Evaluation of Direct Colony Sampling Compared with Sentinel-Free Soiled Bedding Testing for Murine Quarantine Programs
Sentinel-free soiled bedding (SFSB) is a form of environmental health monitoring that is an efficient method for monitoring rodent colony health. In contrast to direct colony sampling (DCS), when using PCR testing, SFSB can detect both active and past infections and is a less invasive method, classifying it as refinement. In this study, we compared DCS and SFSB for quarantine health monitoring in terms of their effectiveness in detecting pathogens during a 14-day quarantine program. In addition, we performed a time and motion study to examine the time required for each sampling method. We hypothesized that SFSB testing for quarantine would exhibit a greater degree of sensitivity than DCS for the tested pathogen list and take less time to perform. Eleven shipping containers, each containing 4-5 male or female mice aged 6-11 weeks, were subjected to simulated shipping stress as would occur during importation. During the 14-day quarantine, DCS samples included oral swabs, adhesive pelt swabs, and fresh fecal pellets that were pooled per cage and collected on days 0 (baseline) and 7. Each cage was given its own soiled-bedding sampling system, and SFSB samples were comprised of 3 groups: day 0, day 7, and a combination of days 0 and 7. There were no statistically significant differences between the 3 different SFSB sample time points (day 0, day 7, and days 0 and 7 combined) for all pathogens evaluated (P > 0.05). In addition, there were no statistically significant differences between the DCS day 7 and the days 0 and 7 combined SFSB time point for all pathogens evaluated (P > 0.05). Furthermore, SFSB was shown to be less time-consuming than DCS. Thus, SFSB sampling should be considered for quarantine health monitoring programs, as it has similar sensitivity to DCS, is a refinement, and offers a time-saving benefit.
Introduction
Environmental health monitoring (EHM) has emerged as a practical alternative to soiled bedding sentinel-based health monitoring. As defined by the 3Rs Collaborative, EHM is any type of health monitoring that does not require the use of live animal sentinels.1,2 EHM has multiple advantages over soiled bedding sentinel programs including increased sensitivity,1,3–20 reduced cost and labor,2,21 and, most importantly, it is 3Rs compliant, as it replaces the use of live animals.2,21 There are 4 types of EHM with exhaust dust testing and sentinel-free soiled bedding (SFSB) sampling as the 2 major types used. Direct colony sampling (DCS) and room and equipment monitoring are used as supplemental techniques.1,2
SFSB procedurally resembles a soiled bedding sentinel program, as it involves serial pooling of soiled bedding from rodent colony cages into a separate cage or bin that is then routinely sampled with media and/or swabs for particulates and nucleic acids and submitted for PCR testing.1 SFSB has been shown to be highly effective for routine quarterly colony health monitoring.1,5,6,14,17,19 SFSB can identify active and past infections in mice due to the fact that it collects particulates and nucleic acids over a 3-month period.
DCS involves the collection of feces, fur and oral swabs, and/or blood, which provide minimally-invasive samples of specific animals or their cage microenvironment.1 When performed on colony animals, DCS is a form of EHM, as it monitors colony health without the use of additional live animal sentinels.1,2 This is commonly used for quarantine testing of new arrival rodents2 and what was performed at our institution prior to this study. In fact, as of 2021, a survey performed showed that most institutions (63%) used a form of EHM for their quarantine testing.2 This is most likely due to the fact that DCS is superior to soiled bedding sentinels in detecting multiple pathogens.22,23
Many of the studies that assessed the use of SFSB for routine colony health monitoring also compared this sampling method to DCS. Two studies found agreement in sensitivity between DCS of colony animals and SFSB results for common laboratory animal pathogens.5,19 However, another study found that the detection of astrovirus-1 and segmented filamentous bacteria by SFSB was significantly greater than DCS of colony mice.17 In that study, however, detection of Proteus mirabilis, Staphylococcus xylosus, and Staphylococcus aureus was not significantly different between SFSB and DCS of colony animals.17 These differing results may be due to the fact that while using PCR diagnostic testing, in contrast to SFSB, DCS can only detect active infections in mice, as it is a one-time snapshot of the animal’s pathogen status. Therefore, DCS may have a higher chance of false-negative results over SFSB for pathogens that shed intermittently.22 In addition, there appear to be other advantages of using SFSB over DCS for quarantine programs, as SFSB is less invasive than DCS since it does not involve taking samples directly from the animal, which would classify it as a refinement. SFSB may also be less time-consuming for the veterinary technical staff due to a decrease in sample collection time.
