1. IntroductionInsect production offers a sustainable source of nutrients [1] as insect larvae can efficiently convert a broad range of biomass to valuable proteins (including essential amino acids), fats, vitamins and minerals. According to the International Platform of Insects for Food and Feed (IPIFF), by 2025, most of the demand for insect meal will lie in the pet food sector (approximately 40–50% of the insect meal produced). Subsequently, the growth in use of insect derived processed animal proteins (PAPs) as aquaculture feed will continue reaching 25–35% in terms of market share. Similarly, the market share for insects as feed for poultry (to 20–30%) and for pigs (to 5–15%) are also expected to increase rapidly [2], following the end of the ban (in the EU) on using PAPs from insects in these sectors [3]. However, still, only a limited range of substrates can be used for rearing of the insects used for feed and food [4], and using waste materials that contain animal byproducts as feed for the insects is not allowed [4,5]. To increase the sustainable circular economy potential of insect production, novel, currently banned [4] sources of substrates for insect rearing should be explored. Such substrates include food waste containing meat, slaughter byproducts and animal manure [1]. In order to assess the safety of insects as food and feed when using such substrates of animal origin, knowledge regarding the fate of different, potentially hazardous, agents from animal products in the insects is required. Such agents could be transmitted to animals and humans when they consume the insects or insect-derived products. These hazards can be of chemical (toxins, heavy metals, pesticides) or microbiological (bacteria, prions, fungi, parasites, viruses) origin [5]. For viruses, special concern can be raised for potential transmission of the agents responsible for serious notifiable diseases that are known to be transmitted efficiently via animal products and to survive within meat for a prolonged time period, including African swine fever virus [6], classical swine fever virus [7] and foot-and-mouth disease virus [8,9]. Currently, African swine fever virus (ASFV), in particular, is of major global concern as this virus has now reached pandemic proportions, affecting a number of countries within eastern and western Europe, Asia, the Caribbean and Sub-Saharan Africa [10]. Hence, even within Europe, there is a risk for this virus to be present within different animal byproducts that could be used as a substrate for insect larvae production if the EU legislation is changed. Experimental exposure studies of insects used for feed and food with animal pathogens, including viruses, can provide knowledge about the survival of these pathogens within production systems. Such knowledge can be used for future risk assessments of insect rearing on substrates containing products of animal origin. Previously, we used virus bioexposure assays to examine the uptake and survival of viruses in larvae of two of the most widely used insect species for the production of food and feed, the mealworm (Tenebrio molitor) and the black soldier fly (Hermetia illucens) [5,11], using porcine respiratory coronavirus as a model [12]. In the current study, similar assays were applied to test the survival of ASFV in larvae of these two insect species. Insects containing ASFV, following oral uptake, were subsequently fed to pigs in order to investigate potential transmission of the virus to susceptible host animals via these larvae. 4. DiscussionWe describe here the use of oral virus bioexposure assays for T. molitor and H. illucens larvae [12] using ASFV. Meat and slaughter byproducts could potentially, in the future, be used as a substrate for insect production as this comes with a huge potential for increasing the circular economy of this production system [1]. Given the current world-wide distribution of ASFV [10], it cannot be ruled out that infectious ASFV could be present within some of these products, as it is known to be a very stable virus, e.g., within meat from infected pigs [6]. The fate of ASFV in insects used for feed is of particular interest. It is the only known DNA arbovirus and can replicate within tick vectors, from genus Ornithodorus [20,21]. Furthermore, infectious virus has been detected in insects, i.e., in adult Stomoxys calcitrans, for up to 12 and 48 h, respectively, following feeding on blood containing ASFV [22,23]. Additionally, it has been shown that infectious ASFV can be mechanically transmitted to pigs via oral uptake of S. calcitrans fed on viremic blood [14]. It cannot be ruled out that the same mode of transmission would be possible when the virus is fed (inadvertently) to insects used for feed—including T. molitor and H. illucens larvae, that are then subsequently fed to pigs. In this study, following feeding on serum from pigs infected with ASFV POL/2015/Podlaskie with a titer of 103.3 TCID50/5 μL (equivalent to 105.6 TCID50/mL), viral DNA was detected for the entire study period, up to 