There are around 1,400 bat species grouped in 19 families in the Chiroptera order (23). Based on phylogenetic analysis, bats are currently grouped as Yinpterochiroptera and Yangochiroptera (24, 25). Bats differ from other mammalian species in their capacity to fly and use echolocation (22, 26). The places where bats live, mate, give birth to offspring, hibernate, rest, and protect themselves from adverse climatic conditions are called roosts (15). Bats can build roosts in sheltered areas (e.g., inside a cave or a building) and also externally (e.g., branches of a tree, tree cavities, or foliage) (9, 15). For instance, the common vampire bat (Desmodus rotundus) roosts in caves, house roofs, runways, tunnels, abandoned mines, wells, and hollow trees (27). In roosting sites, D. rotundus typically occupies the most elevated and darkest locations (28). Bats are known as nocturnal creatures; however, foraging times of bats vary among species. Some species leave the roost immediately or a bit late after sunset for foraging (e.g., Pteropus medius), whereas others leave the roost before sunset (e.g., Nyctalus azoreum) (29–32). Foraging time depends on weather, feeding behavior, and food preference (30, 31), and should inform sampling design. Bats have significantly different reproductive strategies than other mammalian species, which increases successful births and the fitness of both the mother and offspring in terms of lifespan, reproduction, and mortality rate (14, 16). The reproduction of bats can be characterized as multiple reproductive events with a delayed onset of sexual maturity and low litter size. With a few exceptions, bats usually produce only one offspring in each gestation (16).

Research requiring bat handling needs a meticulous sampling design that accounts for site selection, a statistically robust sample size, and sampling bias mitigation (33). Inadequate mitigation of biases in sampling design can lead to difficulties in data analysis, potentially misleading the performance of analytical models and interpretation (34). To mitigate sampling bias, study areas could be stratified into different sampling sites based on habitat type to account for the potential effect of environmental configuration as a confounding factor. Considering the distance among sampling points could help increase spatial independence among bat roosting sites when such information is needed (35). Studies should account for data requirements of the expected analytical method, logistics in the study area, study duration, species population dynamics, and budget (36).

Because bats are small, nocturnal, cryptic, and conspicuous when active, estimating the sampling size of bats is challenging (37). The objectives of the study, target species, capture approach, and data precision needed all play a role in establishing the sample size for bat surveys. Based on the study design, power analysis, and simulation studies aid in providing more precise sample size estimations (37–40). For some research questions, the adequate sample size and duration of the research project may not be viable. Thus, researchers should plan to generate alternative approaches, such as collecting a pool of environmental samples from the bat roosts, developing models or simulations, or exploring complementary data and sample repositories.

Every team member (i.e., researcher, student, or technician) participating in bat sampling should complete training requirements to engage in the field activities related to bat research. Every research organization or academic institute should have its own policies and rules regarding research training. For example, training is generally available through the Institutional Animal Care and Use Committee (IACUC), occupational health, and environmental health and safety offices, among others. In the absence of institutional training opportunities, the team leads or principal investigators can organize training sessions to educate project members about biosecurity and biosafety practices at different stages of the research project. It is the responsibility of both team leaders and members to complete the minimum training required to implement biosecurity and biosafety measures to reduce harm to humans and bats. The team lead can follow an operational risk assessment matrix to evaluate the knowledge of team members and take appropriate action (Supplementary material S1).

During the implementation stages of bat research, local wildlife, health, or environmental protection agencies may require certain documents to be completed before sampling can be initiated. The team should have copies of these documents in the field to access when there is limited internet access or even a lack of laptop devices. For example, local police or rangers may ask the team to show permit documents for bat sampling. Furthermore, it is suggested to carry the data collection form or survey questionnaire to be used to collect data from the field. In summary, the field team should bear copies of critical documents during fieldwork.