Even though SFSB is increasing in use for routine colony health monitoring, there is only one other study24 supporting its use for quarantine health monitoring of newly imported animals. Therefore, due to the presumed benefits of SFSB over DCS, this study aimed to compare DCS and SFSB for quarantine health monitoring in terms of their effectiveness in detecting various pathogens. In addition, we performed a time-and-motion study to examine the amount of time each sampling method took, which to our knowledge has never been published. We hypothesized that SFSB testing for quarantine health monitoring would demonstrate a higher degree of sensitivity than DCS for the tested pathogen list and that it would be less time-consuming than DCS.
Ethical review.
All animal care and use were conducted in accordance with federal polices and guidelines and were approved by the University of Chicago’s IACUC. The University of Chicago has a Public Health Service Assurance with the Office of Laboratory Animal Welfare and is accredited by AAALAC International.
Materials and Methods
Animals and husbandry.
A total of n = 11 cages of mice, aged 6-11 weeks, were used for the study for a total of 54 mice. Mice were housed in the University of Chicago Animal Resources Center facilities (RRID:SCR_021806). Cages of immunocompetent, adult male and female mice from the program’s training colony were used for this study, which included the following strains: C57BL/6, Crl:CD1(ICR), CFW, Crl:NIHBL(S), and C57BL/6-Tg(TcraTcrb)1100Mjb/J. Cages with 4-5 mice each, housed by sex, were included in the study (5 cages contained only females, 6 cages contained only males), and the housing density was static throughout the study. All of the cages housed 5 mice except 1 housed 4 mice. The experimental unit was designated at the cage level for this study. Cage densities of 4-5 mice per cage were used to ensure that the highest caging densities were included while using the largest possible sample size.
Mice were housed in solid-bottom polysulfone IVCs (194 × 144 × 398 mm; NexGen MAX high-density mouse IVCs; Allentown, Allentown, NJ) with the IVC rack set at negative pressure and 50 air changes per hour per the manufacturer’s instructions. All cages and cage components (wire bar lids, filter tops, water bottles, and tunnels) were sanitized using a tunnel washer (Basil 6000; STERIS, Mentor, OH) with detergent (Labsan 120; Sanitation Strategies, Holt, MI). To ensure that an appropriate sanitation temperature (180 °F [82.2 °C]) was achieved, a temperature-indicating strip (Temp-Tape 180; Pharmacal Research Laboratories, Naugatuck, CT) was run through the tunnel washer at the start of each day. All cages, cage components, bedding, and enrichment were then autoclaved prior to use (autoclave job no. 971290; Primus, Orlando, FL) with a sterilization time of 20 minutes at 252 °F (122 °C). Cages contained virgin paper pulp cellulose bedding (ALPHA-dri Plus; Shepherd Specialty Paper, Watertown, TN), and for enrichment, each cage contained ∼4 g of specialty shredded paper (Bed-r’Nest; Lab Supply, North Lake, TX). A small amount of the shredded paper enrichment was moved during cage change to ensure scent transfer from the old cage to the new cage. A clear circular tunnel (Mouse Tunnel no. K3487; Bio-Serv, Flemington, NJ) was provided in the cage to perform nonaversive handling.25 All mice received acidified tap water (pH 2.8-3.2) via water bottles. All mice were fed irradiated rodent diet (Teklad 2918; Envigo, Indianapolis, IN) before day 0 of the study. To replicate our standard quarantine practices for mice at our institution, mice were fed Fenbendazole medicated rodent diet (150 ppm, Teklad 2918S, Envigo, Indianapolis, IN) starting on day 0 and were administered 3 μL of moxidectin (Cydectin 5 mg/mL; Boehringer Ingelheim, Ingelheim, Germany) topically on the dorsum on days 0 and 14. Cage change of all cage components was performed every 7 days in a class II type A2 biosafety cabinet (NuAire, Plymouth, MN) with the use of chlorine dioxide (C-Dox, Labsan; Sanitation Strategies, Holt, MI) as a disinfectant. During all procedures throughout the study, personal protective equipment included facility dedicated scrubs or a disposable gown, a hair bonnet, Tyvek sleeves, and examination gloves. Animal rooms were maintained on a 12-hour light/12-hour dark cycle with humidity ranging from 30% to 70% and temperatures ranging from 68 to 76 °F (20 to 24 °C) in compliance with the Guide for the Care and Use of Laboratory Animals.26 Mice were checked daily by the animal care staff to assess their health status and the availability of appropriate food, water, and cage conditions.
