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Vol 54, No 6
November 2015
Pages 745–755
Journal of the American Association for Laboratory Animal Science
Copyright 2015
by the American Association for Laboratory Animal Science
745
The use of evidence-based standard operating procedures
in animal resource centers is crucial to cost containment and
sustainable energy use. Understandably, biosecurity is a major
driver of procedures and processes in rodent facilities and
permeates virtually all aspects of animal resource operations,
making it necessary to balance the cost: benefit of detection,
prevention, and control of infection. As an example, recent
publications that suggested that mouse parvovirus (MPV)
infection can be caused by MPV-contaminated grains that
were not inactivated during their processing into pelleted ro-
dent feed25,36,47 have led to changes in husbandry procedures
including the use of autoclaved or irradiated food. Although
the successful control and prevention of MPV infections in our
facilities has been attributed to the practice of autoclaving mouse
cages preassembled with bedding and food prior to their use
in the facility, this procedure was not definitively proven to be
the sole factor in MPV eradication.30 Because wash centers are
historically the largest utilities consumer in animal facilities,15
this practice of autoclaving cages prior to use is not only labor-
intensive but also energy-intensive, and the labor and energy
consumption is amplified in facilities that lack a bulk autoclave
and in which the cages must be transported to be autoclaved.
In addition, during outbreaks with MPV and other infectious
agents, our long-standing standard operating procedure for
soiled cages is to autoclave them to inactivate infectious agents
prior to removing the soiled bedding and cage washing. This
labor- and energy-intensive practice of using autoclaving to
decontaminate cages has historically been justified because MPV
is a nonenveloped virus that is highly stable in the environment
and difficult to inactivate.5,6,16,27,38,42,49 Despite the environmen-
tal stability of the virus, infections with MPV are difficult to
detect because the amount of virus shed is low; transmission
can be inefficient, resulting in inconsistent seroconversion of
mice housed in the same cage; and PCR analysis can detect
low levels of MPV DNA in the feces of mice that are unable to
transmit virus to contact sentinels.4,30,31 We recently demon-
strated that cage washing alone removed or inactivated MPV
from 14 cages that had housed outbred mice acutely infected
with MPV; these findings are not surprising when the inefficien-
cies of MPV transmission are considered.11 These initial results
challenge the cost:benefit ratio of autoclaving cages as a means
to decontaminate them prior to cage washing during an MPV
outbreak. Our findings with MPV led us to question whether
cage washing alone might be effective for decontaminating
cages after exposure to other infectious agents that are stable
Effect of Cage-Wash Temperature on the Removal
of Infectious Agents from Caging and the
Detection of Infectious Agents on the Filters
of Animal Bedding-Disposal Cabinets
by PCR Analysis
Susan R Compton1,* and James D Macy1,2
Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within
animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission.
Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180
°F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages
seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia
obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained
pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing
at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR
analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the
prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples
collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive
for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected
mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and
Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening.
Abbreviations: ABDC, animal bedding disposal cabinet; MAV, murine adenovirus K87; MHV, mouse hepatitis virus; MNV, mu-
rine norovirus; MPV, mouse parvovirus; MVM, minute virus of mice; MMBTU, million British thermal units; SW, Swiss Webster
Received: 19 Nov 2014. Revision requested: 24 Dec 2014. Accepted: 30 Jan 2015.
1Section of Comparative Medicine, Yale University School of Medicine, and 2Animal
Resources Center, Yale University, New Haven, Connecticut
*Corresponding author. Email: susan.compton@yale.edu
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Journal of the American Association for Laboratory Animal Science
November 2015
virus, MHV, MPV, minute virus of mice (MVM), murine noro-
virus (MNV), pneumonia virus of mice, reovirus, Sendai virus,
Theiler encephalomyelitis virus, and Mycoplasma pulmonis and
were free of bacterial and parasitic infections at the time of
shipment. Random-bred female mice were obtained from 3 local
pet stores (4 mice per store) to serve as sources of Helicobacter
spp. and pinworms. Mice were housed in IVC (70 cages per rack)
under positive pressure (ACE MicroVent,Allentown, NJ). Cages
containing corncob bedding (Harlan Teklad, Indianapolis, IN),
rodent chow (Global 2018S, Harlan Teklad) and nesting material
(Cotton squares,Ancare, Bellmore, NY) were preassembled and
autoclaved. Mice had unrestricted access to hyperchlorinated
(4 to 6 ppm) water delivered by water bottle. The mice were
housed and husbanded according to standard biocontainment
procedures. The animal room had a negative pressure differen-
tial relative to the corridor, a 12:12-h light:dark cycle, 10 to 15
air changes hourly, room temperature of 22.2 ± 1.1 °C, and room
humidity of 50% ± 10% and was used exclusively for this study.
All animal care and experimental procedures were performed in
an AAALAC-accredited animal facility, were approved by the
Yale IACUC, and adverse events (described later) were reported
to the IACUC.All animal care followed the Guide for the Care and
Use of Laboratory Animals,24 and experimental procedure were
in accordance with federal policies and guidelines governing
the use of vertebrate animals.