Protocols for data collection, biosecurity and biosafety plan, and sample storage are fundamental for successful field expeditions. Bat sampling sites are generally located in remote areas. For international fieldwork, it may be challenging to find the necessary supplies or equipment at study sites. Therefore, it is important to learn about the target bat species and study location to purchase supplies, materials, and equipment well in advance. Broadly, equipment, materials, and supplies necessary during the sampling can be classified into two groups: for capturing bats and for sampling bats. The logistics for capturing and sampling bats should cover Personal Protective Equipment (PPE), instruments, materials and consumables, processing station (i.e., portable tables and chairs), and storage (e.g., tanks and coolers). Tasks can be organized by teams; for example, the bat capturing team could focus on purchasing mist nets and traps. Additionally, a portable centrifuge machine with batteries and cables is necessary to separate serum from blood samples. For chemical restraint and euthanizing the bats, the team needs to procure all necessary chemicals. In some cases, locally made utensils for net setting have been found successful for the easy execution of bat capturing. For example, a modified form of mist net is made locally to capture Pteropus bats in Bangladesh, and researchers are utilizing that successfully for captures (41). As for pre-field logistics, team members should focus on making risk assessments to determine which vaccines or pre-exposure medications the team members may require (e.g., rabies, yellow fever, and malaria treatment), as well as other post-exposure or palliative drugs (first aid kit) to address any field incident.

To control and prevent the spread of zoonotic diseases, it is crucial to implement proper biosecurity and biosafety precautions during interactions with animals (42). During fieldwork, biosecurity aims to reduce pathogen transmission from wildlife to humans, while biosafety refers to efforts to reduce pathogen transmission from humans to wildlife. Biosecurity has been defined elsewhere as the measures to avoid the transmission of animal pathogens or parasites to the general public and the environment by using containment equipment (42). Biosafety has been defined as the set of measures taken when handling biological material to prevent the introduction of novel pathogens into new populations (43). Both biosecurity and biosafety require the use of PPE.

During fieldwork, team members need PPE, which includes respirators, gloves, face shields, coveralls, dedicated long-sleeve clothing, gowns, and goggles, as standard infection control precautions (20). PPE in bat-borne disease research creates barriers to protect personnel and bats from pathogens and hazardous materials through various entry points like skin, mouth, nose, and eyes (44). The necessary PPE equipment depends on the biological agents being handled and the research settings (Table 1) (Supplementary material S2). During the removal of PPE, researchers should take extra precautions as all the PPE is assumed to be contaminated and there is a chance of exposure to contaminants. Therefore, we suggest a step-by-step sequence to decontaminate and remove the PPE after completion of designated work (Supplementary material S3).

Beyond the use of PPE to reduce health risks, effective risk management should include diversity, equity, and inclusion (DEI) practices, which can help increase the well-being and safety of the team in field sites. DEI practices, however, require a basic understanding of the team and local culture and may involve previous training to take the necessary steps to prevent, prepare for, and respond to risks.

In a remote area, an unwanted situation may arise. For example, team members may suffer unexpected injuries while in the field. For the fast and proper management of any incident, researchers should gather and organize the addresses and contact information of local police stations, sample collection permitting authorities, and acquire detailed knowledge of the fastest evacuation routes and secure transportation to reach the nearest hospital or healthcare centers rapidly by effectively engaging with local stakeholders and emergency support organizations. Based on incidents and the surrounding environment, we provided a protocol to follow in emergency situations (Supplementary materials S4, S5).

To reach field sites and carry the logistics, the team may need to travel to local, national, or international areas. Transportation arrangements are necessary before the expedition starts. We can broadly classify the transport arrangements into two groups: short-term long distance (e.g., another state or country) transportation and long-term short distance transportation (e.g., systematic surveillance of a colony requiring continuous storage of samples and supplies), so that the necessary vehicles should be secured before the expedition.

Researchers use various techniques to capture bats, including mist nets, harp traps, hand nets, and direct hand capturing of different bat species (51, 52). These capture methods have both pros and cons. Researchers must decide which is most suitable and available for their research purposes. Sometimes, researchers may use a combination of different techniques based on the bat-capturing sites and bat species of interest. For instance, if a study requires a specific bat species from caves and foraging areas, researchers can use mist nets at foraging sites and cone traps at roosting sites. To capture small bats, a group of two people should be enough to set the net. Nevertheless, if the mist net is large (e.g., 20 ft), more than two people may be required in areas with high bat activity. Net-setting techniques and logistics may differ (height and length of the net, pocket size of the net, etc.) based on the study location and target species (Supplementary material S6). Here, we describe some capture techniques that have been used for different bat research purposes.

