Edited by: Erin Katherine Lipp, University of Georgia, USA
Reviewed by: Sofia Kottou, National and Kapodistrian University of Athens, Greece; Edith Mariela Burbano-Rosero, Universidad de Nariño, Colombia
Specialty section: This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
We detail a five-stage protocol to address physical barriers and experimental limitations that have hindered routine pathogen monitoring of wild rats in urban settings. New York City potentially harbors from 2 to 32 million rats among its 8-million people. However, at a time, when people are most vulnerable to disease from over-crowdedness brought on by increased urbanization of society, the difficulty of studying wild rats has led to a paucity of ecological and epidemiological research. Challenges of safely handling animals and the difficulties of identifying individual animals and the emergence of their respective pathogen loads (timing of infection) have impeded progress. We previously reported a method using radio frequency identification paired with load cell and camera traps to enable the identification of individual animals and subsequent monitoring of the animals’ weights (an indicator of health). However, efficient pathogen surveillance requires repeated captures of the same individual in order to isolate and document the emergence of new pathogens, or variations in pathogen load, over time. Most of these barriers are now addressed in our protocol, which is aided by the use of a mobile, outdoor laboratory, followed by incorporation of pheromone-based lures to attract individuals back to active sensors, within a camera trap. This approach allows for the assessment of individual animal health, behaviors under camera, and changing pathogen loads and weights in most urban environments (e.g., financial district, docks, sewers, and residential). Five phases are described and presented: (1) site selection and urban trapping, (2) anesthetization, (3) serological and ectoparasite collection, (4) microchip implantation, and (5) retrapping and luring animals back to active remote sensors. In order to fulfill the unmet call for preemptive pathogen surveillance, public health officials and researchers may wish to adapt, or modify, similar protocols to ensure early detection and monitoring of rat-borne zoonoses, before they become problematic.
Seventy-five percent of the world’s human population is expected to live in urban settings by 2050. With the average rate of population growth in urban cities almost twice as high as the overall growth rate (
It would be inappropriate to address the physical barriers to research without first acknowledging the complex social issues that influence urban rat studies. Rats are associated with filth, disease, and poverty (
Conversely, it has been suggested that in New York City, a population exceeding 8 million people, there are fewer than 10 institutional researchers actively pursuing urban rat research (Corrigan, personal communication, New York City Department Health and Mental Hygiene). Therefore, the social constraints to studying wild city rats are even more daunting because they are magnified by several physical challenges.
Urban rodents are elusive, subterranean, and often go unseen, making knowledge about them prohibitively difficult to ascertain. It is common knowledge that rats detected during daylight hours imply that unnaturally high populations already persist in the immediate environment. Yet, most perceptions about rats are based on a disproportionately few individuals that are detected in the daytime in public, leading to over-generalizations based on more gregarious animals or migrating individuals. The vast majority of rats are not seen by human eyes. The non-independence of most observations leads to misinformation that is propagated by anecdotal accounts, hearsay, and the media. The primary means to combat this growing problem is to overcome the barriers necessary to study urban rats
Remote sensing with common tools, such as radio-telemetry or GPS, is often used to identify individual animals, and thus, ensure independence of a randomly selected sample size (
Feng and Himsworth (
With public health on the line and humans only living more densely packed together in the coming decades, this begs the question as to how many more pathogens will be discovered once rodents are more regularly monitored? And what are the benefits of understanding pathogen toxicity and exposure
We have recently learned that rodent scents are effective (
We have provided a few solutions to some of the barriers prohibiting urban rat research. Thus, by offering our five-step protocol, our objectives are to encourage and enable researchers to more easily undertake systematic pathogen surveillance of rodent-borne pathogens in the built environment. We are now detailing this protocol along with an assay for ectoparasite removal, anticipated results based on the recent literature and consultation with local experts, and a brief discussion of the social and research-based implications inherent in these activities.