Routine colony health monitoring is performed quarterly by exhaust dust testing via PCR. The pathogen testing list includes: pneumonia virus of mice, mouse hepatitis virus, mouse parvoviruses, reovirus, mouse rotavirus, mouse theilovirus, lymphocytic choriomeningitis virus, murine adenovirus, LDH-elevating virus, mouse cytomegalovirus, Giardia muris, fur mites (Myobia musculi, Myocoptes musculinus, and Radfordia affinis), and pinworms (Syphacia spp. and Aspiculuris tetraptera). The following additional agents are excluded from the facility and tested at quarantine and/or biologic materials: Sendai virus (murine respirovirus), ectromelia, hantavirus, polyoma virus, mouse thymic virus, Filobacterium rodentium, Mycoplasma pulmonis, Salmonella spp., Citrobacter rodentium, Clostridium piliforme, Streptobacillus moniliformis, and Corynebacterium kutscheri. Murine norovirus, Rodentibacter spp., and Helicobacter spp. are endemic in the training colony housing room.
Study design.
As shown in Figure 1, 11 cages of mice were moved from their original cages in the training colony housing room to 11 autoclaved shipping containers (The Jackson Laboratory, Bar Harbor, ME) on day −1 while keeping the same mice cohoused. This was performed in a class II type A2 biosafety cabinet with the use of chlorine dioxide as a disinfectant. Examination gloves were changed between each cage to ensure no cross-contamination. A small amount of the shredded paper enrichment and the irradiated rodent diet were moved to the shipping containers. Gel packs (Napa Nectar; Systems Engineering Lab Group, Hickory, NC) were placed in the shipping container for hydration support. Each shipping container was then handled as if there were 11 unique shipments of mice to our institution. On day −1 (at 9 am and 1 pm) and day 0 (at 9 am), the shipping containers were covered with a black plastic bag and placed on a plastic transport rolling cart. They were then moved back and forth through the underground tunnels connecting 2 animal facilities that had corrugated rubber flooring for 15 minutes (Figure 2A). This rubber corrugated flooring resulted in the highest average and peak noise and vibration based on a previous study (data not shown). The shipping containers were then pushed outside for 15 minutes (Figure 2B). This was performed in June 2024 in Chicago, IL, and the outdoor temperature ranged from 75 to 82 °F (24 to 28 °C) during the simulated shipping events. This was done 3 times each with the intention of simulating shipping conditions to produce shipping stress.27,28
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-099
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-099
After the day 0 (9 am) attempt to simulate shipping stress and ∼30 hours after being packaged in the shipping containers, the mice were moved to the quarantine facility. The animals were rehoused in standard caging using barrier procedures described above, while diagnostic samples were collected for DCS and SFSB. The animals were housed in the quarantine facility for the 14-day duration of the study.
The decision was made to simulate shipping stress rather than using scheduled imports to control for variability from multiple shipments so that the study could occur at the same time and from mice with known pathogen status, which would allow us to decrease our total number of mice. Using mice from multiple shipments would increase the variables due to varying pathogen statuses at different institutions and conditions during shipment (for example, weather, shipment method/husbandry, shipment length).
DCS.
Swabs of the oral cavity (Critical Swab, slim cotton double-tipped swab, fine paper handle; VWR International, Radnor, PA), an adhesive swab rubbed along the entire pelt (Puritan, Guilford, ME), and fresh fecal samples were collected from each mouse. All samples collected from each mouse in the cage were pooled. The DCS samples were collected at 2 time points: day 0 (baseline samples) and day 7 (experimental samples) during the weekly cage change. All samples were submitted to an external diagnostic laboratory for quantitative PCR (qPCR) testing within 2 days after the day 7 samples were collected (Charles River Laboratories Research Animal Diagnostic Services, Wilmington, MA).
SFSB sampling.