7 and 9 days, respectively, in exposed T. molitor larvae. The length of these studies was based on previous exposure studies with T. molitor using a porcine coronavirus in our laboratory. In these earlier studies, two different porcine respiratory coronaviruses (PRCVs) were detected within the T. molitor larvae for up to three days post exposure only [12]. As observed for PRCV RNA [12], the amount of ASFV DNA in the sampled larvae was low (Cq values of >30) from the time of exposure, and the number of larvae in which it could be detected decreased subsequently. However, the level of viral DNA within the larvae in which the virus could be detected remained relatively stable, which contrasted with previous findings [12] in which the level of PRCV RNA decreased rapidly and declined to an undetectable level after 3 days. Hence, ASFV DNA seems to be more stable within these larvae than PRCV RNA. Following exposure of H. illucens to chicken feed spiked with spleen suspension derived from pigs infected with ASFV POL/2015/Podlaskie, with a titer of ~105 TCID50 ASFV/g feed, viral DNA was detected for up to 3 days post exposure in the rinsed larvae and their wash fluids. After this, the level of viral DNA within the samples was below the detection limit of the qPCR assay. Cq values were high (>35) in all sampled larvae and wash fluids from the earliest time point, but the levels remained relatively stable within both materials in which the virus could be detected for up to 3 dpe. Using PRCV in the earlier exposure studies, viral RNA was also detectable in H. illucens larvae for up to 3 days post virus exposures [12]. It should be noted that for the PRCV studies using H. illucens larvae, virus suspension containing serum was used for these virus exposures [12]. As discussed later, this could potentially have an effect on the stability of the viruses used. In both T. molitor and H. illucens larvae, the absence of an increase in ASFV DNA over time suggests that these larvae are not biological vectors for the virus, since there was no indication of virus replication within them. Similar conclusions were made from studies with ASFV using adult S. calcitrans [22] and blow fly larvae [24], in which no signs of virus replication were detected within the insects, but they can act as mechanical vectors [14,23].

In the H. illucens ASFV exposure studies, it was observed that extending the exposure period of the larvae to the spiked feed from 2 h to 5 h or 24 h did not increase the number of larvae in which ASFV DNA could be detected. Indeed, the number of rinsed larvae and wash fluids in which viral DNA was detectable decreased markedly when the exposure periods were extended. Interestingly, Cq values were higher by 3.5 (study H1) and 6 (study H2), respectively, (i.e., lower amounts of DNA were present) in the spiked feed used for the two exposures of the larvae at 24 h (i.e., spiked feed with larvae) when compared to spiked feed kept in the insect incubator for 24 h (i.e., spiked feed without larvae).

It has previously been observed, that H. illucens larvae are able to reduce the level of bacteria present in pig manure [25]. The same seems to be true for the level of ASFV genomic material in spiked feed in this study. Previous work has indicated that blow fly larvae can inactivate ASFV within organ material [24]. In that study, viral DNA could be detected in blow fly larvae of the two species, Lucilla sericata and Calliphora vicina, after feeding on spleen material obtained from ASFV infected pigs, but no infectious virus could be recovered from the spleen after the larvae had fed on it. Infectious virus was, however, recovered from spleen that had not been exposed to the larvae [24]. At 5 h into ASFV exposures using spleen supernatant (study H1), the number of rinsed H. illucens larvae and wash fluids in which ASFV DNA could be detected was considerably lower than the proportion of RT-PCR positive H. illucens larvae that had been exposed to PRCV virus suspensions (containing 10% serum) for 6 and 8 h, respectively, in an earlier study [12]. This could perhaps be attributed to the different materials used for exposures. Based on the results obtained in this study, serum could be hypothesized to have a stabilizing/protective effect on ASFV. In serum and clarified spleen suspensions kept at 27 °C in this study, both in the insect incubator and in the laboratory, the level of infectious ASFV remained stable for a considerably longer time period in serum (48 h and beyond), than in a spleen suspension, in which the level of infectious virus decreased markedly within the first 24 h. A stabilizing effect of serum on ASFV during exposures to different inactivation methods has previously been reported [26]. In a previous insect virus exposure study with adult S. calcitrans exposed to ASFV with a titer of 105.2 TCID50/mL, considerably lower Cq values (below 25.5) were detected in flies sampled immediately after exposure [22] when compared to T. molitor larvae and H. illucens larvae in this study. The S. calcitrans