Abstract
Introduction
Bats have gained much attention, given their role as reservoir hosts of pathogens of human importance,1,2 and interest in studying the biology of bat diseases in controlled settings and establishing captive colonies for this purpose
3
has increased. The common vampire bat (
Recently, our research groups at the University of Wisconsin-Madison and the U.S. Geological Survey (USGS) National Wildlife Health Center (NWHC) developed a rabies vaccine candidate for bats.5,6 The vaccine candidate, a recombinant, viral vectored, mosaic glycoprotein vaccine, provided protection against rabies in big brown bats (
This article describes the design of a container to safely transport wild-caught vampire bats in a trip requiring >40 h of driving and one feeding session. We considered the following for the design: 1) compliance with biosafety and animal welfare regulations, 2) ability to feed bats during transport while maintaining biocontainment in the containers, and 3) custom dimensions to fit in the available vehicle designated for transport. We drove the bats across the U.S.–Mexico border at Nuevo Laredo, Mexico–Laredo, Texas, United States, to the NWHC. The trip required a total of ∼2,816 km and one stop overnight to rest and feed the bats.
Materials and Methods
Ethics Statements and Required Permits
Our capture activities were conducted under permits from the Mexican Secretariat of Environment and Natural Resources (SEMARNAT, permit number: SGPA/DGVS/003242/18). Permits for exporting bats from Mexico to the United States were issued by SEMARNAT (permit number 44333). All protocols for capturing, husbandry, and transporting vampire bats complied with NWHC Institutional Animal Care and Use Committee (protocol number EP180418) and followed standard guidelines for work with wildlife. 7
To legally import vampire bats into the United States, we complied with the Centers for Disease Control and Prevention (CDC) requirements to transport the bats and maintain them long-term in NWHC’s BSL-3 animal facility (CDC, permit number 2018-04-108). For the purposes of transport, live vampire bats are considered vectors of human or animal disease and potentially infected with pathogens (e.g., viruses, bacteria, fungi). In addition to rabies, Venezuelan equine encephalitis virus, once isolated in the common vampire bat, 8 was also of concern; hence, special safety measures were required during transport to avoid accidental exposure 9 and at final disposition. The U.S. Fish and Wildlife Service is responsible for inspecting shipments into the United States and verifying the conservation/protection status of the type of wildlife being transported. We declared and filed form USFWS 3-177 to import vampire bats at the designated port of entry. We also notified the Division of Wildlife for the Wisconsin Department of Agriculture, Trade, and Consumer Protection (permit number 356MO7168-BPI) of the transport of bats to their final destination.
Vampire Bat Captures and Husbandry Practices During Temporary Captivity in Mexico
In July–August 2018, we captured 123 common vampire bats (of mixed sex and age) from 7 sites throughout the state of San Luis Potosí, Mexico. 10 The bats were trapped using mist nets in the field, received a health inspection, and were given an individual metal forearm bat band (4.2 mm, aluminum alloy, Porzana, Inc., E. Sussex, United Kingdom). We placed the bats in small mesh cages to drive them to their temporary holding facility in a restricted building, property of the Universidad de Matehuala, in Matehuala, Mexico. The animal room at this holding facility had natural light and ventilation, and we maintained humidity to recommended levels (>40% relative humidity) with humidifiers.11–13 Bats were kept on a diet of citrated bovine blood obtained from a local abattoir and water ad lib. 13 During temporary captivity, bats were housed in custom-made fabric mesh cages of ∼152 cm (long) by 50 cm (wide) by 127 cm (height) in groups of no more than 20 individuals, separated by sex and, as cage availability allowed, by location of capture. Captivity in Mexico lasted approximately 1 to 6 weeks, depending on their capture date, before departing to Madison, Wisconsin, United States.10
Ectoparasites in vampire bats are common.14,15 We treated all bats topically with Selamectin (dose 6 mg/kg, Zoetis, Florham Park, New Jersey, United States) as a preventative measure upon placement into the holding facility and 1 week later if ectoparasites were observed after treatment.