Stainless steel lab cart (flat top required)
Lab bench liner
Digital scale
Chlorhexidene
Betadine
Vetbond
Alcohol preps (200)
Calipers
Wahl cordless razor
Nalgene lab notebooks
Qwik Stop® syptic powder
Sterile, razor-sharp surgical scissors
7.5″ utility scissors
Metal ectoparasite “nit” comb
Square grid Petri dishes
8″ Pyrex roasting pan
Dry ice
Heat bead sterilizer
Ear punch w/tags (optional)
Biohazard bags
Body fluid swabs (Swabs)
DNA collection tubes
DNA preservative
Safeguard® rear-release animal traps #52818 (18″ × 5″ × 5″)
Detex BLOX (non-toxic fluorescent) (optional)
Trap catch emitters (×12)
Hydrogel packs (box)
Dark, standard sized pillow cases (×4)
30% Isoflurene (250 ml bottles) (×3)
USP-grade oxygen refillable e-cylinder w/regulator
Oxygen tank cart
Induction chamber
Matrix VIP 3000 (or similar) vaporizer
Flow meter
Scavenger system
Nose cone (rodent/feline mask)
Reader/antenna/SCU/control block (×2)
12 mm chips (30) + retractors (×3)
Pheromones (pooled scents from lab rats)
Cameras ScoutGuard SG550V (×6)
Batteries AA Panasonic (×3)
Memory SD (×12)
Security locks
Hard hat/vests
N95 masks, or charcoal masks for organic agents
Disposable gloves
Kevlar gloves
Hand sanitizer
Study sites should be identified from a combination of city blocks, public park spaces, and/or residential areas. Researchers should be prepared to extensively search for appropriate research sites. The most usual site configuration includes moderate-to-high rat-infested areas (whether rats are observed during daylight hours) in potentially underserved and potentially dangerous areas of the city. Factors to include are the level of infestation, researcher safety, and ability to undertake research in highly populated areas, but without public knowledge of activities. This discretion will help ease the social anxiety of property owners and willingness to share their properties. This information can be determined by calling the local health department or by assessment of “look listen and learn” protocol, commonly used by pest-management professionals. See Figure S1 in Supplementary Material for a printable, distributable depiction of this information.
Trapping. Common knowledge suggests rats should be trapped using local foods that rats have previously become accustomed to. However, foods do not often result in “retrapping of the same animal” that may learn to associate danger with a food item, and food baits alone could be biased toward males ( Rats often travel “blindly,” using their guard hairs and vibrissae in contact with one or more surfaces to guard them. A high-lumen flashlight with ultraviolet (365 nm) backlight (such as the Nitecore® P20UV) will help expose chemically marked areas where rats repeatedly touch their guard hairs to the wall, while navigating blindly. This leads to sebum pheromone trails and also microdroplets of urine. These marks glow blue–white if fresh or yellow–white if old (96 h or longer). Therefore, trap strategically. Place approximately five rear-door release, small mammal traps such as the Safeguard® 18 × 5 × 5 model #52818, spaced a minimum of 1 m apart, close to the one or more edges of a wall, for every suspected colony assessed. Bait traps with fresh mixed scents obtained from soiled rat bedding, pooled from multiple male and female rats. It is common to use rats from a pet store or university-owned lab rats. Some care should be taken to minimize the age of the scent. To be safe, the scents should not be more than 48 h old when commencing the study.
In order to minimize further aging of pheromones, store scents in an airtight bag under minimal headspace (minimal air-pocket), ensuring the bag is closed tightly and stored in a cool, dry place. Install alert-sensors (emitters) into the trap (widely available on the Internet) along with hydrogel (food and water packs), so that an alert will be transmitted by cell phone to the principal researchers, once an animal has been caught. Rats should never be left alone for longer than 8 h in a trap. Not only is this inhumane but a stressed animal will take longer to anesthetize and may develop infection, rendering it unlikely to survive long enough for data collection. Following captures, cover active traps with a dark pillowcase or neoprene cover to minimize the startle response when picked up and transported by humans.