Each cage was assigned its own soiled-bedding sampling system (PathogenBinder kit; Charles River Laboratories, Wilmington, MA). On day 0, 2 pieces of media (PathogenBinder Kit; Charles River Laboratories, Wilmington, MA) and all the soiled bedding from each shipping container were placed into their respective soiled-bedding sampling systems. The media were in contact with the soiled bedding for a duration of 15 seconds while the sampling system container was rotated and agitated several times to maximize exposure to the media per the manufacturer’s instructions. Once complete, the media were transferred into conical tubes using new, clean gloves. One piece of media was submitted to the diagnostic laboratory (Charles River Laboratories Research Animal Diagnostic Services, Wilmington, MA) as the day 0 sample. The second media were stored to be used as a day 0 and 7 combined sample. Soiled bedding was then discarded. This SFSB sampling was performed again as described above at the day 7 cage change, where all bedding from each cage was placed in the respective soiled-bedding sampling system. The collected SFSB samples consisted of 3 different samples, which represented day 0, day 7, and a combined days 0 and 7, which were media that were agitated with soiled bedding from both time points. The combined days 0 and 7 sample was used to determine whether media exposed to bedding at 2 separate time points and therefore collected over a longer duration (from the shipping container and after 1-week cage change) would increase diagnostic sensitivity. This approach aimed to account for intermittent shedding of certain pathogens and potentially elevate the nucleic acid burden available for qPCR detection. All samples were submitted to an external diagnostic laboratory for qPCR testing within 2 days after the day 7 samples were collected (Charles River Laboratories Research Animal Diagnostic Services, Wilmington, MA).
Negative control group.
A negative control group was included for both sampling methods. For DCS, on day 0, an adhesive swab (Puritan, Guilford, ME) was used to swab the interior of a clean, autoclaved cage at the level of the clean bedding. For SFSB, on day 0, media were placed in a soiled-bedding sampling system (PathogenBinder kit; Charles River Laboratories, Wilmington, MA) with autoclaved bedding and agitated as described. Both samples were submitted to the diagnostic laboratory for qPCR testing (Charles River Laboratories Research Animal Diagnostic Services, Wilmington, MA).
PCR testing pathogen list.
All samples were tested for the pathogens included on the routine health monitoring testing list described above to ensure that the animals remained negative for these known excluded pathogens. In addition, all samples were tested for a list of agents that were not routinely monitored or excluded from this housing room, the non-excluded agent (NEA) list (Table 1).
| Viral agents | Bacterial/fungal agents | Parasitic agents |
|---|---|---|
| Astrovirus 1 and 2 a | Beta-hemolytic Streptococcus groups A, B, C, and G | Chilomastix muris |
| Murine norovirus a | Bordetella bronchiseptica | Cryptosporidium spp. |
| Rodent chapparvovirus 1 | Bordetella hinzii and pseudohinzii | Demodex musculi a |
| Campylobacter spp. | Eimeria spp. | |
| Chlamydia muridarum | Entamoeba spp. a | |
| Corynebacterium bovis | Hexamastix spp. a | |
| Helicobacter spp. a | Spironucleus muris | |
| Klebsiella oxytoca | Tritrichomonas spp. a | |
| Klebsiella pneumoniae | ||
| Pneumocystis spp. | ||
| Proteus mirabilis | ||
| Pseudomonas aeruginosa | ||
| Rodentibacter heylii and pneumotropicus a | ||
| Segmented filamentous bacteria a | ||
| Staphylococcus aureus | ||
| Staphylococcus xylosus a | ||
| Streptococcus pneumoniae |
The table lists the agents that were tested for during this study that were not normally monitored and excluded as part of the health monitoring program for this housing room, that is, the non-excluded agents.
Agents that tested positive using direct colony sampling and sentinel-free soiled bedding (positive non-excluded agents).
Time and motion study.
To quantify the time required by the veterinary technician for each sampling method, a study was performed using 5 cages of mice each housing 5 mice. Setup times to label and prepare diagnostic collection tubes were not included in this study, as they are comparable between the 2 sampling methods. Therefore, for DCS, the time required to collect swabs of the oral cavity and pelt and fresh fecal samples from each mouse in the cage was quantified. For SFSB, the time required to perform this sampling method was calculated by measuring the following steps: removing the soiled bedding from each cage, placing it into the soiled bedding sampling systems, adding media, and rotating and agitating the kit for 15 seconds.
Blinding.
Internal accession numbers were assigned to each sample, so that the diagnostic laboratory was blinded to the sampling type and time point of the samples analyzed.
Statistical analysis.