During the capture period and containment in Mexico, we experienced the deaths of bats due to rabies, as published elsewhere. 10
Vampire Bat Care Requirements and CareDuring Transit
Adult vampire bats feed solely on blood and need to eat daily. Fasting periods of >24–48 h can result in mortality. 16 Because vampire bats rarely drink water, prolonged periods without feeding lead to rapid dehydration. In captivity, blood meals are offered once a day, usually at night. Vampire bats ingest an average of 15–30 mL of blood per day. The blood, generally from cattle and obtained in established abattoirs, is in liquid form and treated either manually to defibrinate or chemically to prevent coagulation. 12 During feeding, the blood is quickly digested, and excess water is eliminated in urine for vampire bats to take flight once they are finished. Their feces contain an excess of ammonia, which produces a strong, characteristic smell and can be a respiratory irritant if not adequately ventilated. 17 For this reason, after feeding the bats overnight, we needed to keep the container as clean as possible to avoid additional irritants in the enclosed environment. In addition, uneaten blood spoils quickly and can be a source of bacterial infection or intoxication for vampire bats. Thus, we considered it necessary to remove uneaten blood within 7 h (i.e., the following morning).
Typical behavior of vampire bats includes congregating in groups of the same sex or females with their offspring. 18 Vampire bats rest by hanging from their hind limbs and actively move, stretch, and groom. The average wingspan of a vampire bat is 30–40 cm. We allocated the bats into the transport containers in same-sex, small groups (no more than 10 individuals) to not only avoid disrupting their natural behavior but also provide enough space for comfortable movement.
Transport Container Design
For the container prototype, we followed the International Air Transport Association (IATA) general regulations for the transport of bat species. We made some modifications according to our needs for vampire bats. IATA requires a primary and a secondary containment device to prevent the escape of animals and ensure that handling during transport does not affect the shape of the containers and harm animals inside.19,20 Additional requirements include adequate ventilation, access to food, appropriate dimensions to allow the animals to move and turn naturally, and proper labeling for identification.
Primary container
To comply with the requirements for the primary container, we searched for durable and nontoxic materials, resistant to cleaning and disinfection, of appropriate dimensions to allow the shipping of 10 bats per container. We used Taconic Transit Cages (TTC), ordinarily used to transport laboratory rats and mice (Taconic, Rensselaer, New York, United States, www.taconic.com). With some modifications to these cages, we built 10 primary containers. The TTCs are made of molded polypropylene #5 plastic and have air vents on the sides made of a filter material that allows airflow while also preventing transmission of most pathogens. Although the TTCs have demonstrated effectiveness in preventing contamination of animals with murine virus (as designed for rats and mice), pore size of the filter mesh is not of sufficient size to provide biocontainment of all airborne viruses. Since rabies (a pathogen transmitted by direct contact) was our major concern in transporting bats, we believe the TTC cages were ideal for our purposes. The cages are autoclavable and reusable. The cage dimensions are 55.9 cm (long) by 40.6 cm (wide) by 17.8 cm (height). However, these dimensions were not large enough to let 10 bats move freely inside the primary container (e.g., open their wingspan, perch upside down with room to flip onto the floor). Thus, we used two TTC bottoms, one inverted and on top of the other, for a total height of ∼33 cm. We joined the two bottoms by placing a set of hinges on the long side, allowing us to open/close the container up to 180°. We glued a waterproof, high-density foam tape strip (Frost King, Mahwah, New Jersey, United States) along the rim of each container to provide a sealing surface when the cage was closed. To fasten the container closed, we drilled two holes through the longer rim (opposite to the hinged side) to insert two 3-inch screws with butterfly wing nuts (Figure 1). We used a thermoplastic film tape (Parafilm M, P7793, Millipore Sigma, Burlington, Massachusetts, United States) to wrap around the junction of the two container parts to provide an additional layer for sealing.
Vampire bat (
To allow for the inspection of the bats during transit, we placed a window on the side of the wide face of the container′s top part by cutting a 10-cm diameter hole (Figure 2). We covered the window hole by gluing a square of clear polycarbonate plexiglass, fastened with four screws on each corner. As per welfare requirements, we provided the bats with a surface to hang from the inside of the top section (i.e., the ceiling). We cut a piece of a 1/4-inch galvanized hardware cloth to the dimensions of the ceiling and attached it with screws and washers, trimming and smoothing the edges off to avoid harming the bats while perching.
