Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) approval was obtained from Colorado State University prior to the initiation of this study. Participants were WDs and their handlers from military, law enforcement, or SAR backgrounds. Permissible breeds were German, Belgian (e.g., Groenendael, Tervuren, Malinois, Laekenois), and Dutch Shepherds, or mixes of the aforementioned breeds. The canines were 1–10 years of age, either male or female, neutered or intact. The canines could be excluded from the IACUC protocol based on aggressive temperament, active dermatologic conditions, or subjective exclusion by their attending veterinarian, unit commander, or supervisor for any reason without explanation or fear of retribution.

The simulant contaminant used was a commercial product, GloGerm™. GloGerm™ is available in powder, spray and gel formulations which are well known in the safety training community and have been employed by other working dog research teams (3, 14, 16, 19, 20). In this study, colorless, GloGerm™ MIST Spray droplets were used to simulate a potentially lethal toxin exposure in a safe non-threatening environment for the canine handler team. The simulant was applied in the presence of the handler, however the GloGerm™ was not visible to the unaided eye once applied, making the exact location of the contamination less obvious. GloGerm™ MIST Spray (Figure 2) was applied topically to each canine at three anatomical locations: dorsal midline behind the shoulder blades (“back”); the inner aspect of either hindlimb above the knee where the abdomen meets the inner thigh (“inguinal”); and the cranial aspect of either forelimb between the carpus and elbow (“forelimb”). A plastic barrier with a cutout (circle, 3.2-cm diameter) for the application of the GloGerm™ (Figure 2) was utilized to prevent overspray and standardize the area of GloGerm™ application. The plastic template was held against the dog and GloGerm™ was sprayed once over the cutout to produce consistent application size at all three locations on the individual dog as well as for each canine participant in the study. Before the application of GloGerm™, a pre-contamination photo was obtained under ultraviolet (UV) illumination in a darkened room. After the application of GloGerm™, photographic images using UV illumination were obtained in a darkened room before and after the handler performed the decontamination procedure using the provided prototype kit. A camera standoff (Figure 2) provided for a consistent 13-cm distance between the camera lens and the dog when each photograph was taken as well as a consistent 1-cm reference measurement within each photograph in order to quantify the area of simulant fluorescence during analysis. After initial photography and simulant application, the handler was instructed to conduct a full canine decontamination while being observed and timed by a research team member. After the handler completed the decontamination procedure, the locations where simulant had been applied were re-photographed and analyzed to determine effectiveness of the kit materials and the procedure to remove the simulant.

The waterless, non-reactive decontamination procedure recommended in the prototype canine field decontamination kits uses a sequence of three microfiber towel types: (1) dry to initially remove gross contaminants from the fur (e.g., package labeled Wipe A Dry Wipe); (2) wet which is saturated with a surfactant to clean away residual contaminants on the fur (e.g., package labeled Wipe B Wet Wipe); and, (3) dry to remove final contamination and residual surfactant (e.g., package labeled Wipe C Dry Wipe; Figure 1). The procedures recommended in the kit instructions were designed to transfer contaminants from the canine’s fur to the microfiber towels while keeping contaminants off and away from the canine’s skin.

This study was conducted in small groups at individual canine handler team locations based on their schedule and availability. Each participating canine handler team was randomly assigned to one of three possible form factors: single towel, multi-towel small, and multi-towel large. Each handler was randomized into a trained or untrained category and one of the three possible form factor subcategories. The trained group participants received approximately 15 min of interactive instruction from a research team member in addition to the kit use instruction sheet and specific instruction on how to implement the “pinch and pull” technique (Figure 3). The untrained group participants received just the kit use instruction sheet to read at their own pace with no additional interactive training from the research team. All participants received identical instruction sheets.

