti-BTV antibodies[9, 24]. It was apparent that the severity of clinical signs (elevated temperature and respiratory distress) together with the level and period of viremia (when infectious virus can be isolated) was much greater in the BTV-2 inoculated sheep than in the BTV-8 infected sheep. It may be that these features are relevant to the probability of transplacental transmission. However, it is noteworthy that similar levels and maintenance of BTV RNA in the blood, as measured by RT-qPCR, were observed for all of the viruses tested and each virus also induced seroconversion. Thus the level and maintenance of BTV RNA within the blood are clearly not indicative of the ability of BTV to cross the placenta.
There has been some discussion previously about the possible role of co-infection of BTV with pestiviruses (e.g. bovine viral diarrhea virus (BVDV) and border disease virus (BDV)) in transplacental transmission. Backx et al. found a possible association between the timing of seroconversion to BVDV in a single dam and the production of a BTV positive calf. However, Zanella et al. showed that 128 (16%) fetuses/calves analyzed in their study were BTV-8 positive but only 2 out of the 763 calves from dams that were tested were found to be coinfected with BTV-8 and BVDV while 9 fetuses/calves were exclusively BVDV positive. Thus these authors concluded that “BVDV did not play an important role as a cross-barrier enhancer”. We are unaware of any studies demonstrating a link between pestivirus infection and BTV transplacental transmission in sheep. In Denmark, the sheep are free from BDV and BVDV infection and, indeed, all sera from each of the ewes were seronegative in a BVDV ELISA (which cross reacts with antibodies against other pestiviruses) at the start of the experiment (day 0). For one group of animals (sheep 5–10), inoculated with BTV-2 wt, it has been shown that these animals seroconverted against pestiviruses by 29 dpi (data not shown). Based on a set of differentiating neutralization tests, it appears that the antibodies detected in these 6 animals were generated against BDV rather than BVDV. All of the other ewes remained seronegative for pestiviruses. Thus it seems that the BTV-2 wt inoculum was contaminated with BDV. We do not believe this has influenced the results significantly for several reasons. As indicated above, sheep 4, one of the control sheep, unexpectedly became infected with BTV-2 without receiving the same inoculum, however, this ewe did not seroconvert against pestiviruses but produced a BTV positive lamb (Table 2). Furthermore, 50% of the BTV-2 rg inoculated group (as with the BTV-2 wt group) also produced BTV infected lambs but none of these animals seroconverted against pestiviruses (data not shown). Thus there was no apparent linkage between pestivirus infection and BTV-2 transplacental transmission in this study.
Oral transmission of BTV-2 in sheep?
An interesting feature of this study was the observation that one ewe (sheep 4), which was in the control group, became infected with BTV-2 (see Figures 2,3 and4). This sheep shared the same airspace with the sheep inoculated with BTV-2 wt but was in a different pen separated by a 70 cm wide corridor. The infection in sheep 4 was delayed by approximately one week compared to the directly inoculated animals and thus it appears that transmission of the virus has occurred between the infected animals and this control animal. The one week time delay does not seem sufficient for this transmission to have occurred through midge vectors since it takes approximately 2 weeks for ingested virus to replicate sufficiently within midges to enable efficient transmission to occur. Furthermore, the animal isolation facilities should not allow access to midges but their introduction with the sheep, or maybe straw, cannot be completely excluded although the experiment was performed outside of the normal vector season within Denmark. It seems more likely that transmission occurred by oral ingestion of virus, e.g. on straw contaminated either during the inoculation procedure of the test group (by leaking of inoculum from inoculation site) or from virus shed during the early stages of infection and transferred, inadvertently, to the control group. Previous studies have also provided some evidence that transmission of BTV can occur between cattle and goats independently from midges[13–16] and it seems that this has occurred by oral transmission. It is also interesting to note that sheep 1, which did not show any evidence for being infected with BTV during the course of the studies presented here, had BTV RNA in its blood at 16 days after lambing of sheep 4, and in organs at autopsy, during a follow-up study (note the control animals, 1–4, were not euthanized with the sheep 5–28). Thus sheep 1 may also have become infected through oral transmission of the virus (likely via sheep 4 since the placenta of the lamb born to this ewe was not found in the pen). Infection via contact with an infected placenta has been described previously. It is clear that a more focused analysis of oral transmission of BTV would be useful.