Use of the side windows to facilitate inspection of the vampire bats at the Mexico–U.S. border,
Secondary container
IATA requires a secondary containment unit as a second line of defense. We built two wooden containers using commercial plywood sheets. The dimensions of each allowed us to stack six primary containers (two columns of three containers) in one (Figure 3) and three containers in the other. Each primary container was placed over a stainless steel tray (commercially available for kitchenware) as a liquid-proof divider surface between them. To provide adequate ventilation and access the primary container during feeding, we included two lateral windows and a front door into the wooden box (Figure 4). All were shielded with a 1/4-inch galvanized hardware cloth mesh. We also installed a 12-volt fuse plug electric fan on the back face of the larger secondary container that served as an air extractor. The tenth single container was placed inside a commercially available pet carrier with dimensions 91.4 cm (long) by 71.1 cm (wide) by 81.3 cm (height) made of plastic and metal wire (Petmate Traditional Vari kennel).
Final setup of the transport cages inside the minivan during transport within the United States. The largest secondary container (shown here) held six primary containers. A smaller secondary container held three primary containers, and a tenth primary container was placed inside a commercial dog kennel cage.
Secondary container featuring the placement of an extraction fan to aid in ventilation during transit (unfinished version of the container).
Feeding systems
The most critical aspect of transporting vampire bats over the ∼48-h planned travel time was to have the capacity to feed the bats while maintaining biosecurity measures (e.g., not opening the primary container and avoiding spillage or aerosolization of particles from inside the cage). To overcome this challenge, we devised a feeding system using enteral feeding bags (The Kangaroo Gravity Feeding Bag set, Product #8884702500). We made some modifications to the primary transport container that allowed the delivery of blood into a feeding receptacle (i.e., trough) placed inside. To build the trough, we cut in half (transversally) a 2-inch PVC pipe, measured it to fit within the length of the container, and glued it fixed onto the bottom section (i.e., floor) by the opening side. The size of the trough allowed for a filling capacity of ∼200–250 mL of liquid without spilling. We drilled a small hole in the bottom section of the container (same side as the window) as close as possible to the center bottom of the trough. We then inserted an “L”-shaped hard plastic tube with proper diameter to fit tightly into the container and allow the flow of blood or liquids from the outside into the trough. We glued and sealed the joining of the tube to the container wall using silicon glue. The tube faced downward into the trough and protruded ∼1/2 inch to the outside, to which end we connected an extension line connector set with a male Luer lock end (container’s connector). This connector was the only port to move liquids (blood or cleaning solutions) into or out of the trough and always remained attached to the container, tightly capped. To fill the trough with blood and to clean/rinse it after the feeding session, we used two sets of three-way stopcock valves, two extension line connectors with double male Luer lock (Jorgensen Laboratories, Loveland, Colorado, United States), one feeding bag set, and two 60-mL needleless syringes that would connect to the main tube/connector, per container. Each cage was lined with disposable pet training pads to absorb any liquid spilled during the transport (e.g., urine, blood) and prevent leakage.
Feeding and Cleaning Process
We fed the vampire bats with citrate-treated bovine blood, 13 following the same procedures as during captivity in Matehuala. The blood had been previously packed in the feeding bags or in 1-L high-density polyethylene (HDPE) bottles (United States Plastic Corp., Lima, Ohio, United States) and stored frozen at −20°C until use. A few hours before the feeding session (and resting stop) overnight, we thawed the blood under warm water to ensure a liquid state and easy flow through the feeding bag set lines and the connectors (Figure 5).
Feeding session. Bovine blood (thawed and placed in the feeding bag set) was delivered into the container's trough using the extension lines. Parts are
After inspecting the bats inside, we accessed the primary containers and began the feeding process. First, we hung the feeding bags at an elevated height, allowing gravity to facilitate blood flow. We removed the caps from each container’s connector and attached a three-way stopcock valve onto the male Luer lock. We then connected the feeding bag line to the second port of the three-way stopcock valve and opened it to allow the blood to flow into the trough. The filling time per container was approximately 5–7 min (for ∼200 mL of blood). Once each trough was filled to capacity, we turned the stopcock valve closed, disconnected the feeding bag line, and either filled another cage or discarded the empty feeding bag. Because the blood was considered “clean,” we only spray-cleaned the lines and bags with 70% ethanol. When finished, each container extension line was secured again with the male Luer lock cap. No leaking or blockage of blood occurred using this tubing system, and the process was relatively easy and fast to complete. A video of the feeding and cleaning process is available as a supplemental file.