Analysis of photos was performed with FIJI image software (based on ImageJ, originally developed by the National Institutes of Health). The “particle count” tool was used with a mask subtraction technique to objectively quantify the quantity of fluorescent particles removed by the handlers using the prototype canine field decontamination kits. Three photos from the same dog and same anatomical location (one pre-contamination photo, one post-contamination photo, and one post-decontamination photo) were captured and uploaded to FIJI. In order to measure particle count, these three images were cropped to include only the simulant contaminated region of interest (e.g., excluded the 1-cm fiducial for photographic scaling). These images were then compiled into a ‘stack’ on FIJI and converted from color to 8-bit black and white images. From there, the threshold of all images was adjusted at once. The ‘pre’ image was selected to view as the threshold is adjusted, as there should be no contamination visible on the ‘pre-contamination’ image. The threshold was adjusted so that there were no visible particles (e.g., dust and dander can fluoresce under UV light) in the ‘pre-contamination’ images, and that same threshold was applied to all the images in the stack. This allowed the images to be analyzed by comparing the quantified particle count for the ‘post-contamination’ and ‘post-decontamination’ images. The fluorescent particle counts were calculated by selecting ‘analyze particles’ on FIJI. This computes a fluorescence particle count for each image, and these values were recorded for further analysis to calculate the percent particle count decontamination. Similarly, the FIJI software “size” tool was used with a mask subtraction technique to objectively measure the area (cm²) of fluorescent simulant reduction in the same images. The results from the area reduction method did not differ substantially from the particle count method and were excluded from this report in support of continued use of the quantitative particle count method in this and future studies.

Early in this investigation, a mitt form factor was evaluated. Despite positive feedback from end users, the mitt form factor was found to be underperforming compared to the other form factors (single towel, multi-towel small, multi-towel large) and presented logistical burdens in both manufacturing and utilization. Due to the static position on the hand, the surface area in direct contact with the dog’s coat was limited. Only the area between the fingertips and the palm on one side of the mitt made direct contact with the fur during the “pinch and pull” method and the back of the mitt never contacted the fur. The additional thickness of the mitt increased the size and weight of the kit which was an important consideration raised in feedback from all end users. Due to poor early performance, difficulties in manufacturing, and limited adaptability for use with different personal protective equipment (PPE) and individual hand sizes, along with the increase in kit size/weight, the mitt form factor was excluded from further canine handler team evaluation and from data analysis.

Descriptive statistics were computed for the percentage of decontamination for both “size” (area; cm²) calculated with the FIJI software area tool and “particle count” (FIJI software image subtraction tool) reduction considered across all variables (trained versus untrained, towel form factors, anatomical location). Histograms and dot plots were constructed to visualize the distribution of percentage decontamination “particle count” and “size.” For each anatomical location and form factor combination, the distribution of the number of dogs in the quartile categories was compared between the trained and untrained groups using Fisher’s Exact Test. Similarly, for each location and training group, the distribution of the number of dogs in the quartile categories was compared among the kit combinations using Fisher’s Exact Test. When differences in either percentage “particle count” or “size” decontamination between training groups were not found, training groups were combined to compare the kit combinations at each location using Fisher’s Exact Test. A significance level of 0.05 was used for all tests of significance. Data was also evaluated using the above methods as “pass” versus “fail,” with <75% of contaminant removed denoting a failure, in order to consider areas that were overlooked by the handler or inadequately decontaminated by the wipe procedures and not substantially contacted with the decontamination wipes.

The highest decontamination efficacy based on mean particle count reduction was noted on the back (91.38% ± 0.18). The guard hairs at this anatomical location are longer, which allows for more efficient performance of the recommended “pinch and pull” decontamination technique, are the most easily accessible for the handler, and have more intrinsic protective qualities when compared to other hair types. The forelimb and inguinal areas had less contaminant removed across the form factors and both training groups. The forelimb had a mean fluorescent particle count reduction of 82.04% ± 0.26. Due to the short guard hairs at this anatomical location, decontamination using the “pinch and pull” method is difficult and less practical in a moving canine. Some handlers disregarded the recommended “pinch and pull” technique in the instructions and utilized a “circumferential rub” technique. Observationally, this may have improved their efficiency of GloGerm™ removal and time spent decontaminating the limbs. This technique may be added to future kit instructions and dry wipe decontamination procedures. When spraying GloGerm™ on the shorter or thinner haired areas on the limbs of some dogs, the GloGerm™ droplets penetrated the fur and directly contacted the skin. This is noted because the goal is to avoid skin contact with the contaminant since this is the point at which the canine could become poisoned. Also, contaminated skin is anticipated to be the most challenging location to decontaminate because the overlying hair may block direct physical contact with the wipe.

