Materials and Methods

Study overview—Rubber boots were contaminated for use in the study by contact with used animal bedding following a standardized procedure. One boot in each pair remained untreated (untreated control boots) and the other was treated in a disinfectant-filled footmat, disinfectant-filled footbath, or water-filled footmat (treated control boots). The relative efficacy of each treatment was evaluated by comparing numbers of bacteria (that could be cultured under aerobic conditions) recovered from treated and untreated boots.

Boots—Fifteen pairs of rubber overboots^b were used in the study. As previously described,¹ a template was used to delineate 4 sampling zones (each a 20 × 1-cm area along the long axis of the boot, designated 1 through 4 on the left boot and 5 through 8 on the right boot) with paint on the sole of each boot. Soles of these boots were generally smooth with minimal tread. The exteriors of boots were scrubbed with a soap solution,^c thoroughly rinsed with water, and disinfected by soaking for 5 minutes in a 70% ethanol solution. After airdrying, pairs of boots were stored in new plastic bags^d until used. Each pair of boots was used in the study twice. After each use, boots were cleaned and disinfected as described.

Contamination process—The goal of this process was to obtain uniform bacterial contamination on each boot that would mimic contamination typically encountered on footwear of personnel working in large animal hospitals. A mature cow (owned by the JLV-VTH) was housed for 3 days in a straw-bedded stall in the JLV-VTH. Fecal material and soiled bedding were not removed from the stall during this stabling period, but additional straw bedding was added daily. Experimental contamination of rubber boots was achieved by staff who wore the boots while walking through the contaminated stall in a serpentine pattern for 2 minutes.

Footmats—New footmats^e were used for the project. Each footmat was filled with an appropriate solution (disinfectant or water) until the solution was no longer retained by the footmat (approx 2 gallons [7.6 L]).

Footbath—One widemouthed, plastic tub^f was used as a footbath (capacity, 7 gallons [26.5 L]). Prior to use, the tub was thoroughly scrubbed with a detergent solution,^c thoroughly rinsed with tap water, and disinfected with 70% ethanol solution. After air-drying, the footbath was filled with 4 gallons (15.1 L) of fresh disinfectant solution.

Disinfectant—The disinfectant used was a peroxygen based disinfectant containing potassium peroxymonosulfate.^a At the time of the study, this disinfectant was used in all disinfectant footbaths and footmats at the JLV-VTH as part of footwear hygiene protocols. Tap water was used as a control treatment for comparison. Fresh disinfectant solution (1% aqueous solution) was prepared according to manufacturers' instructions just prior to use. The same batch of disinfectant solution was used to process all boots used in this study.

Boot disinfection—Protocols for boot disinfection were rigorously standardized and timed, and all experiments were conducted at ambient environmental temperature (approx 20°C [68°F]). Briefly, after the contamination process, 1 boot (right or left) was randomly selected by coin toss to remain as an untreated control boot, and the other boot was treated either by stepping on a footmat or by stepping through a footbath. The untreated boot was removed, and the investigator stepped with the other boot into the footbath or onto the footmat for 2 seconds and then stepped out. The treated boot was removed, and both boots were placed on their sides; samples were collected 10 minutes after the treatment. The order for experimental application of treatments (disinfectant-filled footmat, disinfectant-filled footbath, or water-filled footmat) was predetermined by use of a random numbers table. The person walking in the bedded stall was unaware of the ensuing treatment until the end of the contamination process. Replicate number, boot identification (right or left), treatment, and sample zone (1 to 8) were recorded for each sample.

Sampling process—Sterile cotton swabs^g were premoistened with Dey-Engley (neutralizing) broth,^h which contained neutralizers for common disinfectants. A different swab was used to vigorously collect a sample from each prelabeled sampling zone on the sole of each boot; 4 samples were obtained from each boot. Each swab was placed in 10 mL of neutralizing broth and kept on ice until processed in the laboratory.