After the bats had fed on the blood, the remaining blood was considered potentially contaminated with pathogens (e.g., rabies virus). The cleaning process after the feeding period included rinsing the lines and the trough with saline solution and a disinfectant (2% chlorhexidine, Nolvasan, Fort Dodge, Iowa, United States), retrieving the liquids using suction, and discarding them into waste containers. For the rinsing, we reattached the three-way stopcock valve to the container’s connector, with one of its ports attached to an extension connector line. On the other end of this extension connector, we attached a second three-way stopcock valve. The two male Luer lock ports of this second valve were each attached to a 60-mL needleless syringe and the line of a 1-L HDPE waste bottle container. We delivered the rinsing solution into the trough using the same procedure to dispense the blood. After filling the trough, we redirected both three-way stopcock valves and applied suction using the 60-mL syringe to retrieve the rinsing solution into the syringe. When the syringe was full, we redirected the second valve only to push out the recovered solution into a waste bottle containing 100 mL of bleach. To avoid pressurizing the waste bottle, we modified the bottle caps by placing and sealing a 0.22-µm filter (Cole-Parmer, UX-02915-60, Vernon Hills, Illinois, United States) through the bottle cap that let air out. This process was repeated 2–3 times until the saline solution was retrieved clear. In the final rinse step, we used a 2% chlorhexidine solution to disinfect the trough and the extension lines. Upon completing the cleaning, we closed the first three-way stopcock valve and secondarily occluded the line using the attached pinching device to avoid liquid entry into the trough. Using a syringe, we passed a solution of 10% bleach through the connectors and extension lines into the waste bottle, disinfecting the interior of the extension lines through which uneaten contaminated blood had passed. Last, we disconnected all lines and disinfected them by spraying with 70% ethanol. We placed all waste into biohazard-labeled plastic bags first and then into screw-top buckets before being autoclaved at the NWHC for final disposition.
Transport Description
We transported bats to the NWHC from Matehuala on September 5, 2018. We fed the bats 2–3 h earlier than usual the night before the trip to ensure that all ate completely. At 2:00 am, we began placing bats in cages for transport. All bats received a health check and a subcutaneous fluid administration of lactated Ringer’s solution (∼0.5–0.7 mL per 20–30 g of weight). One of the authors, a licensed veterinarian in Mexico, performed such checks and provided care during the temporary housing, in guidance from the USGS NWHC attending veterinarian. We distributed the bats into the travel containers and tried to maintain the groups already formed during captivity to avoid stress; males and females always remained separate.
We rented a 15-passenger van to drive the bats from Matehuala, Mexico, to the International Bridge in Laredo, Texas, United States (∼531 km), where Mexican and U.S. authorities inspected the export/import of the vampire bats. Once in Laredo, TX, we transferred the containers into our agency vehicle (minivan) to complete the rest of the trip. Driving and all the processes involved were performed by two people. During the trip, the temperature was controlled with the vehicle A/C system at an ambient temperature of 25°C; we kept the back windows open to allow airflow. Twenty-two hours after placing the bats in the travel containers, we stopped and fed them overnight for ∼6 h. It took about 1 h to complete the feeding session, while the cleaning process lasted ∼1.5 h the next morning. After visually inspecting the bats through the container windows, we resumed driving to the NWHC.
During most of the trip, the bats remained apparently calm and roosted as a single group in each transport container. Bats were observed moving around the container upon stopping in the evening. Once beef blood had been provided, the bats immediately approached the trough to feed. All bats appeared to be eating. Bats could be heard vocalizing during and after eating, but no significant aggression or food guarding was observed.
One male bat was observed roosting outside the group the following morning and appeared lethargic. Because complete containment during transport was required, the bat could not be examined or treated. It was observed that he appeared dead during a visual check later that afternoon. The bat was confirmed dead upon arrival at NWHC and later confirmed as positive for rabies by the Wisconsin State Laboratory of Hygiene. No other bats showed signs of rabies or were injured. However, later in our captive study at the NWHC, we experienced a rabies outbreak 2 months after arrival, linked to the bat that died in transport. 10 Upon arrival at NWHC, the remaining 92 bats were visually examined and placed in mesh housing cages. None appeared significantly dehydrated or lethargic. The bats were fed more citrated beef blood shortly after arrival, and some were observed eating immediately. Over the following week, bats were examined in groups of 20–40 per day. One female bat was humanely euthanized for a chronic injury. It was submitted for rabies testing and was also positive, despite not showing any clinical signs typical of rabies infection. This bat had not been cohoused with the first rabies case before or during transport, and thus, it was likely infected before capture.