The inguinal area had a mean reduction of 70.15% ± 0.31 for fluorescent particle count across all form factors and training groups. This represents failed decontamination of this anatomical region based on the 75% cutoff used in the study. The hairs in this anatomical location are considered undercoat, lacking the protective quality of guard hairs present in other areas. The inguinal area is also the most difficult to reach and most easily overlooked by the handler. Additionally, the hairs vary in length and density and in some individual dogs there was GloGerm™ penetration to the skin. Some members of the canine community hypothesize the inguinal area is less likely to be contaminated due to its anatomically protected location. However, discussion with subject matter experts (SME) during military decontamination training events provided observational evidence to the contrary. For example, a dog walking in tall grass or lying down on a surface will easily have droplets transfer from the contaminated vegetation or surface to their ventrum. Peer-reviewed literature has also shown the ventral surfaces of the dog are more likely to be contaminated and more likely to be missed during wet decontamination (16). While the Perry et al. (16) study focuses on environmental contamination from standing liquid hazards which may differ from the dispersion of a chemical warfare agent, the findings reinforce the need for proper decontamination of ventral anatomical areas. In other decontamination training events, lifting of the canine onto a table or tailgate improved the handler’s ability to access and decontaminate the ventrum and inguinal areas of the canine. For these reasons, kit use instructions and training recommendations must highlight frequently encountered human errors in order to optimize decontamination efficacy. Proper restraint techniques to ensure the physical safety of the handler and other personnel assisting with canine decontamination as well as proper wear of PPE must be emphasized to avoid contaminant transference to additional personnel and equipment. Finally, sedation may be required in cases of medically symptomatic or overtly aggressive canines to allow a handler to implement the “pinch and pull” and rub or wipe techniques in difficult to reach and often overlooked anatomical areas.

There was no statistically significant difference between the single towel, multi-towel small, and multi-towel large form factors in percentage of fluorescent particle reduction, percentage of area reduction, or quartile reduction categories (e.g., 0–25, 26–50, 51–75, 76–100%). Combining objective data collection with handler and SME feedback informed several kit improvement recommendations. For example, the multi-towel large form factor was much larger in size and weight, and there was not a significant improvement in handler decontamination efficacy, therefore this kit form factor was excluded from production consideration. The larger towels were often noted to be unwieldy as handlers tried to use the clean portions of the towel without cross contamination of the dog or themselves from unclean portions of the towel. Handlers preferred to use and discard multiple small towels. The single towel and multi-towel small kits both had advantages: the single towel was the most compact and lightweight of all the evaluated form factors, giving it a significant advantage in handler feedback; however, one towel was not adequate for whole body decontamination (e.g., quickly covered with dirt, loose fur) and shared similar cross contamination issues described for large towels. Mass animal decontamination in disaster response preparedness is a growing area of concern. The single large towel form factor may be the most beneficial in mass casualty scenarios where kits could be provided to pet owners prior to going through wet decontamination. This would allow for earlier contaminant removal, decreased wicking of contaminants to the dog’s skin, and decreased cross contamination. For individual canine handler use at the point of exposure, the multi-towel small form factor has the advantage of using a separate towel for specific regions of the body to avoid tracking contamination to clean and sensitive areas. The multi-towel small form factor was more favorably viewed by medical personnel for this reason. Training handlers in a decontamination pattern based on anatomical region may improve the percentage removal of contaminants. For example, use the first towel on the head and neck region and discard the towel, use the second towel on the back and discard, etc.

There was no statistically significant difference found between the “trained” and “untrained” variables. The lack of significant difference between these two groups provides evidence that the dry wipe decontamination procedures are intuitive and that the written instructions were clear, easy to understand, and easy to implement by working dog handlers.

The average time for decontamination using the prototype canine field decontamination kit was 9 min 24 s (range = 3 min 4 s to 26 min 0 s) across all handler backgrounds, trained and untrained groups, and kit form factors. However, handlers were not wearing full Mission Oriented Protective Posture (MOPP) gear or PPE. This average time can be used as a baseline for planning purposes in a hasty decontamination or limited exposure scenario. A significant time difference is expected for decontamination in lethal environments with personnel operating in Joint Service Lightweight Integrated Suit Technology overgarments or Advanced Chemical Protective Garments.