Laboratory processing—Samples were processed in the laboratory within 3 hours of collection. Swabs in neutralizing broth were vortexed before plating with a spiral platerⁱ on 2 types of media: BA (trypticase soy agar with 5% sheep RBCs^j) and MAC.^k Plates were incubated aerobically for 18 to 24 hours at 37°C. Bacteria were enumerated by counting CFUs in specific zones on each plate according to the manufacturer's instructions. According to the manufacturer's specified limits of detection, plates with < 30 CFUs/plate were considered to have numbers of viable bacteria that were below the limit of reliable quantification and were therefore assigned a concentration of ≤500 CFUs/mL (≤250 CFUs/cm²). Plates with > 300 CFUs in the most dilute counting zone were considered to have numbers of viable bacteria greater than the limit of reliable quantification and were therefore assigned a concentration of ≥ 5.0 × 10⁶ CFUs/mL (≥2.5 × 10⁶ CFUs/cm²). The final results were presented as CFUs per squared centimeter of boot surface.

Data analysis—Bacterial counts were transformed to log₁₀ values to facilitate parametric analyses. Generalized linear modeling was used to analyze differences in bacterial counts; log₁₀ bacterial counts were included as the dependant variable, and treatment (disinfectant-filled footmat, disinfectant-filled footbath, water-filled footmat, or untreated control) was included as the independent variable of interest. Separate analyses evaluated bacterial counts estimated on BA or MAC plates. Statistical analyses used generalized estimating equations to control for the hierarchical and repeated nature of the data^l; boot identification (right or left) was nested within replicate identification (1 through 30). Boot identification (right or left) was also forced into models as a fixed effect. Although contamination was not considered likely to vary between right and left boots or among different sample zones on boots, these variables were controlled in models to account for unforeseen differences. Least square means for log₁₀ bacterial counts and variance estimates were determined from these models and used to compare differences associated with the experimental treatments. A critical a of 0.05 was used in evaluating all statistical comparisons.

Results

After the experimental contamination process, moisture and small amounts of bedding were frequently adhered to the soles of boots but heavy fecal contamination was not found. The results obtained with a peroxygen-based disinfectant–filled footmat were comparable to results obtained with a peroxygen-based disinfectant–filled footbath (Table 1). Both peroxygen treatments resulted in a statistically detectable (P < 0.001) reduction of 1.3 to 1.4 log₁₀ (95.4% to 99.8%) in bacterial counts, compared with the count from untreated boots. As expected, minimal differences in bacterial counts were detected following treatment via a water-filled footmat. Mean bacterial counts in cultures grown on MAC were approximately 1 log₁₀ lower than counts in cultures grown on BA.

Discussion

Results of the present study have suggested that footmats and footbaths containing peroxygen-based disinfectant were highly effective in reducing bacterial contamination (by 95.4% to 99.8%) on the soles of boots when used in conditions representative of large animal hospitals and that similar results were achieved regardless of whether footmats or footbaths were used. The present study was designed to assess the effectiveness of disinfectant-filled footmats versus disinfectant-filled footbaths that are typically used in veterinary hospitals; therefore, footwear was not scrubbed before treatments, and the time of contact between footwear and disinfectant was brief. Scrubbing the boots before disinfection and prolonging the duration of contact between boots and disinfectant would have likely increased the efficacy of the disinfectant treatments. In similar evaluations of footbath efficacy,^1–5 reductions in bacterial counts of this magnitude were not identified, which is probably attributable to differences in the duration of disinfectant exposure prior to culture of samples and differences in the bacterial load and the amount of organic material that was applied to boots as part of the various study protocols. In a previous study¹ conducted by our group, brief (2-second) treatment of boots with the peroxygen-based disinfectant followed by a 7-minute contact period resulted in approximately 75% reduction in mean bacterial concentrations. The present study incorporated the same method and duration of boot immersion but allowed a 10-minute contact period prior to collection of samples. This difference of 3 minutes in contact time was associated with a large apparent difference in disinfectant efficacy (ie, 75% vs 95.4% to 99.8% reduction in CFUs) and highlights the importance of allowing appropriate contact time between footwear and disinfectant to achieve optimal disinfectant efficacy. In theory, this disinfectant activity would continue after stepping out of footbaths or footmats, unless the disinfectant solution was rinsed from footwear surfaces.