To reduce stress on bats entangled in the nets, regular monitoring is required after setting the net or trap. Additionally, some bats may chew the net, make a hole, and escape. The team should check the nets at 15-min intervals (60, 68). When a bat becomes entangled in the net or trap, ideally, two individuals must be actively involved in removing the bats from the net immediately after a tangle. The first individual holds and untangles a bat using their left and right hands, while the second individual assists in removing the bats; experienced people can remove the bats alone (51). Bats should be removed promptly from mist nets to prevent stress, injury to delicate wing bones, and patagia. A badly tangled bat can be removed by cutting strands of the mist net to prioritize the welfare of the animal. In the case of a harp trap, one person removes bats from the harp trap, with another person accompanying them. When a team plans to set nets at resting sites of bats (e.g., cave or old building), several team members may be required to remove the bats, as many bats start to move or leave the resting sites at the same time and become entangled in the nets. All bats are securely wrapped in a sterile cotton cloth bag with drawstrings or ties at the top (Supplementary material S7C) (52, 69).

The most crucial step after selecting a bat-capturing site is to determine the most suitable and safe capture site and laboratory setting. The laboratory setup should be conducted under the supervision of an experienced wildlife health researcher, particularly a veterinarian, to ensure standard procedures for animal handling and sample collection. To process the collected specimens and store them properly at the field site, maintain proper biosecurity and biosafety for both team members and captured bats. The goal is to enhance the laboratory environment for optimal performance and achievement of research goals with no risk of pathogen exposure to team members. To achieve these goals, several points should be followed.

After the bat capture, cloth bags containing bats are brought to the field laboratory and hung in the designated space sequentially with enough distance to avoid contact between bats. To reduce bat stress and accelerate the release of animals during sampling, we propose a minimum of four members at the field laboratory bench (Supplementary material S9).

Proper restraining of bats is important to reduce the risk of accidents to team members (e.g., bites), as well as to minimize stress, pain, and the escape of bats, and to ensure the collection of quality samples. Bats can be restrained using chemical or physical methods, with handlers using leather gloves and double nitrile or latex gloves to prevent bites or scratches. There are different chemicals available to safely restrain bats. Table 3 offers an example of chemicals used to restrain D. rotundus. In the field, we have successfully used Isoflurane in a soaked cotton swab placed in a plastic container (e.g., Ziploc bag) with the bat for a few seconds with a dose of 0.1-0.2 mL (2 drops) (Figure 4). During physical restraint, the handler should be aware of stress and pain, and monitor respiration to prevent harming the animal (e.g., choking, fractures).

Team members should have a clear understanding of the type, amount, number of aliquots, collection method, and storing media of samples before starting the sampling. Here, we provide an overview of sampling methods.

Researchers may need to perform euthanasia on bats that are injured (51). Researchers may also collect a representative sample of animals captured to deposit their carcasses and skins for further examination and study (voucher specimens). Lethal sampling, however, must adhere to local laws and regulations regarding euthanasia and lethal sample collection. Usually, a veterinarian directly performs euthanasia, following animal welfare and biosecurity guidelines, specifically the “American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals” (latest edition) and the “Guidelines for Euthanasia of Nondomestic Animals” of the American Association of Zoo Veterinarians (76). In special situations, personnel trained by veterinarians can perform the euthanasia of bats in the field. The protocol for field euthanasia involves administering an overdose of isoflurane gas anesthesia, followed by cervical dislocation of the bat (77), as described by the United States Geological Survey-approved euthanasia methods for small bats (Microchiroptera) (78). First, use a Ziploc bag as a chamber and a stainless-steel mesh tea ball infuser with a ball of cotton containing 0.5–1.0 mL of isoflurane for a 500 mL volume chamber (79) (Figure 4). Bats are then introduced into the sealable bag and remain there until no respiration is observed or detected by the veterinarian. If isoflurane fails to euthanize the bat, cervical dislocation is performed by applying pressure to the neck to dislocate the spinal column from the skull (76). Nevertheless, performing cervical dislocation on bats could expose researchers to the rabies virus (80). As an alternative to cervical dislocation, researchers can consider the use of carbon dioxide and exsanguination of an anesthetized or already unconscious bat.