PCR analysis. Individual fecal pellets were collected from the
anus of each mouse, and pools of 6 to 10 pellets were collected
from soiled bedding. The nape, abdomen, and rump of mice
were swabbed by using sterile Hydraflock swabs (Puritan, Guil-
ford, ME) for detection of fur mites. Cage swabs were collected
by swabbing the perimeter of the cage just above the bedding
and then passing the swab through the soiled bedding in an X
pattern from cage corner to cage corner to detect viruses, Heli-
cobacter spp., and pinworms. Cecal samples were collected from
mice after carbon dioxide euthanasia. All samples were frozen
in 1.5-mL tubes at –20 °C prior to PCR analysis. DNAand RNA
were extracted from samples by using DNeasy or RNeasy kits
(Qiagen, Valencia, CA) according to the manufacturer’s instruc-
tions. PCR assays were performed by using iTaq Universal SYBR
Green kit (BioRad, Hercules, CA), and RT–PCR assays were per-
formed by using iScript One-Step RT–PCR kit with SYBR Green
(BioRad) and a thermocycler (CFX Connect, Biorad). Primers
specific for cilia-associated respiratory bacillus, Helicobacter
spp., MHV, parvovirus (MPV and MVM), murine adenovirus
K87 (MAV), MNV, murine rotavirus, Myobia musculi, Myocoptes
musculinus, Pasteurella pneumotropica, pinworms (Aspiculuris
tetraptera and Syphacia obvelata), Pneumocystis murina, reovirus,
and Theiler encephalomyelitis virus were used as previously
described.12,20,21,44 RT–PCR analysis for lymphocytic choriomen-
ingitis virus was performed by using the primers LCMVS710
(5′ AGG CTC AGA TGG CAA GAC C 3′) and LCMVS1641 (5′
GCC CAA ATG TTG TGA CAC TCT 3′). RT–PCR analysis for
pneumonia virus of mice was performed by using PVM1616 (5′
CCCAACATG GAG GTCAAG CAG 3′) and PVM1987 (5′ GCA
TTG CCAAGCACAACACTG 3′). RT–PCR analysis for Sendai
virus was performed by using SEN399 (5′ GGAGTAAAC GCC
GAT GTC AAA 3′) and SEN1274 (5′ CCC TTG GCT GTA TCC
GTC ACT 3′). Mycobacteria spp. PCR analysis used MYB404 (5′
TTT CTC GGA TTG ACG GTA GG 3′) and MYB1235 (5′ TGA
GAC CGG CTT TAA AAG GA 3′). Mycoplasma pulmonis PCR
analysis used MYPUL180 (5′ TTA GAT CGC ATG ATT TAG
AT 3′) and MYPUL875 (5′ TGC GAG CAT ACT ACT CAG 3′).
Serology. Cardiocentesis was performed after carbon dioxide
overdose. Sera were tested in immunofluorescent antibody
in the environment, even if they are shed for longer periods of
time or at higher levels than is MPV.
In our previous study,11 we showed that cage washing was
effective at preventing fomite-based transmission of MPV by
cage components, and we postulated that water temperature
and detergent type contribute to MPV decontamination either
directly through inactivation of the virus and/or indirectly
through effective mechanical removal of organic waste and
residual virus from the cage. The Guide for the Care and Use of
Laboratory Animals24 states that “disinfection from the use of hot
water alone is the result of the combined effect of temperature
and the length of time at a given temperature” and that “effec-
tive disinfection can be achieved with wash and rinse water at
143–180 °F or more.” Theoretically, contact times to disinfect
equipment at these temperatures would need to be 1800 s at
143 °F (61.7 °C) and 0.1 s at 180 °F (82.2 °C).46 Contact times
of 6 s or more at 168 to 180 °F (75.6 to 82.2 °C) killed 3 types
of bacteria (Pseudomonas aeruginosa, Salmonella cholerasuis, and
Staphylococcus aureus) in one study,45 and contact times of 2 min
or more at 160 °F (71.1 °C) killed 5 types of bacteria (Escherichia
coli, Klebsiella pneumonia, Proteus mirabilis, Providencia rettgeri,
and Staphylococcus epidermidis) in another.39 However, these 2
studies were performed by using hot water in a test tube and,
therefore, detergent and mechanical spraying action that occur
within rack washers was not a factor.
Traditionally, the temperature used for the wash and rinse
water in our facilities has been 180 °F (82.2 °C) with a belt speed
of 2 to 3 ft./min (0.6 to 0.9 m/min). We postulated that if the
volume and force of the wash water, combined with detergents,
consistently diluted or removed infectious agents to below the
level necessary for the transmission of infection, then wash
temperatures high enough to inactivate the agents would be
unnecessary. Given the energy usage and infrastructure neces-
sary to boost ‘domestic’ hot water to 180 °F, the purpose of this
study was to compare the efficacy of cage washing by using
the domestic hot-water temperature (110 °F [43.3 °C]) and the
traditional steam-boosted wash temperature (180 °F [82.2 °C])
on preventing the transmission from contaminated caging of 3 of
the most prevalent viral agents (MNV, MPV, MHV; prevalence,
32.6%,1.8%, and 1.6%, respectively ) and bacterial and parasitic
agents (Helicobacter spp. and pinworms; prevalence,15.9% and
0.3%, respectively).35
Effective and efficient decontamination of caging goes hand-
in-hand with effective and efficient detection of infection.
