Technical and Anatomical Considerations for Reproducible Inactivation of Large Animal Carcasses by Steam Sterilization

Abstract

Introduction: The complete inactivation of infectious tissues of large animal carcasses is one of the most challenging tasks in high-containment facilities. Steam sterilization is a method frequently in use to achieve biological inactivation of liquid and solid waste.

Objective: This study aims to highlight parameters most effective in creating reproducible cycles for steam sterilization of pig and calf carcasses.

Methods: Two pigs or 1 calf were sterilized by running a liquid cycle (n = 3) at 121°C for at least 120 minutes in a pass-through autoclave. To assess the physical and biological parameters, temperature data loggers and biological indicators (BIs) with spores of Geobacillus stearothermophilus (ATCC 7953) were placed at defined positions within animal carcasses. After completion of each cycle, data loggers were analyzed and BIs were incubated for 7 days at 60°C.

Results: Initial testing with an undissected pig carcass resulted in suboptimal temperatures at the tissue level with growth on 1 BI. After modifications of the used stainless-steel boxes and by placing the reference probe of the autoclave in the animal carcass, reproducible cycles could be created. A complete inactivation of BIs and a temperature profile of >121°C for at least 20 minutes could be achieved in almost all probed tissues.

Conclusion: Only minor modifications in carcass preparation and the used sterilization equipment resulted in effective and reproducible cycles to inactivate large animal carcasses by using a steam autoclave.

Introduction

Research on select agents or emerging infectious diseases is often performed in high- and maximum-containment laboratories of biosafety level (BSL)-3 and BSL-4. To work safely with hazardous pathogens, many constructional and technical controls are in place to guarantee occupational, environmental, and public health. Within laboratories, a biosafety cabinet or isolated caging systems usually serve as primary containment barriers. Thus, infectious cell cultures and infection studies in small animals can be performed efficiently and safely. In addition, small animal models require less housing capacity, produce less waste to be treated, and are available with a wide array of genetically modified animal lines and molecular biological tools.^1,2 Therefore, in most containment laboratory facilities, small- to medium-sized animal models such as mice and rats and, to a lesser degree, ferrets or nonhuman primates (NHPs) are in use.

In veterinary research, in contrast, the use of large animals as natural animal models has a long history. This is exemplified by the discovery of the agent that causes foot-and-mouth disease by Friedrich Loeffler and Paul Frosch during animal studies in 1897,³ who thereby accidentally established the field of virology. In recent years, large animal models have found their way into translational research, as seen in the large animal modeling of tuberculosis (TB)^4,5 or the modeling of human disease-relevant conditions in genetically modified pigs.⁶ These alternative models are costly and time-consuming but can answer distinct scientific questions more reliably than rodent models that are widely used in basic research to study general principles of diseases.⁷ However, they lack specific details such as lung lesion heterogeneity in TB⁸ or coagulation disorder in Ebola virus infection,⁹ to name just 2 examples.

With larger animal species such as pigs and ruminants, housing in individual containment cages is impossible, and therefore, animals are maintained with the room serving as primary containment. This increases the challenges in terms of biosafety significantly. In general, experiments with infectious diseases in susceptible animal hosts are prone to produce high titers in excretions and tissues during viral¹⁰ or bacterial¹¹ replication. During animal experiments and at the time of necropsy, infectious air is cleared by HEPA filters, and infectious blood and feces are inactivated in the effluent decontamination systems (EDS). Normally, inactivation of infected large animal carcasses is done by alkaline hydrolysis,¹² in a tissue grinder (renderer), or by incineration. However, not all containment facilities have direct access to these kinds of cost-intensive infrastructure. In the event of a malfunction in this dedicated equipment, scheduled necropsies in a study cannot be performed. Animals would have to be euthanized and infectious carcasses would have to be frozen and maintained in the laboratory until the equipment is repaired. A backup strategy for other effective inactivation methods for infectious animal carcasses must be in place.¹³ The efficient inactivation of rather small animal carcasses (0.5-10.0 kg weight per individual) via steam sterilization in an autoclave chamber is widely described.^13-15 To our knowledge, the largest volume sterilized by this method so far comprised 6 NHPs with a maximum load of 60 kg.¹⁵ Here we describe a method to safely inactivate large animal carcasses by using a pass-through autoclave.