Eliminate many of the hazards of rats by following a safe-handling protocol.
For ease of mobility across a wide range of urban environments, prepare a mobile indoor/outdoor lab that can be transported to multiple study sites. A stainless steel lab cart covered with lab bench liner (Figure Pre-workup (5–10 min)
At least two researchers will need personal protective equipment (PPE; masks, gauntlets), before beginning. One worker will be required to maintain hold on the animal, while the other adjusts the isoflurane flow, oxygen flow, and mask. Sterilize table, check hoses, and connections for isoflurane unit. Isoflurane unit is on, all tubes connected. Table is clean. Instruments, microchips, and collection tubes on table. Scan a selected microchip (Trovan® 12 mm chip is appropriate) to provide initial data point. Place dry ice on two Pyrex 8″-roasting pans. One for ectorparasite collection and the other for body fluid collections. Anal swab (Swubes®) should be accessible. Update water-proof lab notebook hanging nearby. Initially, set isoflurane to 5% with an oxygen flow rate of 2–3 ml O2/min to flow into the Tupperware induction chamber. Animal handling on surgical table (15–20 min)
Make way to trapped animal carrying a dark pillow case that will be used to place over cage, ideally, prior to animal spotting human presence. Gently transport the covered cage to the modified Tupperware induction chamber, while minimizing any anthropogenic noise (Figure Dip entire locked cage into chamber for a minimum of 15 min until animal shows signs of slowed movement (partly sedated). Open induction chamber with one researcher lifting the cage up at a 45° angle and released the rear release trap door gently freeing the animal onto the base on the chamber. The second researcher will use the toe-pinch method to determine level of sedation. While the animal is mostly unconscious, use the claw method of a gloved hand (first two fingers over the shoulders, thumb and fourth finger gently constricting the diaphragm) and firmly lift the animal out of the cage and place onto the surgical area. Animal physical examination
The feline/rodent mask should be immediately placed over the animal, completely covering the nostrils and mouth (Figure The second researcher will reset the flow meter to the second part of the dual circuit (mask) and switch off petcock to the induction chamber. Ether flow should be re-adjusted to 5% isoflurane and 2–3 ml/min O2. Within 2–5 min, the animal should now be completely sedated. The physical characterization may now commence. At this stage, the first researcher can open the lab notebook and place microchip identification (usually a sticker) in the appropriate lab entry form. Record sex, length, weight, and notes on appearance: porphyrin stains around head area, mottled or greasy appearance of hair, evidence of wounds or fighting, and general health (any breaks) of guard hairs and vibrissae. With the mask still in place, the animal may be placed ventral side down, take sharp surgical scissors and snip 2.5–5 mm from end of tail, add to a marked DNA collection tube filled with preservative. Save marked tube for transportation back to laboratory.
Body fluid and fecal samples for blood-borne and systemic bacterial and viral pathogens ( Collect rectal swab, seal, and set aside to return to lab. Blood can be collected from the tail by squeezing/massaging the area around the DNA clipping into a capillary tube, or by cannulation of the lateral tail vain. Pressure and Kwik-stop styptic powder can be used to cauterize tail in case of bleeding. Ectoparasite collection
Place animal ventral side down. Grasp animal by abdominal and thoracic region. Forceps can be used to lift fur, while stainless steel nit/louse comb to brush animals’ hair both directions over square Petri dishes filled with dry ice ( Ectoparasites can be sorted visually, stored on dry ice, and transferred to −80°C for storage, until further analyses can be performed. Animals are now ready for pathogen detection assays.
Serological and tissue-based analyses of bacteria and viruses common to rodents in Manhattan are well described (
Most rats from 80 g and above can readily accommodate a 12-mm Trovan® passive chip, roughly the size of a grain of rice.