Timely detection of infection is important to biosecurity, and
environmental sampling is a promising adjunct to sentinel
exposure programs for the early detection of infectious agents.
PCR analysis of cage and rack components, including the out-
flow prefilter of ventilated racks, has been shown to be of use
for several infectious agents including MPV, MHV, Helicobacter
spp., and fur mites.10,26,31 The ability to reliably detect infectious
agents from a site where soiled bedding debris is aerosolized and
concentrated might provide an efficient adjunct for infectious
agent screening. In the current study, we determined whether
monitoring by PCR analysis of dust and debris collected from
the animal bedding disposal cabinet (ABDC) prefilter could be
used as an efficient adjunct to sentinel programs to screen for
contamination by infectious agents.
Materials and Methods
Mice. Female Swiss Webster mice (Crl:CFW [SW]; age, 4 to 6
wk) were obtained from Charles River Laboratories (Kingston,
NY). Vendor reports indicated that mice were seronegative for
ectromeliavirus,murinerotavirus,lymphocyticchoriomeningitis
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Effect of cage-wash temperature on pathogen removal
removed by ‘dumping’ the soiled bedding from the contami-
nated cages into the ABDC; cages were then scraped prior to
loading into the tunnel washer (Better Built Metal Wash Tunnel
Washer, Northwestern Systems, Delta, Canada), which had a
belt speed of 2.4 ft/min (0.74 m/min). Cages were exposed to
hot water in the wash and rinse cycles for a total of 4.6 min. The
chemicals used in the tunnel washer were Enviro-Kleen 1500,
GLPC7, and Acidulate 150 (Quip Labs, Wilmington, DE). After
the soiled bedding from all 36 cages was dumped, 4 approxi-
mately 1-mL samples of dust and debris were collected from
the prefilter of the ABDC, and PCR analysis was performed
to determine whether detectable amounts of MPV, MHV, or
MNV had been deposited on the prefilter during dumping of
soiled bedding. Then, the first 16 cages, with filter tops and
wire bars, were washed at a wash temperature of 110 °F (43.3
°C), which was confirmed by using a chromel–alumel type K
thermocouple connected to a Meter (Fluke, Everett, WA). The
thermocouple was located in the rinse tank, which stores and
maintains the preset water temperature for this tunnel washer.
Before the second set of 16 cages was washed, the steam system
was activated, and the water in the tunnel washer tank was
allowed to heat to 180 °F (82.2 °C). The wash temperature was
confirmed to be 180 °F by using temperature-sensitive tape
(Thermolabel, Paper Thermometer, Greenfield, NH), and then
the second set of 16 cages was washed. The final 4 cages were
not washed and served as positive-control cages to confirm that
the amount of virus present on the soiled cages was sufficient
to cause infection of naive sentinel mice. After cage washing at
either 110 or 180 °F, cages were reassembled, transported back
to the animal room, and placed on the IVC rack to dry for 2 h.
The efficacy of cage sanitation was assessed by ATPase testing
(Accupoint 2 Sanitation Monitoring System with surface sam-
plers, Neogen, Lansing, MI).
Autoclaved food, autoclaved bedding, autoclaved nesting
material, and an autoclaved water bottle filled with hyper-
chlorinated water were added to each washed cage, and then
a 4-wk-old female SW sentinel mouse was placed into each
cage. One week after the addition of sentinel mice to the cages
(that is, 1 wk after exposure to the washed cages), feces were
collected from sentinel mice for PCR analysis. At 2 wk after
exposure to the washed cages, sentinel mice were moved to
clean cages. At 3 wk after exposure to the washed cages, mice
were euthanized, and feces and blood were collected for PCR
analysis and serology, respectively.
Helicobacter spp. and pinworm contact-infection study. The
study design included a 2-stage approach to produce SW mice
infected with Helicobacter spp. and pinworms because stocks of
these infectious agents were not readily available. Initially, 12
random-bred female mice were obtained from 3 local pet stores
(4 mice per store); 9 of these mice were housed individually. We
added 4-wk-old female SW mice (n = 4 per cage; 36 SW mice
total) to each of the 9 cages to initiate infection with Helicobacter
spp. and pinworms, understanding that other infectious agents
harbored by the pet-store mice would also be transmitted to
the SW contact mice. The extra 3 pet-store mice were housed
separately as replacements for pet-store mice that might become
ill during the study. Feces from pet-store mice were tested for
Helicobacter spp., pinworms, lymphocytic choriomeningitis vi-
rus, MAV, MHV, MNV, parvovirus, murine rotavirus, reovirus,
and Theiler encephalomyelitis virus by PCR analysis on their
arrival to our facility. Pet-store mice were rotated among the 9
cages of contact SW mice on days 5, 10, 15 and 20 to produce a
more uniform infection profile than would be obtained if each
cage of contact SW mice were exposed to only a single pet-store
assays as previously described40 for antibodies to one or more
of the following agents: ectromelia virus, lymphocytic chori-
omeningitis virus, MAV, MHV, MPV, MVM, murine rotavirus,
pneumonia virus of mice, reovirus, Sendai virus, Theiler en-
cephalomyelitis virus, and M. pulmonis.