Data were considered significant for P values ≤ 0.05. Power calculations for group size were made using data from previous literature assessing the use of SFSB for routine health monitoring. Power analysis suggested groups of 8-10 to reject the null hypothesis with 95% probability and a power of 80%. Data we rerecorded into spreadsheets for recordkeeping (Excel; Microsoft, Redmond, WA). Data were analyzed using the statistical program R (R version 4.3.2 [R Core Team (2023)]) or Excel. Comparisons of DCS compared with SFSB for each positive NEA (posNEA) and each timepoint were done using a McNemar test. A χ2 test and Kruskal–Wallis test were used to compare SFSB collection days to each other. Analysis of the infectious agent taxonomic classification totals was performed using the Wilcoxon signed rank test. Analysis of the time and motion study was performed using a one-tailed t test.
Results
PCR testing pathogen list.
All DCS and SFSB samples at all times points were negative for the agents on the routine health monitoring testing panel (see Materials and Methods). Of the agents on the NEA list (Table 1), there were only positive results using SFSB and DCS for the following agents: viral: astrovirus 1 and MNV; bacterial: Helicobacter spp. (H. hepaticus, H. ganmani), Rodentibacter heylii, R. pneumotropicus, S. xylosus, and segmented filamentous bacteria; and parasitic: Demodex musculi, Entamoeba spp., Hexamastix spp., and Tritrichomonas spp. Data analysis was only performed on the agents with posNEA results from the list.
DCS.
PosNEAs were evaluated for samples collected via DCS at day 0 and day 7. On assessment of DCS day 0 compared with day 7, there were no significant differences in positive testing results for the posNEAs (P > 0.05), with the exception of Hexamastix spp. There was a significantly decreased number of positive results for Hexamastix spp. at day 7 compared with day 0 (P = 0.023; Table 2).
| DCS (%) | SFSB (%) | ||||
|---|---|---|---|---|---|
| Sampling day | Day 0 | Day 7 | Day 0 | Day 7 | Days 0 and 7 |
| Viral agents | |||||
| Astrovirus 1 | 55 | 64 | 27 | 46 | 46 |
| MNV | 36 | 36 | 18 | 36 | 27 |
| Bacterial agents | |||||
| Helicobacter spp. | 91 | 91 | 91 | 91 | 91 |
| Helicobacter hepaticus | 46 | 46 | 36 | 46 | 46 |
| Helicobacter ganmani | 55 | 55 | 55 | 55 | 55 |
| Rodentibacter heylii | 46 | 36 | 18 | 36 | 27 |
| Rodentibacter pneumotropicus | 27 | 36 | 9 | 18 | 18 |
| Staphylococcus xylosus | 27 | 36 | 9 | 46 | 36 |
| SFB | 36 | 36 | 9 | 36 | 27 |
| Parasitic agents | |||||
| Demodex musculi | 27 | 36 | 18 | 27 | 27 |
| Entamoeba spp. | 18 | 0 | 18 | 18 | 18 |
| Hexamastix spp. | 73 a | 9 a | 73 a | 55 | 55 |
| Tritrichomonas spp. | 36 | 46 | 46 | 46 | 36 |
DCS sampling was assessed at day 0 and day 7. SFSB testing was assessed at day 0, day 7, and days 0 and 7 combined. Data were considered significant if P ≤ 0.05. There was a significantly decreased number of positive test results for Hexamastix spp. at DCS day 7 when compared with both DCS day 0 and SFSB day 0. No other significant differences were seen when comparing testing groups of the specific agents.
Abbreviations: DCS, direct colony sampling; posNEA, positive non-excluded agent; SFB, segmented filamentous bacteria; SFSB, sentinel-free soiled bedding.
Significant results.
SFSB sampling.
The posNEAs were assessed to evaluate the timing of SFSB by comparing sample collecting at day 0, day 7, and day 0 and day 7 combined. There was no significant difference in positive pathogen results seen between any sample collection days (P > 0.05). This indicates there is no difference seen in sensitivity of SFSB testing based on the day of sample collection (Table 2).
DCS compared with SFSB sampling.
Both DCS time points were compared with all time points of SFSB to determine whether there existed any differences in sensitivity between the 2 collection methods. When comparing all collection methods at each time point to each other, there were no significant differences seen for any of the agents, except Hexamastix spp. (P = 0.018). Each sample collection method and time point were also compared with each other individually (for example, DCS day 0 compared with SFSB day 0, DCS day 0 compared with SFSB day 7). There was a significantly decreased number of positive results for Hexamastix spp. at DCS day 7 compared with SFSB day 0 (P = 0.023). All other posNEAs had no difference seen in sensitivity when comparing DCS day 0 with any SFSB time point (Table 2).