Amass et al^4,5 evaluated the efficacy of footbaths by immersing boots that were heavily contaminated with pig manure in various disinfectants for 2 minutes. Under those conditions, there was no decrease in bacterial counts unless boots were scrubbed with peroxygen-based disinfectant^a and no difference in bacterial counts among boots treated with different disinfectants with or without scrubbing. The results of the present study have suggested that a peroxygenbased disinfectant reduces bacterial counts on boots even without scrubbing. In our study, we attempted to mimic the conditions under which disinfectant footbaths and footmats are typically used in large animal hospital settings; as such, both the contamination and disinfection processes differed from those described by Amass et al.^4,5 However, the results of the present study may be more applicable to conditions encountered in veterinary hospitals.

Samples were collected from boots 10 minutes after treatment on the basis of an assumption that some disinfecting action would continue after stepping out of a disinfectant solution. Although the boots were no longer in contact with disinfectant in the footbath or footmat during the 10 minutes following treatment, trace amounts of disinfectant may have still been present on the soles. Therefore, the results obtained from sample collection 10 minutes after treatments would more likely resemble the effects that the use of footbaths or footmats may have on infection control efforts. As mentioned, the reduction of bacterial counts determined after peroxygen based disinfectant–filled footbath treatment was greater than that reported previously when samples were collected from boots only 7 minutes following treatments,¹ and disinfection efficacy would likely further increase by extending the time to sample collection after treatment.

Typically, a reduction of bacterial counts of ≥3 log₁₀ is considered the minimum needed to identify effective surface disinfectants.⁶ Thus, 1.3 to 1.4 log₁₀ reduction achieved in the present study may not seem adequate to affect infection control efforts in a large animal hospital. However, this still represents a reduction of 95.4% to 99.8% in bacterial counts, which should decrease the probability of transmitting agents susceptible to the peroxygen-based disinfectant.^a This would seem to be especially true if footbaths or footmats were frequently used as personnel moved through hospital environments. For example, these results suggest that the bacterial load on footwear of people would generally be decreased by at least 10 fold each time that they walked through footmats or footbaths filled with peroxygen-based disinfectant. The cumulative effect of bacterial reduction following multiple treatments has the potential to substantially reduce environmental buildup and trafficking of infectious agents.

To achieve maximal decontamination, it is also typically recommended that surfaces be scrubbed with a detergent, rinsed, and treated with an appropriate disinfectant, allowing a minimum of 15 to 30 minutes of contact time.⁷ Instead, in the present study, disinfectants were applied for approximately 2 seconds without prior cleaning of boots and samples were taken after only 10 minutes of contact time. The 2-second immersion time was chosen because it was considered representative of the duration of direct boot contact with disinfectant footbaths and footmats achieved by personnel at the JLV-VTH when routinely moving through the hospital. The 10-minute contact time was arbitrarily selected as being an intermediate but reasonable estimate of what might occur as personnel moved between stalls during routine activities. The immersion and contact times are less than optimal for disinfection but were considered typical of conditions used in veterinary hospitals. Personnel in veterinary hospitals typically do not scrub boots or other footwear before using a footbath or footmat, nor do they typically use a lengthy soaking process or consistently allow the frequently recommended 15- to 30-minute contact time to elapse before moving through the hospital. Thus, the expectations for efficacy of disinfectants are fairly rigorous when used in footbaths in typical animal husbandry environments, and in this regard, the results of this study should be considered reasonably conservative.