Methods

Biological Indicators and Temperature Measuring Devices

Measuring of physical parameters was performed by placement of data loggers (EBI-12 Probes; EBRO, Weilheim, Germany) for high-precision temperature measurement, each equipped with 2 flexible probes at distinct positions within animal tissues. Corresponding to probe positions, biological indicators (BIs, initial run = ProSpore; all validation runs = BI, MagnaAmp; Mesa Laboratories, Chassieu Cedex, France) with up to 1.9 × 10⁶ spores of Geobacillus stearothermophilus (ATCC 7953) were placed inside the tissues. Resulting openings were sutured with single or continuous staples. After completion of each sterilization cycle, data loggers and BIs were removed. BIs were incubated for 7 days at 60°C, and data loggers were analyzed to obtain temperature values. Because of the documented caramelization effect of spore media¹⁶ in extended steam sterilization cycles, we decided to double check each spore ampoule. Therefore, on the seventh day of the incubation period, the glass vials were opened and 20 μL of a vortexed solution was transferred aseptically into 180 μL of fresh trypticasein soy broth (TSB; Carl Roth, Karlsruhe, Germany) inside a 96-well microtiter plate that was incubated for a further 7 days at 60°C. Untreated biological indicators serving as positive controls were incubated during each sterilization procedure or verification of viability of spore ampoules.

Animal Ethics Statement

All animals involved in this study were used after euthanasia in animal studies approved by the institution's ethics review committee at Friedrich-Loeffler-Institute (FLI) or were purchased from a local abattoir.

Initial Trial

A frozen pig (49.6 kg) that had been thawed overnight was prepared with BIs, a negative spore control, and data loggers; wrapped in 2 autoclave bags (Figure 1A,B); and placed on wood chips as condensate-absorbing material (ALLSPAN classic; Allspan GmbH Spanverarbeitung, Karlsruhe, Germany) inside a stainless-steel box (length = 1.00 m, width = 0.60 m, height = 0.60 m). Finally, the prepared pig (inside the box) and a stainless-steel vessel filled with ∼50 L of water thought to simulate the pig carcass volume were placed inside the autoclave chamber (Figure 1C). The vessel was covered by a stainless-steel lid with the autoclave-reference probe fixed in the middle of the lid to exclude any contact except with water during the liquid steam sterilization cycle. The reference probe and the physical parameter inside the autoclave chamber were cross-checked with 2 data logger probes each. At this time point, the lowest temperature measured was inside the abdominal cavity (−1.92°C). A preformatted carcass sterilization cycle was applied with the parameters shown in Table 1.

Figure 1.

Pig carcass preparation and temperature graphs at the tissue level during the initial run. (A) The pig carcass was left intact and biological indicators (BIs) and data loggers were placed inside the head (1), the neck (2), Musculus (M.) triceps brachii (3), the abdominal cavity (4), and in subcutaneous (5) and, respectively, intramuscular (i.m.) position (6) of M. biceps femoris. (B) The prepared pig carcass is wrapped in 2 autoclave bags, while data loggers are still visible and placed inside a stainless-steel box before adding embedding material. (C) The box is placed inside the autoclave chamber, and the autoclave-reference probe (white arrow) is injected into a stainless-steel box filled with 50 L of water. (D) After artificially cutting through and removing the autoclave bags, a minorly disintegrated pig carcass within a fully soaked embedding material and considerable amounts of condensate and meat fluid are visible at the end of the sterilization cycle. (E) Subcutaneously placed BI at M. biceps femoris (5) is still in its position. (F) Deep inside the musculature of the neck (7.7 cm) and adjacent to a vertebral process, a spore-containing ampoule is still in its initial position. (G) BIs were recovered directly after the end of the sterilization cycle with spore media of BIs 2, 3, 5, and 6 and the negative control (asterisk) showing a caramel-like discoloration, while the BI of the abdominal cavity (4) still has its purple color. (I) Complete sterilization cycle with vacuum and heating phase indicated by an asterisk. (J) Focused data representation showing the failure to reach 121 °C for most data loggers inside animal tissues.

Table 1.