Comparison of infectious agent taxonomic classification.
Looking categorically at the posNEA data, there were a total of 2 viral, 6 bacterial, and 4 parasitic agents assessed in this study. In addition to looking at results for each individual agent, we assessed the number of positive results within the infectious agent group categories evaluated in this study: viral, bacterial, and parasitic. There were no significant differences in the number of positive results for any of the infectious agent groups between DCS and SFSB testing. There were also no significant differences when assessing the total percent positives for all results for each collection method and time point compared with each other (Figure 3).
Citation: Journal of the American Association for Laboratory Animal Science 64, 5; 10.30802/AALAS-JAALAS-25-099
Negative control group.
The negative control groups for both DCS and SFSB were negative for all pathogens tested in the excluded and NEA lists.
Time and motion study.
For DCS, the time required to collect swabs of the oral cavity and pelt and fresh fecal samples from each mouse in the cage was averaged for the 5 trials and was 8 minutes 54 seconds (minimum, 7 minutes 46 seconds; maximum, 10 minutes 26 seconds) per cage. For one SFSB time point, the time required to perform this sampling method was calculated for 5 trials and averaged by measuring the following steps: removing the soiled bedding from each cage, placing it into the soiled bedding sampling systems, adding media, and rotating and agitating the kit for 15 seconds. The mean for SFSB was 61 seconds (minimum, 54 seconds; maximum, 67 seconds) per cage. Therefore, for the combined days 0 and 7 SFSB sampling, the total mean time would be double this at 122 seconds per cage. Overall, SFSB was significantly faster to perform than DCS (P < 0.0001). DCS took on average 7 minutes 53 seconds more per cage than did an SFSB single time point, and DCS took 5 minutes 51 seconds longer per cage than did the combined SFSB sampling.
Discussion
This study sought to compare the efficacy of SFSB compared with DCS as forms of EHM for quarantine health monitoring programs. As SFSB can identify active and past infections through its ability to collect particulates and nucleic acids over a period of time, we hypothesized that this would be more sensitive than DCS for the tested pathogen list. This is also due to the fact that when using PCR testing, DCS only allows for detection of active infections.22 Previous studies have shown that SFSB is more sensitive than DCS when used for routine colony health monitoring over a 3-month period for certain pathogens17; however, there are few studies assessing sensitivity when the time period for the SFSB collection decreases.24 Because our study sought to assess health monitoring for a quarantine program, we used SFSB over a 7- or 8-day period based on the day 7 sample or combined days 0 and 7 sample, respectively. The day 7 sample collected nucleic acids accumulated in the bedding for a 7-day period between day 0 and day 7, whereas the combined day 0 and day 7 sample was exposed to nucleic acids over this 7-day period plus the 1-day period during the simulated transport. Our results showed that DCS and SFSB were equally sensitive for 11 of 12 of the posNEAs detected; however, for Hexamastix spp., there were statistically significant differences found. In addition, we performed a time-and-motion study to examine the amount of time that each sampling method took. We hypothesized that SFSB would take less time to perform than DCS for a veterinary technician, since DCS requires taking samples directly from the animals, including swabs of the oral cavity, the entire pelt, and fresh fecal samples. For SFSB, the time involvement includes removing the soiled bedding from each cage, placing it into the soiled bedding sampling systems, adding media, and rotating and agitating the kit for 15 seconds. In the study, SFSB was shown to be substantially less time-consuming than DCS.
For Hexamastix spp., statistical significance was found when performing 2 comparisons. When comparing the DCS sampling time points, there was a significantly decreased number of positive results for Hexamastix spp. at DCS day 7 compared with DCS day 0. In addition, when comparing DCS to SFSB, there was a significantly decreased number of positive results for Hexamastix spp. at DCS day 7 compared with SFSB day 0. Overall, these differences may be due to the fenbendazole and moxidectin treatment provided to the animals, which led to a smaller percentage of positive results at day 7 compared with day 0 in both testing methods. It is theorized that no significant difference was seen between SFSB day 0 and SFSB days 0 and 7 combined or SFSB day 7, as nucleic acids from Hexamastix spp. were still present in the bedding from the presence of the protozoa in the feces, which would not be detected in feces collected directly from animals at day 7 in DCS sample collection. While these are not the typical recommended treatment options for protozoa, there may have been some effectiveness; however, overall, treatment recommendations for Hexamastix spp. are lacking.29 Another theory may be that the mice were shedding Hexamastix spp. more at day 0 after the simulated shipping stress compared with day 7.