Waterless decontamination with the prototype canine field decontamination kits used in this study and the “pinch and pull” technique was developed and studied under laboratory conditions for the removal of sulfur mustard (HD; blister agent) and venomous agent X (VX; nerve agent) from canine cadaver skin/fur samples (7). The results of handler GloGerm™ decontamination efficacy on the canine back were similar to the live agent laboratory decontamination results. The lower mean particle count reduction on the back by the handler (91.38%) versus mean reduction of VX (97.9%) and HD (99.6%) from canine cadaver fur samples may be attributed to the research team’s visual observation of 11% of handlers omitting an area when decontaminating the entire dog versus conditions in a controlled laboratory setting where the location of contaminant was known. This inter-study comparison lends strength to the necessity and ability of a canine handler to initiate decontamination at the point of exposure. Additionally, the levels of effective decontamination demonstrated by the handlers in this study provides evidence that untrained individuals can effectively utilize the dry wipe decontamination method in a realistic scenario.

The training guidelines were created early in the study design based on published canine decontamination literature and laboratory studies. As the study progressed, discussions with professional working dog personnel and observations of handler kit use and effectiveness led to productive insights. This information was not implemented during the study to avoid affecting data collection. However, new recommendations were discussed with handlers and SMEs after data collection at each testing site with favorable feedback. Additionally, handlers were not required to wear full MOPP gear or PPE during decontamination procedures. In real-world scenarios when wearing this gear, it is expected that performing decontamination procedures and canine handling will increase in difficulty, increasing the time required to fully decontaminate a WD. Individual handler experiences with other decontamination methods was unknown in this study. Each handler received instructions as described but then were allowed to conduct the decontamination procedure to their satisfaction without further guidance from the research team. Future experimental designs could evaluate handler-related variables like prior training and deployment experience as well as the conditions necessary to improve efficiency and efficacy in trained decontamination procedures. This study included only German, Belgian, and Dutch Shepherd breeds. Future research should include breeds with a wider variety of fur textures and thicknesses.

Using the canine field decontamination kit allows for debulking prior to and in conjunction with soap and water decontamination protocols. Dry wipe decontamination leverages the intrinsic protective mechanisms of the canine’s fur coat, and prevents initial wetting of the coat which has been shown to cause transport of the contaminant to the skin and transdermal absorption. Field decontamination compliments current water-based decontamination protocols by reducing aerosolization risks, reducing excessive contaminated water, and reducing environmental and cross contamination through its more controlled collection of contaminants within the microfiber towels. The contaminated wipes can then be disposed of as a solid waste whereas soap and water wash decontamination methods create a significant logistical challenge to dispose of up to 40 L of contaminated water per canine (8). The dry wipe decontamination technique limits contact with the skin and the prototype kit contains a proprietary surfactant (not a soap) proven to be non-irritating for both canine and human skin. These factors limit the impact of decontamination on the natural skin barrier, making dry wipe decontamination more suitable when repeated decontamination procedures are necessary (e.g., daily SAR work in a contaminated environment) (13, 21).

The prototype canine field decontamination kit has been designed and tested for single handler use, reducing stress on the canine and preventing additional personnel exposures. It allows for canine decontamination to be performed at the point of contamination and, when possible, away from large crowds and loud equipment. By reducing stress of the canine, a more thorough decontamination can be performed. This reduces the human health hazards associated with an incomplete decontamination of the canine, re-exposure of personnel to contaminants, PPE suit tear risks, and/or physical injury to the canine or personnel.

In a mass contamination scenario, using the prototype canine field decontamination kit, handlers can simultaneously decontaminate their canines as opposed to a one at a time system of a wet soap and water lane. This allows early decontamination, early medical intervention, decreased cross contamination, and overall improvement in canine and human outcomes and survivability. In civilian canine contamination scenarios, field decontamination can be performed by untrained personnel like pet owners or disaster relief volunteers.

The canine field decontamination kit is non-reactive, surfactant-based, and may be effective for a variety of chemical, biological, and nuclear contaminants, but because of their inherent dangers, individual contaminant types must be specifically tested in controlled laboratory studies. Individual training at the unit-level as well as complex multiagency events should include hands-on decontamination scenarios using GloGerm™ or other traceable simulants to allow handlers to practice a variety of decontamination techniques while they experience and discover their own lessons learned (e.g., circumferential rub technique for the forelimb, elevation of the canine to reach the ventrum, a step-wise approach to the multiwipe kits, commonly missed anatomical areas) while improving their confidence, efficiency and efficacy.