Autoclave cycle parameters during sterilization of animal carcasses

Cycle	Run	Weight, kg	T0, °C	Tinc, °C	P, mbar	Vacuum, min	Heating, min	Incubation, min	Evacuation, min	Cooling, min	Σ min	Σ ∼ h
Initial	—	49	15.94	134	3344	5	30	300	547	231	1113	18.5
Pigs	1	42/37	23.32	121	2269	8	471	120	274	386	1259	21.0
	2	39.5/34.5	24.29	121	2228	7	382	120	264	337	1110	18.5
	3	39.5/25.5	22.41	121	2236	8	351	120	263	302	1044	17.5
Calves	1	135	34.07	134	3318	9	341	240	545	224	1359	22.5
	2	123	32.02	121	2245	9	401	120	270	352	1152	19.0
	3	101	35.02	121	2259	9	688	120	270	381	1468	24.5

P, pressure (maximum); T₀, temperature in the water-filled vessel (initial run) or in the abdomen of pigs and calves (validation runs) before starting steam sterilization cycles; T_inc, temperature (minimum) during incubation phase.

Validation Runs with Pig and Calf Carcasses

An autoclave load of each validation run consisted either of 2 pigs (6 pigs in total, mean carcass weight = 36.33 ± 5.89 kg) or 1 calf (3 calves in total, mean carcass weight = 119.67 ± 17.24 kg). Each pig was partially dissected by removing the limbs and the head from the torso. Limbs, head, and torso of 1 carcass were placed on a steel grid to lift all parts 10 cm above the floor of the stainless-steel box (Figure 2A). The heaviest pig carcass (designated as pig 1) was always placed inside the bottom box, which was moved on a roller frame. The autoclave-reference probe for the liquid steam sterilization cycle was placed inside the abdomen of pig 1 without suturing the abdominal opening. The lighter pig carcass (designated as pig 2) was subsequently placed in a separate stainless-steel box and lifted on top of the bottom box. The mean load weight for both pigs was 72.67 ± 7.09 kg.

Figure 2.

Pig carcass before and after steam sterilization with temperature graphs at the tissue level during validation run 1. (A) Partially dissected pig carcass with limbs, head, and torso separately placed on a robust steel grid, and positions 1 to 6 of temperature probes and biological indicators are indicated. (B) After the end of the sterilization cycle, the reference probe (white arrow) is still deep inside the abdominal cavity of the progressively disintegrated pig carcass and considerable amounts of liquids are accumulated under the steel grid. Sterilization cycles of run 1 for pig 1 (C, D; pig in lower box) and pig 2 (E, F; pig in upper box) with the vacuum phase indicated by an asterisk (C, E) are depicted.

Each calf was partially dissected with the head and limbs placed on a steel grid in the upper box (Figure 3A). The heavier torso with the reference probe localized inside the abdomen was placed on a steel grid within the bottom box (Figure 3B). Supplemental Figure S1 shows the arrangement of the 2 stainless-steel boxes inside the autoclave chamber. The applied parameters of all 6 runs are shown in Table 1, and all positions of BIs and data loggers in pigs and calves are shown in Table 2.

Figure 3.

Calf carcass before and after steam sterilization with temperature graphs at the tissue level during validation run 3. (A, B) Head, limbs, and torso of a calf were placed separately on robust steel grids in 2 boxes, and positions 1 to 9 of temperature probes and biological indicators are indicated. (C) At the end of the sterilization cycle, almost all bones are freed from musculature and skin. (D) A progressively disintegrated calf torso with a ruptured and displaced rumen and considerable amounts of liquids accumulated under the steel grid are obvious. (E, F) Sterilization cycle and focused data of run 3 showing the successful achievement of 121°C of all data loggers inside animal tissues with the vacuum phase indicated by an asterisk.

Table 2.

Positions of biological indicators and data loggers inside animal tissues

Cycle	Head	Neck	Musculus triceps brachii s.c.	Musculus triceps brachii i.m.	Thoracic Cavity	Abdominal Cavity	Back	Musculus biceps femoris s.c.	Musculus biceps femoris i.m.
Initial	DL	BI/DL	n.t.	BI/DL	n.t.	BI/DL	n.t.	BI/DL	BI/DL
Pigs	BI/DL	BI/DL	BI	BI	BI/DL	BI/DL	BI	BI/DL	BI/DL
Calves	BI/DL	BI/DL	BI/DL	BI/DL	BI/DL	BI/DL	BI/DL	BI/DL	BI/DL

BI, biological indicator; DL, data logger; i.m., intramuscular; n.t., not tested; s.c., subcutaneous.

Steam Sterilization Processing

Steam for sterilization purposes is centrally generated at FLI. Three steam generators each have the capacity to produce 6 tons of steam per hour (equals 13 megawatts of power) with a steam pressure of 800 kPa. The daily consumption of steam for the whole institute ranges from 3.0 to 7.5 tons.