Previous studies assessing the use of SFSB for routine colony health monitoring found comparable sensitivity between DCS of colony animals and SFSB results for common laboratory animal pathogens as shown in this study.5,19 The pathogens evaluated in those studies were comparable to those in this study as they included murine norovirus, Helicobacter spp., R. heylii, R. pneumotropicus, P. mirabilis, Entamoeba spp., Spironucleus muris, S. xylosus, and Tritrichomonas spp.5,19 Another study found that the detection of astrovirus 1 and segmented filamentous bacteria by SFSB was significantly greater than DCS of colony mice.17 This is consistent with our hypothesis that SFSB is more sensitive than DCS due to the fact that it can detect active and past infections. However, in our study, we did not see a difference in sensitivity for these 2 pathogens when comparing DCS and SFSB. In the one study comparing DCS to SFSB for quarantine health monitoring assessing multiple agents, SFSB was found to have equivalent or improved detection as compared with DCS.24 When we compared the total number of positive tests for each taxonomic classification (virus, bacteria, and parasite), we again did not find any statistical difference in DCS compared with SFSB. Ultimately, as shown in previous publications and in this study, SFSB health monitoring is efficacious for viral, bacterial, and parasitic agents. Finally, in the recently published study assessing DCS compared with SFSB for a quarantine program, we found comparable results, as they concluded that SFSB provided equal or improved detection for multiple pathogens as compared with DCS.24 This study also resolved some of the limitations of the current study, as they had a larger sample size and did not have to simulate shipping stress.24
We performed assessments at 3 different time points using SFSB (day 0, day 7, and days 0 and 7 combined) to determine whether the timing of the SFSB collection method affects sensitivity. The combined day 0 and 7 sample was used to determine whether media exposed to bedding collected over a longer duration (from the shipping container and after 1-week cage change) would increase diagnostic sensitivity. We did not find a statistical difference when comparing these 3 time points for all pathogens tested. Based on these data, we plan to use the days 0 and 7 combined time point moving forward at our institution for quarantine health monitoring to provide the most comprehensive SFSB testing. If multiple cages arrive in a shipment, the soiled bedding from these cages would be pooled into one soiled-bedding sampling system. Overall, this would then allow for a 14-day quarantine program at our institution, as the animals can be released once negative PCR testing results are available. To ensure sufficient length of treatment of fenbendazole diet for pinworm treatment30 due to the chance of false-negative PCR results,31 we transport cages to the destination housing rooms and maintain fenbendazole feed for an additional 3 weeks for a total of 5 weeks of treatment. This is performed based on a risk assessment for importations from institutions that provide proof of no pinworm outbreaks for at least 12 months.
For the time and motion study, there was a substantial difference in time it takes to perform DCS over SFSB. DCS took on average 5 minutes 51 seconds longer than the combined SFSB for the veterinary technician to perform for each shipment. For this study, each shipment was only comprised of 4-5 animals; however, actual imports to an institution could include additional animals, which would increase the time required. Depending on the number of imported shipments an institution receives each year, this time savings could be beneficial. For example, our program has an estimated 55 imports each year, and this could save at least 5 hours each year. More importantly, even though DCS involves minimally invasive procedures such as swabs of the oral cavity, the entire pelt, and collecting fresh fecal samples, this involves restraint of the animal, which is known to be stressful and may cause harm to mice.32,33 In contrast, SFSB does not involve touching the animal, as the soiled bedding from the standard cage change is used for the diagnostic testing. Therefore, SFSB is a clear refinement compared with DCS.
Conclusions
SFSB sampling should be considered for quarantine health monitoring programs as it has similar sensitivity to DCS, is a refinement, and offers a time-saving benefit. In addition, it allows for a 14-day quarantine program.
Experimental Timeline.
Attempts to Recreate Shipping Stress.
Number of Positive Results Categorized within Infectious Agent Taxonomic Classification: Viral, Bacterial, and Parasitic.
Contributor Notes