Identification and labeling are crucial steps for successful sample collection, laboratory testing, data analysis, and interpretation. Inaccurate labeling and misidentification of specimens can result in project failure, incorrect lab results, inappropriate data analysis, and misleading interpretation. Therefore, we recommend identification numbers at two different stages. First, assign a specific identification number for a bat as an individual. In the second stage, assign a specific identification number for each specimen collected from an individual bat. For example, if a team decides to capture bats in Argentina, they can start the identification number as A0001 for their first bat. The team will collect and record all the information or data (e.g., age, sex, body condition, and different measurements of that bat) under the identification number A0001 (Table 4). In the second stage, the team can use a syntax that describes the specimen type and storage media used. For example, if a team collects an oropharyngeal swab and stores the swab in Trizol, they can assign the identification number of the specimen as A0001.OPT. Here, A0001 refers to the individual bat number from which this specimen originated, OP means oropharyngeal swab, and T means Trizol. Similarly, A0001.BSN indicates blood serum with no media from the bat identified as A0001. Sometimes, the label of specimens is lost or erased for various reasons. In that situation, it is difficult to trace back the specimen’s source and identification. Without labeling, a specimen becomes useless and should be discarded in the interest of the rigor of the study.

To identify bat species, it is necessary to have bat species identification guides during field sampling (Supplementary material S12). For this, different morphological measurements such as weight, head, nose-leaf, body, tail, forearm, and hind foot length are collected from captured bats and compared with the previously reported species to determine species (Supplementary materials S12B, S12C) (51). Furthermore, researchers use different temporary and permanent marking systems to monitor bats and avoid resampling during a single sampling event, as well as to follow up on a specific bat at different time intervals. The recommendation is to avoid permanent marking of bats for successful research if a temporary marking system can be employed. For temporary marking, people use clipping of dorsal fur from different body areas and pinpricking the wing membrane to create unique tattoos for bats (57). For permanent marking of bats, light tags, wing bands, and microchips can be used (51). Different kinds of microchip injectors and scanners are available in the market for use in bats. Microchips are injected subcutaneously by pulling up the skin on the back of the bat to insert the needle with the Passive Integrated Transponder (PIT) tag under the skin. Then, the PIT tag is ejected, and the needle is pulled out gently (81, 82). Finally, a drop of surgical glue on the injection point in the skin is recommended to secure the tag after the release of bats back into their habitat. Each tag will have a unique number to identify each individual bat and avoid resampling.

All the field equipment and clothes used during bat capturing and sampling become contaminated with the biological materials of bats. Contaminated equipment and the laboratory environment pose a major source of infection for both team members and bats. Researchers need to spray disinfectants on the tables to clean them and use alcohol on their hands to prevent cross-contamination after each bat sampling. After completion of everyday fieldwork, non-disposable equipment and laboratory floors should be cleaned and disinfected using chlorine bleach or Amphyl (hospital-grade Lysol) (52). A list of reagents that can be used for different equipment is listed in Supplementary material S3.

Biohazards are substances that pose a significant threat to human, animal, or environmental health due to their potential for infectious or dangerous effects (87). Improper disposal of biohazardous material may create public outrage, spread infections, and attract other animals (88). Hence, potentially infectious biological wastes, including body fluids, carcasses, and bedding materials, are considered hazardous or nonhazardous when they come into contact with animals or humans (87, 88). A comprehensive biological waste management and disposal plan is essential for effective biosafety and biosecurity practices in bat sampling. Before going to the field, consider all the options you must follow for waste management guidelines.

Sometimes, researchers need to ship samples from one country to another. During this shipment process, there are different rules and regulations that researchers need to follow. For example, shipping specimens collected from a certain species of bats to the United States requires compliance with guidelines and permits established by the Convention on International Trade in Endangered Species of Wild Fauna and Flora and the Animal and Plant Health Inspection Service of the U. S. Department of Agriculture. Import or export permits vary based on the bat species, types of specimens, source of origin, and the targeted destination of the shipment. Obtaining all necessary permits is often simple, but understanding the process may take time and effort. Therefore, it is wise to be prepared and start the application process for a permit at least six to four months before shipping. All permits need to be included with the specimens’ package during transportation.

In the meantime, researchers should explore and identify a suitable shipping agency to ship the specimens. A shipping company also needs additional documents for the shipment based on its company policy. Before or during the signing of the contract for the specimen’s shipment, carefully and clearly explain the transportation requirements (for example, the need to always maintain the cold chain) to uphold the standards of quality specimens during shipment. Shipping agencies, however, generally fail to sustain the cold chain and adhere to schedules (e.g., World Courier, Escobar per. Comm. 1/8/2025). A monitoring report at the end of the shipment process could help confirm if the temperature chain was maintained.