The pass-through autoclave used (17-9-14 HS2, PST-series; Belimed, Zug, Switzerland) had a chamber volume of 2.63 m³ (height = 180 cm, width = 95 cm, length = 154 cm) with 1 opening inside a necropsy room separated from a side room by a bioseal. Exhaust air was sterilized by incineration, and condensate was captured in a kill tank with secondary sterilization in an EDS. All steam sterilization cycles consisted of a prevacuum (a one-time vacuum drawn to less than 7000 Pa), heating, incubation, evacuation of pressure, and cooling phase (Table 1). The temperatures measured by the reference probe before starting the liquid cycles are depicted in Table 1. To trigger the start of the incubation phase (120-300 minutes) beginning at 121°C (for most cycles), respectively 134°C (initial water and first calf cycle), the autoclave-reference probe was inserted into a vessel filled with water (initial run) or inside the abdomen of animal carcasses (validation runs). The amount of deionized water consumed during sterilization in the liquid cycles ranged from 1 to 4 m³ with the 134°C/240-min cycles as the most demanding ones.

Data and Statistical Analysis

Temperature probes were analyzed using the manufacturer's software (Winlog.validation software; EBRO) and raw data was exported for evaluation and visualization of temperature graphs in Prism 8 (GraphPad Software, La Jolla, CA). Figure panels were generated in Photoshop CC (Adobe Systems Software, San Jose, CA).

Results

Initial Run

We observed considerable amounts of condensate and meat fluid in the fully soaked embedding material while removing data loggers and autoclave bags (Figure 1D). Carcass preparation was very effective in terms of keeping the BIs in place during steam sterilization. Careful dissection of brittle muscle and bone tissues revealed the initial position of BI superficially sutured to Musculus (M.) biceps femoris (Figure 1E) respectively adjacent to the basis of a vertebral process (Figure 1F). Four of 5 BIs and the negative control showed a dark brown flocculent spore media (caramelization effect; Figure 1G), while 1 BI (abdominal cavity) remained purple and showed bacterial growth in the microtiter assay. The reference probe within the water-filled vessel reached the 134°C limit after 35 minutes. Thus, the sterilization phase of 300 minutes started relatively quickly after initiation of the autoclave cycle (Figure 1I). This resulted in a failure to achieve 121°C in almost all probed tissues. The highest temperature was measured subcutaneously (s.c.) at 135.07°C in the M. biceps femoris after 18 minutes. Thereby, only at this location could the targeted 121°C be achieved. Heating of all other probed parts took much longer and reached a maximum in the evacuation phase rather than in the incubation phase. Thus, the second highest temperature was achieved in the head at 119.35°C after 418 minutes ( ∼ 7 hours; Figure 1J). The lowest temperature was recorded at only 107.28°C after 804 minutes (∼ 13.5 hours) inside the abdominal cavity.

Validation Runs with Pig and Calf Carcasses

Failure of the initial carcass sterilization cycle forced us to rethink our general procedure. First, we decided to avoid low starting temperatures at the animal tissue level as described by others.¹⁵ Therefore, the positioning of BIs and data loggers was performed above 20°C. Second, we relinquished the use of embedding material and configurated the stainless-steel boxes by inserting a robust 10-cm-high grid that allowed placing the carcass above the floor of the boxes to keep it above the accumulating condensate and meat fluid. The third modification was to place the reference probe of the autoclave for pig and calf cycles deep inside the abdominal cavity instead of in a water-filled vessel since the abdominal cavity had been the worst-case position with the lowest temperature during the initial run. Based on standard sterilization parameters, 121°C inside the abdominal cavity followed by an incubation time of at least 20 minutes should be sufficient for a 6 log₁₀ reduction of spores. However, an additional safety margin of up to 120 minutes of incubation time was set in place.

The 10-cm-high grid was able to keep the carcasses outside the accumulating condensate and meat fluid, and in comparison to the initial run, most bones and the body cavities were freed from their skin and muscular components (Figure 2B and Figure 3C,D), indicating massive tissue disintegration during steam sterilization. All recovered BIs showed the caramelization effect and were negative in the subsequent microtiter assay.