Conclusion
While our prototype container was efficient, we have suggestions to improve the design. Although our modified TTCs prevented transmission of pathogens via fomites or direct contact, the pore size of the filter material would not have completely prevented airborne transmission of viruses. If that is a major concern, a different type of cage would be needed. We recommend using a softer material for the perching area (ceiling), as the metal mesh may irritate the foot padding of the bats. We also recommend designing the cage opening with a limited range (compared to the 180°). In our case, the two TTC pieces opened wide enough and from all sides that bats could have potentially escaped; however, they were never opened while in transit. Another setback of the transport cages was the susceptibility of some materials (e.g., foam padding, silicon glue around the bolts) to high temperatures during autoclaving. Therefore, these parts must be replaced if the transport cages are reused.
In addition, some beef blood was stored frozen inside the enteral feeding bags. During storage and transport of the frozen bags, the plastic cracked on several bags, leading to the leaking of beef blood during transport. We recommend freezing the blood in hard-sided containers such as HDPE bottles and transferring it to the enteral feeding bags just before use.
The feeding system we designed was effective, and we were able to provide ∼200 mL of bovine blood per cage (of 10 bats maximum) for one overnight feeding session. We successfully rinsed and flushed the trough and tubes with disinfectant using the same tubing system and valves. We did not experience any spills of blood spills (clean or waste) or disinfecting liquid, and the time to complete both tasks was reasonably fast, performed by two people. In our experience, we noticed that vampire bats were very adaptable to a novel surrounding and feeding setting as they readily figured out how to perch and feed on the trough.
To feed the bats during transport, we carried containers of bovine blood, assumed to be noninfectious, in our vehicle. However, once the blood came into contact with the bats, it could be considered potentially exposed to pathogens and therefore subject to specific handling requirements under U.S. Department of Transportation (DOT) Hazardous Materials Regulations (HMR) (e.g., medical waste). All associated waste materials (e.g., gloves, paper towels, feeding tubes, etc. used to handle or dispose of the blood) should be treated similarly. For future work, we recommend developing a simple protocol for classifying and handling research-related waste, including guidance on labeling, documentation, packaging, and training to ensure full compliance with DOT HMR.
Overall, we transported 93 bats from San Luis Potosí, Mexico, to Madison, Wisconsin, United States, by vehicle over a total of ∼2,816 km. The trip was completed in a total of 47 h from the moment the bats were placed in their transport containers until they were placed in cages at the NWHC on September 6, 2018. The transport containers and feeding system described herein provided safe and biosecure transportation for vampire bats during extended land travel.
Authors’ Contributions
E.M.C.C.: Conceptualization, methodology, writing original draft preparation, visualization. M.C.B.: Conceptualization, methodology. E.A.F.: Conceptualization. J.E.O. and T.E.R.: Supervision, project administration, resources and funding acquisition. All authors reviewed and edited the article.
Footnotes
Acknowledgments
We thank Richard Carson (University of Wisconsin-Madison) for providing material to build the primary containers. Emily Russel, Lex Medina, Jordan Mandli, Nichole Kuha, and Dean Dischler helped assemble the transport containers. Carrie Smith (USGS NWHC) provided guidance on biosafety guidelines and requirements. José Ignacio Cárdenas-Canales designed and provided 
. This article is based upon the research conducted for the degree for Doctor of Philosophy and corresponds to a chapter in “Cárdenas-Canales, EM. Vaccinating Vampire Bats against Rabies: Studies of Vaccine Efficacy, Field Application, and Social Perceptions in México [dissertation]. Madison, WI, University of Wisconsin—Madison, 2021.”
Funding Information
This research was funded by the U.S. Geological Survey (T.E.R.), American Association of Zoo Veterinarians (J.E.O.; UW award number MSN209345), a Graduate Student Research Award from the University of Wisconsin-Madison Global Health Institute, and a University of Wisconsin International Division IRIS Incubator Grant for Interdisciplinary Research in International Studies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
Authors’ Disclosure Statement
Use of trade, product, or firm names and commercial sources are for identification only and do not imply endorsement by the U.S. Government.