Parasitology. To detect S. obvelata, cellophane tape tests
were performed by applying tape to the perianal region of
the mouse. The adherent surface of the tape was placed onto
a glass microscope slide, and slides were observed by using a
microscope at 10× and 40× magnification. Slurries were made
in 0.9% saline from pools of 6 to 10 feces collected from soiled
bedding. A small amount of the slurry was placed on a glass
microscope slide, a cover slip was placed on the sample, and the
sample was examined for evidence of ova or adult endoparasites
by microscopy at 10× and 40× magnification. Portions of the
cecum and colon from each mouse were submitted for direct
examination for intestinal parasites. Immediately after collection
of the cecum and colon, approximately 0.5 mL of 0.9% saline
was added to the sample in a culture dish and mixed with a
wooden applicator. A small amount of the sample was placed
on a glass microscope slide, cover-slipped, and examined for
evidence of ova or adult endoparasites by microscopy at 10×
and 40× magnification. Parasites were identified and speciated
morphologically. The cecocolonic contents in the culture dish
were examined again, at 24 h after the initial exam, under low-
power (10×) microscopy for evidence of adult endoparasites.
Fur plucked from the nape and rump of each mouse was placed
in a drop of mineral oil on a microscope slide; a cover slip was
placed on the sample; and samples were examined by using a
microscope under low power (10× magnification) for evidence
of fur-mite ova, larvae, and adults, which were identified and
speciated morphologically.
Statistical analysis. Two-tailed Fisher exact probability tests
were performed by using an online statistics application (vas-
sarstats.net). A P value of less than 0.05 denoted statistical
significance.
Experimental viral infection study. Unanesthetized 4-wk-old
female SW index mice (n = 36) were inoculated orally with 300
ID50
of MPV1d (20 μL of a 10% spleen stock), 36 unanesthetized
4-wk-old female SW index mice were inoculated orally with
3000 ID50
of MHV-Y (20 μL of a 10% intestinal stock), and 36
unanesthetized 4-wk-old female SW index mice were inoculated
orally with 100 ID50
of MNV-G8 (20 μL of a 10% colon stock).
Mice were housed in 36 cages, with each cage containing one
MPV-inoculated mouse, one MHV-inoculated mouse, and one
MNV-inoculated mouse. On day 9 after inoculation, all inocu-
lated (index) mice were removed from their respective soiled
cages and were placed in clean cages (3 mice per cage) for an
additional 2 wk to allow for seroconversion. Feces were collected
from all index mice at 10 d after inoculation, and feces and blood
was collected from index mice at 23 d to test for MPV-, MHV-,
and MNV-specific nucleic acids in feces and antibodies in serum.
On day 9 after inoculation, soiled cage tops and bottoms
were marked with heat-resistant ink. Each cage was swabbed,
and PCR analysis was performed to confirm that all cages had
detectable levels of viral nucleic acids on their surfaces prior
to cage washing. Cages were immediately transported to the
wash center. Prior to removing the soiled bedding from the
cages, approximately 1-mL samples (n = 4) of dust and debris
were collected from the prefilter of theABDC (model NU607400,
Labgard Class 1 Animal Bedding Disposal Cabinet, NuAire,
Plymouth, MN) by using forceps. PCR analysis was performed
on the 4 samples to determine whether infectious agents had
previously been deposited on the prefilter. Soiled bedding was
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Vol 54, No 6
Journal of the American Association for Laboratory Animal Science
November 2015
the prefilter of the ABDC. PBS was added to 5 samples of
dust and debris, and RNA and DNA were extracted from the
debris–PBS mixture (indirect extraction). DNA was extracted
from 5 samples of dust and debris directly after the addition
of lysis buffer (direct extraction). PCR analysis to determine
whether detectable amounts of infectious agents had been
deposited on the prefilter during soiled bedding dumping
was performed on all dust and debris samples.
Cages were washed in a Better Built Metal Wash Tunnel
Washer with a belt speed of 2.4 ft./min (0.74 m/min) by
using EnviroKleen 1500, GLPC7, and Acidulate 150. Cages
were exposed to hot water in the wash and rinse cycles for
a total of 4.6 min. The first 16 cages were washing by using
a wash temperature of 110 °F, after which the steam system
was activated and the water in the tank was heated to 180 °F.
The wash temperature was confirmed to be 180 °F by using
Thermolabel temperature-sensitive tape (Paper Thermom-
eter), and then the second 16 cages were washed. The final 7
cages were not washed and served as positive-control cages
to confirm that the amount of infectious agents present on
the soiled cages was sufficient to infect the sentinel mice.
Cages were reassembled and transported back to the animal
room, where they were placed on the IVC rack to dry for 2
h. Autoclaved food, autoclaved bedding, autoclaved nesting
material, and a clean water bottle were added to each cage,
and a single 4-wk-old female SW sentinel mouse was placed
in each cage.
On days 55 and 56, fecal pellets and fur swabs were collected
from the 78 infected mice for PCR analysis, tape tests were
performed and fur plucks were collected and analyzed. Then
the mice were euthanized by CO2
asphyxiation, and blood
was collected for serology and ceca were collected for direct
observation and PCR.
On days 65 and 78 (days 14 and 27 after exposure to contami-
nated cages) sentinels were moved to clean cages. One month
after exposure to the washed cages (on day 82), sentinel mice
were euthanized, and feces and blood were collected for PCR
and serology, respectively.