In the pig cycles, the duration of the heating phase had a modest variability of 351 to 471 minutes (Table 1), resulting in very homogeneous sterilization cycles of all pigs (Figure 2C-F; Suppl. Figure S2A-H). The steam penetration capacity was highly efficient at all investigated tissue levels, with the M. biceps femoris s.c. as the earliest and the abdominal cavity as the latest point that reached the targeted 121°C (Figure 2C-F; Suppl. Figure S2A-H).

The calf cycles were equally efficient in reaching 121°C for at least 20 minutes in most probed tissues, but the duration of the heating phase showed a considerable variability from 153 to 688 minutes compared to the aforementioned pig cycles. The first run was performed with a heating phase up to 134°C followed by an incubation period of 240 minutes. The higher temperature resulted in a comparably quick achievement of the targeted 121°C in all probed tissues within 162 minutes as the longest time inside the abdominal cavity again (Suppl. Figure S3A,B). At run 2, we just barely failed to reach 121°C inside the thoracic cavity (120.95°C at 529 minutes) and inside the musculature of the neck (120.64°C at 529 minutes), while inside the abdominal cavity, 113.85°C at 651 minutes could be achieved (Suppl. Figure S3C,D). Run 3 had the longest heating phase with 688 minutes, and 121°C was reached at 742 minutes inside the abdominal cavity during the incubation phase for more than 20 minutes (Figure 3E,F). In terms of material compatibility, we observed no damage to the autoclave itself or data loggers when considering the duration of all cycles (17.5-24.5 hours).

Discussion

The use of efficient inactivation of infectious animal carcasses after animal studies is a crucial task to maintain the necessary biosafety conditions in terms of occupational, public, and environmental health. The current literature that deals with animal carcass sterilization is scant, and to our knowledge, studies with turkey and Cornish hen carcasses as surrogates for NHPs^14,15 and ferrets¹³ exist. All animal carcasses used in these experiments had a body weight between 0.5 and 10.0 kg, which overall can be considered a small animal setup for steam sterilization. Our approach described here was to examine the feasibility of steam sterilization as a means to inactivate biohazardous large animal carcasses. By reviewing the literature, it was clear that only a liquid cycle would be appropriate for carcass sterilization to avoid flash vaporization at the end of the cycle.¹⁵ Therefore, we initially placed the liquid cycle reference probe in a vessel filled with water to mimic the volume of a pig carcass to be inactivated. The 50 L of water was quickly heated to 134°C. Thus, the incubation phase started much earlier than expected but subsequently failed to reach at least 121°C for 20 minutes in all animal tissues. As described by Santacroce,¹⁴ water is an inappropriate representation in terms of density and texture, and physical parametrization within the animal tissues itself is more reliable. Among the different tissues, the abdominal cavity turned out to be the most persistent place for achieving temperatures of up to 121°C. Therefore, we used this position as an ideal place for the reference probe, since all other tissues should reach the targeted temperature earlier. In addition, this allows an individual heating phase that considers weight differences of carcasses and, in the subsequent cycle phases, establishes homogeneous temperature values within the animal tissues as shown in this study.

Another aspect that we observed was the considerable amount of condensate that accumulated around the carcasses during the sterilization process. This finding is most likely owed to relatively long cycle durations and the evaporation of liquids from large animal carcasses themselves. Thereby, the accumulated water partially covered the pig carcass, resulting in a slowed heat conduction into deeper animal tissues and an inefficient sterilization cycle.¹⁵ To circumvent this negative effect and avoid contact of the accumulating water with animal tissues, we placed all other animal carcasses in the validation runs on a 10-cm-high robust grid. Furthermore, during the validation runs, we abstained from wrapping the carcasses in autoclave bags, which resulted in their complete exposure to the hot steam and prevented the generation of potential air pockets. Such air pockets are discussed as a cause of insufficient steam penetration and subsequent growth of BIs when ferrets with fur were sterilized inside autoclave bags.¹³ Overall, using the grid and abstaining from wrapping the carcasses significantly improved heat conduction in deeper tissues, resulting in very homogeneous data sets of the pig carcass validation cycles.