Our study was not designed to evaluate the risk of transmitting specific pathogens, such as Salmonella enterica, but to assess treatment-associated reductions in bacteria that can be cultured under aerobic conditions. It was assumed that culture on BA would allow quantification of both aerobic gram-positive and aerobic gram-negative bacteria, whereas culture on MAC would primarily allow quantification of enteric gram-negative bacteria. Comparison of bacterial counts on MAC with those achieved on BA suggested that < 10% of the aerobic bacteria accumulated after contamination in the stall were gram-negative organisms. This is consistent with results of studies regarding gastrointestinal microflora of animals⁸ and data regarding quantification of bacteria in the JLV-VMC environment.⁹ It was not possible to use culture methods that would allow recovery of every type of potentially pathogenic bacteria, as many require special culture enrichments that make colony enumeration difficult (eg, S enterica) or require different environmental conditions for growth (eg, anaerobic and microaerophilic organisms). It was also not feasible to identify all the bacteria that remained viable after disinfectant treatments. However, determination of numbers of viable bacteria is routinely used to evaluate the environmental cleanliness.^10–12 Thus, numbers of bacteria recovered from soles of the boots via aerobic bacterial culture techniques can likely be extrapolated to provide estimations of disinfection efficacy against a broad variety of potential bacterial pathogens, especially those that have been shown to be susceptible to peroxygen-based disinfectant via in vitro testing.^13–15

There are few reports^16–18 of objective investigations regarding the efficacy of footbaths for decreasing the frequency of bacterial infections in animals or reducing bacterial counts on footwear, and most only indirectly relate to footbath use in veterinary hospitals. Antimicrobial footbaths are routinely used successfully for management of infectious hoof problems in ruminants.^19–22 Also, use of disposable boots, hypochlorite (bleach) boot baths, or so-called bag-in-a-box shipping methods were highly efficacious in preventing mechanical transmission of porcine reproductive and respiratory syndrome virus.²³ In Great Britain, the risk of Campylobacter infections in commercial broiler flocks was significantly reduced by the application of effective hygiene barriers, including appropriate use of disinfectant boot dips.^16,17 Similarly, results of an epidemiologic study of Campylobacter infection in broiler flocks in The Netherlands suggested that there was a reduced risk of infection when boots were specifically designated for use in a specific broiler house and when disinfectant footbaths were used on entering the broiler houses.¹⁸

We are unaware of any published studies that examine the health benefits that might be associated with use of footbaths or footmats in veterinary hospitals or large animal production facilities. One recent study²⁴ evaluated boot contamination on dairy operations, and results indicated that S enterica could be routinely cultured from the surface of rubber boots after being worn in cattle-housing areas at a large California dairy. Similarly, antimicrobial-resistant Escherichia coli was isolated from tracks made by experimentally contaminated boots on a plastic surface for a distance of approximately 121.9 m (400 feet) and from a concrete surface for up to 47.7 m (150 feet) on a dairy farm. The amount of bacterial tracking was decreased by use of disinfectant footbaths.²⁵

It is important to note that footwear characteristics likely have a great impact on the efficacy of disinfectant footbaths in field situations. Boots used in the present study had minimal tread, no seams, and were impervious to water. These features allow for less accumulation of dirt and organic material, and the waterproof nature of the boots improves compliance with footbath protocols. If footwear with deep treads is worn, it is more likely that brushing will be necessary to achieve measurable decreases in bacterial counts.^4,5

Our data suggest that peroxygen-based disinfectant–filled footmats and footbaths are similarly effective and that either can be used to aid infection control efforts in large animal hospitals. However, findings of this and other studies^4,5,25 indicate that footbaths should not be relied on as the only method of controlling the trafficking of infectious agents in veterinary hospital environments. General cleanliness of footwear should always be emphasized in hospital settings, and footbaths cannot be relied on as an absolute barrier against spread of contagious bacteria.