could theoretically have consumed up to 11–15 µL of blood [27]. This means that the flies may have contained 103.2 to 103.4 TCID50 ASFV—which compares well with the 103.3 TCID50 ASFV/5 µL used for exposure of T. molitor larva. As previously discussed [12], the higher Cq values in T. molitor (above 30) when compared to S. calcitrans (below 25.5) could perhaps be attributed to less efficient extraction of DNA from the larvae prior to qPCR analysis. Even though homogenates of the larvae were diluted 1:5 prior to extraction, the high lipid content of the homogenates could interfere with DNA extraction [12]. Attempts to isolate infectious virus from six T. molitor larvae was not successful following passaging in PPAM. The six samples had Cq values in the range from 29.8 to 32.8. It has previously been shown that ASFV infectivity assays can be expected to fail (or be unreliable) if Cq values are above 30 [19]. In addition, the six samples were not filtered to remove contaminants. Filtration was attempted, but proved difficult, and for four out of six samples, contamination of the PPAM, e.g., by bacteria and fungi, was observed. It has previously been found that it can be difficult to examine insect samples for infectious virus due to the presence of such contaminators [22]. An additional approach to assess the presence of infectious virus within the T. molitor and H. illucens larvae and the risk of virus transmission via oral consumption was used by feeding pigs with cakes containing larvae exposed to ASFV POL/2015/Podlaskie. Pigs that were allowed to consume 50 T. molitor (euthanized immediately after ASFV exposure or at 2 days post virus exposure) or 50 H. illucens (euthanized at 5 h or 24 h into ASFV exposure) larvae did not become infected with ASFV. It has previously been shown that the same inoculation method, using oral uptake of blood-fed insects (using pig blood containing ASFV) within soft cake dough, can result in ASFV infection [14]. In the current study, the number of T. molitor and H. illucens larvae used for feeding of pigs in all four groups was based on the expected dose in the 50 T. molitor of 5.0 log10 TCID50 ASFV used for inoculation of group 1. Based on the number of larvae of both species that contained the virus at these time points and the level of viral DNA within them in studies T1, T2, H1 and H2, it was expected that the dose used for inoculation of the remaining three pig groups was lower. It was not attempted to establish the infectious dose of ASFV in the larvae used for inoculation. In group 1, however, the expected dose used for the six pigs that were exposed to 50 T. molitor larvae euthanized immediately after feeding on viremic serum did not differ significantly from the inoculation dose previously used [14]. In that study, two out of four pigs that consumed 20 S. calcitrans fed on viremic blood became infected. It was estimated that these 20 flies provided an inoculation dose of 5.1–5.3 log10 TCID50 ASFV. Oral infection of pigs with ASFV can, however, be difficult to establish under experimental settings, e.g., using feed inoculation [28]. Indeed, in that study, oral inoculation of pigs with feed containing a dose of 4.3–5.0 log10 TCID50 given on 14 consecutive days did not result in infection. In our study, two weeks following oral inoculation of the pigs, five selected pigs were inoculated intranasally with 104TCID50 of the ASFV POL/2015/Podlaskie. These pigs did become infected with the virus 4–5 days following the inoculations, demonstrating that the pigs were indeed susceptible to the infection (data not shown). It has been reported that the minimum infectious dose of ASFV in solid matter (compound feed) is higher when compared to liquid [29]. The reason for this is not known, but it has been suggested that liquids provide a suitable substrate for virus contact with the tonsils where the primary virus replication occurs following exposure via the oronasal or intrapharyngeal routes [29]. However, in a previous study, in which two groups of pigs were fed either intact flies (solid matter) or fly homogenates (liquid form) following feeding of these flies on blood containing ASFV, no difference was observed in the establishment of infection between the two groups of pigs [14]. In summary, in this study, we have used established virus bioexposure assays for T. molitor and H. illucens to assess the survival of ASFV within these larvae. In this study, and the earlier study [12], the detection and survival of viruses within insect larvae seems to depend on many factors, including the virus itself, the material used for exposure and the insects. Therefore, care should be taken with extrapolation of results obtained using one pathogen in one insect species to others. Finally, even though we, in our experimental setting, were not able to demonstrate oral infection of a small number of pigs using T. molitor and H. illucens larvae that had been exposed to ASFV, it does not fully rule out the possibility of virus transmission via this route in field settings.