The more variable sterilization cycles in calves are in part addressed by the likely displacement of the reference probe when compared to the cycles of runs 2 and 3 and by the higher temperature of 134°C used in run 1. Based on the pronounced disintegration of abdominal organs seen in calves after sterilization, it can be hypothesized that the special anatomy of the ruminant gastrointestinal system with its higher fluid and ingesta content, when compared to a monogastric system, might have led to the evaporation of ruminal fluids, which displaced abdominal organs, subsequently moving the reference probe. The heating phase in run 3 was considerably longer, indicating proper placement of the reference probe inside the abdominal cavity. To prevent movement of the reference probe during carcass deformation, attaching the reference probe to a rib or to the abdominal wall with a robust suture could be 1 choice. However, carcass measures should be limited to the minimum necessary to minimize the production of infectious aerosols or cutting injuries, especially in a BSL-3/4 containment environment.¹⁷ In our BSL-4 laboratory, we avoid the use of bone saws during necropsies, and as outlined earlier, partial dissection of carcasses is performed with 1 sharp knife.

Our results indicate that a heating phase of at least 700 minutes for partially dissected calves should be sufficient to achieve 121°C at the tissue level. One might argue that 121°C of moist heat sterilization is for many research or industrial applications (eg, sterilizing pipette tips, pharmaceutical products, or precleaned surgical equipment) an overkill process, especially when it comes to enveloped viruses, fungi, and vegetative agents that are least resistant.^18,19 However, the challenges start to rise when nonenveloped viruses, mycobacteria, or spore-forming bacteria are present in large quantities of organic matrices. Large animal carcass sterilization clearly is the most challenging task in this respect. When the nonenveloped hepatitis E virus (present in liver tissue of a naturally infected wild boar) was inactivated by incubation at 95°C for 1 minute in a heating block, just a 4 log₁₀ reduction was possible.²⁰ Even after 90 days of incubation at 80°C, Mycobacterium avium subsp. paratuberculosis was still recoverable from compost mixed with infected tissues.²¹ Bearss and colleagues¹⁵ reported 110°C to 115°C as a peak carcass temperature when they placed a data logger inside the abdominal cavity of NHPs after an 8-hour exposure time at 121°C without growth of BIs. This is in agreement with our findings that all BIs were inactivated even if 121°C was not always reached in all tissue sites. Thus, an overall cycle length of 19.0 to 24.5 hours is sufficient to achieve a 6 log₁₀ reduction of spores in calf carcass sterilization up to 135 kg. However, to exclude any possible occupational and environmental hazards after sterilization of highly infectious animal carcasses, the general rule of thumb in moist heat sterilization (121°C/20 min) should remain in place, especially in large animal carcass sterilization.

Conclusion

This study demonstrates inactivation of infectious agents is possible in large animals, provided strict configurations and sterilization parameters are followed. However, the autoclave chamber must be at least medium sized (chamber volume ≥2.63 m³) and a considerable cycle length must be considered. Although we did not observe any damage to the autoclave itself or the data loggers during the study, such long programs will stress the autoclave and cause wear in various parts of the autoclave. In our case, such extremely long inactivation cycles resulted in an enormous consumption of deionized water (up to 4 m³) for indirect cooling of exhaust air streams. This cooling water has to be collected and inactivated separately under BSL-4 settings and should be considered when planning of the recording volume of the EDS. Therefore, using a conventional laboratory steam autoclave for sterilizing large animal carcasses is uneconomic and cannot be recommended as general procedure. Whether a remarkable reduction of the complete sterilization cycle could be achieved by running a higher cycle temperature of 134°C (as the results of our first run with a calf carcass indicate) will be subject to further investigation. However, the inactivation of larger animals (more than 135 kg) by steam autoclaving seems to be unrealistic with this method, considering the even larger autoclave chamber and the infrastructure necessary for steam generation. Thus, this method should only be used in case of a temporary failure of more suitable methods for reliable sterilization of large animal carcasses such as alkaline hydrolysis, use of a tissue grinder, or an incinerator.

Ethical Approval Statement

Animal care and use were in accordance with the Directive 2010/63/ EU and all experiments were approved by the FLI Institutional Animal Care and Use Committee (protocol number FLI 06/10).

Statement of Human and Animal Rights

No live animals or human subjects were involved in the experiments.

Statement of Informed Consent

Not applicable to this study.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: All equipment, animals, and consumables were financed by the institutions the authors belonged to. There was no third-party funding.

ORCID iD

Jan Schinköthe https://orcid.org/0000-0002-3426-3090

Prior Presentation

Parts of this study were presented by Jan Schinköthe at the 62nd Annual Biosafety and Biosecurity Conference, November 20, 2019, Birmingham, Alabama.

Supplemental Material

Supplemental material for this article is available online.