With animal on the table, dorsal-side up, use a battery-operated razor to shave 2 cm × 2 cm area of hair between the shoulder scapulae, making sure not to burn animal with the razor. Use alcohol or betadine and cotton swab with an inside out circling motion to sterilize shaved area. Pinch skin between fingers to form a tent. Insert needle bevel up, eject chip from retractor (Figure Check for bleeding, use gauze, firm pressure, and veterinary glue if necessary Remove mask and return animal to cage. Place cage near exact spot where it was trapped. Once the animal is moving again (usually 5–10 min, the animal is then ready for release), open door and let animal move out of cage.
The radio-frequency identification (RFID) system will be preassembled in a place within 10 m of rat activity. We used the SA-148, SQID customizable PIT tag system (Seabird model; Figure
Used rat bedding can be collected from a pet store or university housing for lab rats, making sure five or more adult males and females are represented.
Ensure RFID unit is powered on and that camera trap is switched to video mode. Set 100 g of fresh rat bedding on sensors
Check around all other traps for signs of rat activity (Figure S1 in Supplementary Material).
Shut down mobile theater, wipe down table, remove instruments (place scissors in bead sterilizer), replace lab bench lining.
Update lab manual
Re-bait stations with 100 g fresh pheromones (both stations and sensors will have pheromones).
Transport labeled collection vials (DNA, plasma, parasites) on dry ice to −80°-freezer.
Rats are among the most sensitive animals to the smell of conspecifics. Similar to many mammals, they approach scents in a process called “scent inspection,” whereby most scents (even repellents) causes the animal to approach scents closely to ascertain their biological meaning ( Traps can be re-baited continually as long as their participation is required, in order to obtain adequate sample size and sufficient spatial replication.
Pairwise comparisons of visitation or dwell times can be performed between different pheromone types × sex using PROC ANOVA with the Tukey option in SAS v. 9.4 (Cary, NC, USA). Scatter plot of number of visits per animal expressed as days elapsed since first capture. This graph can be generated using the PROC SCATTER. Points may be jittered on the
The downloads from RFID data loggers will indicate the time of day (time stamp), dwell times, frequency of visitation, weight, and all demographic factors for each individual animal over the duration of the study (Table
Tag # | Sex/juvenile | Site/location (a–g) | Weight begin (g) | Length (mm) | Health (1–4) | Marks/wounds | Visits to sensor (#) | Recaptures (#) | Weight change (g) |
---|---|---|---|---|---|---|---|---|---|
J876 | M | b | 448 | 25 | 3 | Scarring/right flank | 1032 | 3 | 20 |
M469 | F | d | 350 | 21 | 4 | 490 | 7 | 120 | |
0B99 | M | a | 610 | 25 | 3 | Mottled coat | 1120 | 2 | −10 |
9639 | M | g | 380 | 23 | 2 | 3.5 mm left dorsal | 886 | 0 | 100 |
9DCA | M | g | 224 | 22 | 2 | Tail lost (8 mm remains) | 429 | 0 | 275 |
B877 | F | a | 389 | 24 | 3 | Porphyrin excess | 0 | 2 | 111 |
7FAO | M/j | a | 202 | 14 | 4 | 1420 | 1 | 340 | |
657C6 | F | d | 490 | 20 | 3 | Damaged vibrissae | 630 | 7 | −15 |
J764 | F/j | c | 96 | 15 | 4 | 553 | 3 | 156 | |
A123 | M | e | 521 | 29 | 1 | Left eye blind | 632 | 4 | 5 |
Male | 6 | 398 | 23 | 3 | 122 | ||||
Female | 4 | 331 | 20 | 5 | 93 |
Tag # | Sex/juvenile | Site captured (a–g) | Pathogens total( |
Ectoparasite vectored ( |
Δ Winter | Δ Spring | Δ Summer |
---|---|---|---|---|---|---|---|
J876 | M | b | Parvovirus; |
Parvovirus; |
|||
M469 | F | d | |||||
0B99 | M | a | |||||
9639 | M | g | Parvovirus; |
||||
9DCA | M | g | |||||
B877 | F | a | |||||
7FAO | M/j | a | |||||
657C6 | F | d | |||||
J764 | F/j | c | |||||