Results
Experimental viral infection study. To determine the effect of
water temperature during cage washing on the removal or inac-
tivation of viruses, cages were washed when peak levels of virus
were expected to be present in the cages. Specifically, 9 d after
experimental inoculation of mice with MPV, MHV, and MNV,
by which time a substantial amount of each virus should have
accumulated in the soiled bedding and on the cage surfaces, 16
soiled cages were washed at 110 °F, and 16 soiled cages were
washed at 180 °F. In addition, 4 cages from which the bedding
was removed, the surfaces scraped to remove adhered bedding,
but not washed were used to confirm that the amount of virus
present on the soiled cage surface was sufficient to infect the
sentinel mice (positive control).
Experimental viral infections of mice. PCR analysis of feces
collected individually from the 108 index mice, 10 d after ex-
perimental inoculation, detected MPV in 94% and MHV in
98% of the mice, indicating that most of the mice were actively
shedding MPV and MHV when cages were washed (Table 1). In
addition, at 23 d after inoculation, 97% of the index mice were
seropositive for MPV (all 3 mice in one cage did not seroconvert
to MPV), and all index mice were seropositive for MHV (Table
1). These data indicate that all mice inoculated with MHV
became infected, and all but one mouse inoculated with MPV
became infected; thereafter the inoculated mice infected their
mouse. Cages were changed on days 10, 18, 30, 42, 51, 65, and
78. Two SW mice were found dead on day 7 in a cage in which
a water bottle accident had occurred, and 2 of the ‘extra’ pet-
store mice were added as replacements to that cage to restore
the cage census to 5 mice (45 mice total: 11 pet-store mice and
34 SW contact mice). The remaining pet-store mouse was eu-
thanized on day 20, when it was determined that it would not
be needed as a replacement.
On days 22 and 23, feces were collected individually from
pet-store and SW contact mice for PCR analysis, fecal pools were
collected for direct examination, fur swabs were collected for
PCR analysis and tape tests, and fur plucks were performed.
Unexpected adverse events occurred between days 22 and 36.
A total of 5 SW contact mice were found dead on days 23 and
29, despite careful monitoring, and 6 SW contact mice were
euthanized because they were observed to be acutely dyspneic,
dehydrated, and hunched or circling on days 20, 23, 25, and 26.
On day 30, pinworm- and Helicobacter-infected mice (11 pet-
store mice and the 23 remaining SW contact mice) were placed
individually in 34 cages, and a second set of 68 naïve 4-wk-old
female SW mice were distributed (2 per cage) among the 34
cages, resulting in 102 mice total at a density of 3 mice per cage.
Ten more of the original SW contact mice were found dead on
days 30 to 36, despite careful monitoring, and 3 more of the origi-
nal SW contact mice were euthanized because they were noted
to be acutely dyspneic, and dehydrated or hunched on day
35. Therefore, on day 37, the remaining 12 original contact SW
mice were euthanized because of concerns that they also would
develop similar acute clinical disease. The euthanasia or death
of all 36 of the original SW contact mice left 23 of the 34 cages to
be used to generate soiled bedding containing Helicobacter spp.
and pinworms without an infected mouse, and the second set of
68 SW mice had not yet had sufficient time to acquire and shed
both Helicobacter spp. and pinworms by day 37 (7 d after expo-
sure to infected mice). Given concerns that exposing additional
naive SW mice would result in clinical disease or mortality, we
decided that no additional SW mice would be used. One of the
68 SW mice appeared runted and was euthanized on day 42.
Therefore on day 42, the remaining 78 (11 pet-store and 67 SW
mice from the second set of SW mice) animals were distributed
among 39 clean cages (2 mice per cage) to generate soiled cages
to be washed at 110 and 180 °F on day 51. Prior to cage washing
(day 50), pools of feces were collected from each cage for PCR
analysis to determine whether all of the cages contained mice
that were infected with Helicobacter spp., were infested with
pinworms, and were infected with other agents.
On day 51, 9 d after the last cage change and 21 d after
exposure of the second set of 67 SW contact mice to infected
mice, this cohort of contact-infected mice (11 pet-store mice
and the second set of 67 SW mice) were moved to clean cages,
and soiled cage tops and bottoms were marked with heat-
resistant ink. Each cage was swabbed, and PCR analysis was
performed to confirm that all cages had detectable levels of
Helicobacter spp. and pinworm DNA on their surfaces prior
to cage washing. Cages were transported to the wash center.
Prior to removal of the soiled bedding, 3 approximately 1-mL
samples of dust and debris and were collected from the pre-
filter of the ABDC and were submitted for PCR analysis to
determine whether MAV, MHV, MNV, parvovirus, Helicobacter
spp., pinworms or fur mites had previously been deposited on
the prefilter. Soiled bedding was dumped into the ABDC, and
cages were scraped prior to loading into the tunnel washer.
After the bedding from all 39 cages was dumped, 10 approxi-
mately 1-mL samples of dust and debris were collected from
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Effect of cage-wash temperature on pathogen removal
much higher ATP levels (7614; 42,358; greater than 99,999;
and greater than 99,999) were detected on the 4 unwashed
cages, which were visibly ‘dirty.’
Testing of SW sentinels exposed to washed and unwashed
cages. PCR analysis of feces collected from the 32 sentinel mice,
7 d after exposure to washed cages, were all negative for MPV
DNAand MHV RNA, whereas PCR of feces collected from 3 of
the 4 sentinel mice, 7 d after exposure to unwashed cages, were
positive for MPV DNA and MHV RNA (Table 1). Similarly, all
serologic samples from the 32 sentinel mice, 21 d after exposure
to washed cages, were negative for MPV and MHV, whereas 3
of 4 sentinel mice, 21 d after exposure to unwashed cages, were
MPV- and MHV-seropositive (Table 1). One of the unwashed
cages did not transmit MPV or MHV, indicating that the viral
load in this cage was below the transmission threshold. These
results indicate that both 110 and 180 °F were effective at de-
contaminating soiled cages in which mice with active infections
of MPV and MHV were housed.
Helicobacter spp. and pinworm contact-infection study. The
effect of water temperature during cage washing on decontami-
nation of cages harboring common murine bacteria (Helicobacter
spp.) and parasites (A. tetraptera and S. obvelata) of laboratory
mice was determined. To generate the soiled cages to be washed,
SW mice were infected with Helicobacter spp. and pinwormsby
contact with pet-store mice. Then 16 soiled cages were washed
at 180 °F, and 16 soiled cages were washed at 110 °F. The final
7 cages were dumped and scraped clean but not washed and
therefore served as positive control cages to confirm that the
amount of infectious agents present on the soiled cages was
sufficient to infect the sentinel mice.
Infection and testing of mice used as sources for cage con-
tamination. Initial testing of pet-store mice. Pet-store mice (n =
9) were tested, within 1 wk of arrival, by fecal and fur-swab
PCR analysis and by visual observation of pools of feces.
Infection with Helicobacter spp. was detected in all mice (H.
bilis [n = 5 mice], H. typhlonius [n = 2], H. ganmani [n = 1], H.
mastomyrinus [n = 1]). Coinfection with A. tetraptera and S.
obvelata was detected in 2 mice, and infection with A. tetraptera
alone was detected in 3 mice. In addition, pet-store mice also
were infected with Myocoptes musculinus (n = 5 mice), Roden-
tolepis nana (n = 9), Hymenolepis diminuta (n = 5), lice (n = 2),
MPV or MVM (n = 4), MHV (n = 5), and MAV (n = 3). Fecal
PCR did not detect MNV, lymphocytic choriomeningitis vi-
rus, murine rotavirus, reovirus, or Theiler encephalomyelitis
virus in the pet-store mice.
Antemortem testing of SW and pet-store mice. PCR analysis
of feces from individual mice (33 SW and 11 pet-store mice)
on day 22 detected Helicobacter spp. DNA in 98% of mice, S.
obvelata DNA in 93% of mice, and A. tetraptera DNA in 4.5%
of mice. Fur-swab PCR analysis on day 23 detected Myocoptes
musculinus DNA in 93% of mice and Myobia musculi DNA
in 73% of mice. Fur-pluck observation on day 23 detected
mite eggs on 36% of the mice and detected lice eggs on 41%
of the mice.
Postmortem testing of SW mice exhibiting clinical disease. PCR
evaluation of lungs from the 21 SW mice that were euthanized
between days 22 and 37 detected parvovirus in 100%, M. pul-
monis in 82%, and Pasteurella pneumotropica in 23% of mice but
not Pneumocystis murina, Mycobacteria spp., cilia-associated
respiratory bacillus, MHV, Sendai virus, or pneumonia virus of
mice. Visual observation of cecal contents from the 12 SW mice
euthanized on day 37 revealed all 12 of mice had S. obvelata, 5
had A. tetraptera, 5 had R. nana, 2 had Hymenolepis diminuta,
and 2 had Entamoeba muris. PCR assessment of feces and cecal
cage mates. Experimental infection with MNV was attempted
but was unsuccessful.
Testing of cages for viral contamination. On day 9, 36 cages
were swabbed, just prior to removal of the soiled bedding from
the cages. MPV DNA was detected on all but one cage, and
MHV RNA was detected on all cages (Table 1).
ABDC testing. The presence of MPV and MHV in soiled
bedding was confirmed by testing of dust and debris that
was aerosolized during soiled bedding disposal and that
was deposited on the ABDC prefilter. PCR-based testing of
4 samples of the dust and debris collected from the prefilter
of the ABDC prior to soiled bedding disposal from the cages
housing MPV- and MHV-infected mice, indicated the pres-
ence of MNV RNA and Helicobacter spp. DNA in all 4 samples
but not MPV DNA or MHV RNA. This result was expected,
because both MNV and Helicobacter spp. are endemic in this
facility, whereas MPV and MHV are not. In contrast, PCR-
based testing of 4 samples of the dust and debris present on
the prefilter of the ABDC after soiled-bedding disposal from
the cages housing MPV- and MHV-infected mice detected
MHV and MNV RNA as well as MPV and Helicobacter spp.
DNA. The detection of MHV RNA and MPV DNA in all
postdumping samples indicates that the accumulation of
MHV and MPV particles in aerosolized dust and debris from
the 16 cages’ worth of soiled bedding was sufficient to be
detectable by PCR.
Cage cleanliness and decontamination assessment. The
cleanliness of cages after washing at 110 or 180 °F was as-
sessed objectively by measuring residual ATP levels on the
cage bottom to determine whether washing at the lower
temperature was less effective at removing organic material.