A123 | M | e |
Rat | Sex/juvenile | Total visits | Pheromone type | Average visits/day | Sig ( |
Average dwell time ± (s) | Sig ( |
---|---|---|---|---|---|---|---|
J876 | M | 1072 | Sebum | 4.5 | 3.2 | ||
M469 | F | 983 | Sebum | 2.8 | 2.0 | ||
0B99 | M | 796 | Sebum | 1.1 | 1.6 | ||
9639 | M | 557 | Sebum | 0.4 | 2 | ||
9DCA | M | 978 | Sebum | 2.5 | 0.5 | ||
B877 | F | 690 | Sebum | 3.8 | 2.3 | ||
7FAO | M/j | 1235 | Sebum | 4.6 | 4.8 | ||
657C6 | F | 787 | Sebum | 7.5 | 6.25 | ||
Male | 846 | 2.6 | 34.7; |
3.5 | 0.98; 0.245 | ||
Female | 1011 | 4.7 | 2.4 |
Rat research is becoming more important for increasingly urbanized, and thus vulnerable, human populations. However, new methods are required to overcome the physical and social barriers that impede progress. In 2015, Firth et al. studied a population of only 133 rats in New York City, and discovered 18 pathogens previously unknown to science. Similarly, when the international Journal Vector-Borne and Zoonotic Diseases broadcasted a call for research in a special issue rodent-vectored pathogens and influence on human health, there were no scientific entries from North America (
We have shown that careful planning and following or modifying our protocol can yield a wealth of information regarding the ecology, time, and place of disease emergence and pathogenicity of wild rats in the urban environment. In a recent study (
From an ethological perspective, we can deduce different behaviors between males and females, adults and juveniles, including dominant, subdominant behaviors, sexual receptivity, and peak activity times. Most importantly, the data will encode the individual identification of rodents that are assessed for pathogens, and importantly, of repeatedly captured individuals, so that changing pathogen loads can be monitored over time. For instance, we may find a higher incidence of arthropod-borne bacteria, such as
While methodology such as this requires significant preparation and pre-planning of both laboratory and field gear, we note that the overall cost of our purchases was less than $15,000 USD; an insignificant cost considering the implications on our health and estimated $19 billion per annum that rats cost society (
The protocols we recommend are sufficient to provide serum and rectal fluids necessary for targeted molecular analysis and UHTS assays. However, we note that tissue distribution of viruses is not possible without sacrificing the animal. Thus, our assay is intended to enable the documentation of changes in known microbial diversity and distribution, as well as viral incidence, and not to determine the tissue-level sites of viral replication (within the animal).
All procedures are in accordance with the guidelines for ethical conduct in the care and use of non-human animals in research (Hofstra IACUC #0093).
MP and MD conceived and designed the protocol. MP wrote the manuscript with editorial input from RS and MD.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Urshuula Dulakia and Beverly Shelton of Columbia University, Institute of Comparative Medicine (ICM), provided training on rodent handling and surgical procedures. The authors thank the late Wayne Culberth, along with Art Cameron of VANTRO Systems, LLC for assistance with the design, modifications, and support for the RFID system. The authors thank Lawrence Levy and the Center for Suburban Sustainability at Hofstra University (CSS) for helpful support throughout the project. The authors also thank Robert Corrigan (Department Health and Mental Hygiene) for his ongoing support.
This project was self-funded and relied on in-kind donations from Vantro Systems, Inc., Arrow Pest Control (MD’s time) with logistic support from the ICM at Columbia University.
The Supplementary Material for this article can be found online at