The cage-wash temperature (110 °F compared with 180 °F)
did not affect cage sanitation. Specifically, the ATP levels
detected in the 2 groups of washed cages did not differ sig-
nificantly, given that ATP levels of 1 to 64 (mean, 14) were
detected in the 16 cages washed at 180 °F, and ATP levels of
1 to 99 (mean,16) were detected in 16 cages washed at 110
°F and was consistent with visual assessments in which no
differences in cage cleanliness could be detected. In contrast,
Table 1. PCR and serologic analysis for experimental virus infection:
cages, index mice, and sentinel mice
180 °F
wash
110 °F
wash Unwashed
9 DPI MPV cage swab 15/16 16/16 4/4
10 DPI MPV index feces 44/48 45/47 12/12
23 DPI MPV index sera 45/48 48/48 12/12
7 DPE MPV sentinel PCR 0/16a 0/16b 3/4a,b
21 DPE MPV sentinel sera 0/16a 0/16b 3/4a,b
9 DPI MHV cage swab 16/16 16/16 4/4
10 DPI MHV index feces 47/48 46/47 12/12
23 DPI MHV index sera 45/48 48/48 12/12
7 DPE MHV sentinel PCR 0/16a 0/16b 3/4a,b
21 DPE MHV sentinel sera 0/16a 0/16b 3/4a,b
DPE, days postexposure to washed or unwashed cages; DPI, days
postinoculation
Data are given as the number of samples positive for viral nucleic acids
or antibodies / the number of samples tested.
aSignificant (P < 0.005) difference between unwashed and 180 °F washed
cage groups.
bSignificant (P < 0.005) difference between unwashed and 110 °F washed
cage groups
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Journal of the American Association for Laboratory Animal Science
November 2015
contents from the 12 SW mice euthanized on day 37 detected
parvovirus and Helicobacter spp. in 100%, S. obvelata in 85%,
MAV in 50%, and MHV in 41% of the mice.
Antemortem testing of SW and pet-store mice just prior to cage
washing. PCR analysis of fecal pools collected from each of
the 39 soiled cages 1 d prior to cage washing on day 50, when
7 d of fecal accumulation was present in the cage, detected
Helicobacter spp. DNA in 97% of cages, pinworm DNA in 46%,
parvovirus DNA in 100%, MAV DNA in 5%, and MHV RNA
in 77% (Table 2).
Postmortem testing of contact-exposed SW and pet-store mice.
The 78 SW and pet-store mice were euthanized 4 to 5 d after cage
washing on days 55 to 56, and serology, PCR, cecal observation,
and tape tests were performed. Serology detected antibodies
specific for MPV in 82%, MVM in 13%, MHV in 99%, MAV in 4%,
and M. pulmonis in 61% of the mice (Table 3). PCR analysis per-
formed on feces, ceca, or lungs detected nucleic acids specific for
Helicobacter spp. in 97%, parvovirus in 26%, MHV in 47%, MAV
in 7%, M. pulmonis in 44%, and S. obvelata in 92% of mice (Tables 3
and4).SequencingofHelicobacterPCRproductsrevealedthepres-
ence of H. bilis, H. ganmani, and H. typhlonius. In addition, direct
observation of cecal contents detected pinworms in 90% of mice,
with S. obvelata detected in 76% of mice, A. tetraptera detected in
16% of mice, and pinworm larva of unknown species in 8% of
mice (Table 4). In addition, cecal observation detected several
other intestinal parasites (Entamoeba muris, 27%; trichomonads,
13%; R. nana, 13%; and Hymenolepis diminuta, 5%). PCR analyses
of individual fecal pellets and tape tests were less sensitive than
cecal methods (PCR analysis and observation) at detecting S. ob-
velata (P < 0.005; Table 4). Fur-swab PCR assay was more sensitive
than fur pluck analysis for the detection of Myocoptes musculinus
(P < 0.005; Table 4).
Testing of cages for bacterial, parasitic, and viral con-
tamination. We swabbed 39 cages on day 51, just prior to
removal of soiled bedding from the cages. Helicobacter spp.
DNA was detected on 85% of cages, pinworm DNA on 69%,
and parvovirus DNA on 92% of cages (Table 2). MAV DNA
was detected on a single cage and MHV RNA was detected
on 2 cages in the 110 °F wash group but not on any of the
cages in the 180 °F or unwashed cage groups (Table 2). The
efficacy of fecal-pool PCR assay on day 50 (Table 2) and of
cage-swab PCR analysis (Table 2) on day 51 was significantly
different only for MHV (P < 0.005), with MHV detected less
frequently on cages.
ABDC testing. The presence of infectious agents in soiled
bedding was confirmed by testing of dust and debris gener-
ated during the dumping of soiled bedding. Regarding indirect
sample extraction, PCR-based testing of 3 samples of the dust
and debris present on the prefilter of the ABDC prior to soiled
bedding disposal from the cages housing infected mice indicated
the presence of MNV RNA in 2 samples and Helicobacter spp.
DNA in 3 samples but not MHV RNA or MAV, parvovirus,
pinworm, or fur mite DNA. This result was expected, because
both MNV and Helicobacter spp. are endemic in and are not
excluded from the mouse colonies in this facility. PCR-based
testing of 5 samples of the dust and debris present on the pre-
filter of the ABDC after dumping of soiled bedding from cages
housing infected mice was performed by using the indirect
extraction method to extract nucleic acids. Parvovirus DNA,
MNV RNA, and Helicobacter spp. DNA were detected in all 5
samples, indicating that sufficient parvoviral particle accumula-
tion on the prefilter occurred during soiled bedding dumping.
MHV RNA and MAV DNA were not detected in any of the 5
samples collected from the ABDC after dumping of soiled bed-
Table 2. PCR analysis of pools of feces or swabs collected on the day
prior to cage washing from cages housing Helicobacter spp. and pinworm
contact-infection mice
180 °F wash 110 °F wash Unwashed
Helicobacter fecal pool 16/16 16/16 6/7
Helicobacter cage swab 14/16 14/16 5/7
Pinworm fecal pool 7/16 (7S/0A) 10/16 (8S/2A) 1/7 (1S/0A)
Pinworm cage swab 11/16
(11S/0A)
11/16
(11S/1A)
5/7 (4S/1A)
Parvovirus fecal pool 16/16 16/16 7/7
Parvovirus cage swab 15/16 14/16 7/7
MAV fecal pool 0/16 2/16 0/7
MAV cage swab 0/16 1/16 0/7
MHV fecal pool 16/16a,b 11/16a 3/7b
MHV cage swab 0/16 2/16 0/7
Data are given as the number of samples positive for infectious agent
nucleic acids / the number of samples tested (the number of. cages
positive for Syphacia /the number of cages positive for Aspiculuris).
aSignificant (P < 0.05) difference between 110 °F and 180 °F washed-
cage groups.
bSignificant difference between unwashed and 180F washed cage
groups (P < 0.005).
Table 3. Serologic and PCR analysis of Helicobacter spp. and pinworm
contact-infection mice 4 to 5 d after cage washing
180 °F wash 110 °F wash Unwashed
MPV serology 25/31 26/29 10/14
MVM serology 6/31 3/30 1/14
Parvovirus fecal PCR 9/31 7/28 3/14
MHV serology 30/30 30/30 13/14
MHV fecal PCR 16/31 15/28 3/14
MAV serology 0/31 3/30 0/14
MAV fecal PCR 1/31 4/28 0/14
M. pulmonis serology 19/30 18/30 8/14
M. pulmonis lung PCR 13/32 14/32 7/14
Helicobacter cecal PCR 31/32 31/32 14/14
Helicobacter fecal PCR 24/31 24/28 9/14
Data are given as the number of samples positive for infectious agent
nucleic acids or antibodies / the number of samples tested
No significant difference was detected between groups for any agent
Table 4. Testing for parasites in Helicobacter spp. and pinworm contact-
infection mice 4 to 5 d after cage washing
180 °F
wash
110 °F
wash Unwashed
Pinworm fecal PCR (Syphacia) 8/31 7/28 1/14
Pinworm cecal PCR (Syphacia) 31/32 28/32 13/14
Pinworm cecal observation 20/24
(19S,
3A,1L)
24/25
(18S,
6A,3L)
12/13 (10S,
1A,1L)
Pinworm tape test (Syphacia) 14/25 15/25 11/13
Fur pluck observation (Myocoptes) 13/24 15/25 7/13
Fur swab PCR (Myocoptes) 30/32 26/30 13/14
Data are given as the number of samples positive for parasites / the
number of samples tested (the number of. mice positive for S. obvelata
/the number of mice positive for A. tetraptera/the number of mice
positive for pinworm larvae)
No significant difference was detected between groups for any agent
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Effect of cage-wash temperature on pathogen removal
The equation used to determine the steam energy (in 1 mil-
lion British thermal units [MMBTU]) per year required to heat
domestic hot water is:
Note that additional steam is used to maintain approximately
50 gal of water within the wash tank and rinse tank at approxi-
mately 200 °F continuously (8760 h per year.) Although several
reasonable engineering assumptions can be made to estimate the
amount of steam use associated with maintaining the wash- and
rinse-tanktemperatures,itismoreappropriatetometerthissteam
or solicit more detailed information from the manufacturer. No
metering was performed in this analysis, so this additional steam
use was not included in the savings estimates.
Tunnel washer 2 (model 3236LS, Getinge, Rochester NY)
was manufactured in 2009. Calculations were based on washer
operating-time estimates of 8 h daily, 5 d each week, 52 wk yearly
(2080hannually).Steamisusedinthewash,initialrinse,finalrinse,
and dryer cycles at an average rate of 900 lb/h, according to the
operatingmanual.Unlikethosefortunnelwasher1,themanufac-
turer’s data for this newer tunnel washer provided steam-supply
specifications for life-cycle cost analysis. Therefore, steam is
used at
ding. Even though the majority of mice housed in cages were
infested with pinworms and fur mites, DNA from these agents
was not PCR-amplified from these prefilter samples. The 8 dust
and debris samples (3 before and 5 after bedding disposal) had
been extracted by adding 1 mL PBS to the 1.5-mL tube contain-
ing the debris, followed by adding 100 μL of the buffer–debris
mixture to the lysis buffer (indirect extraction).
In an effort to determine whether direct extraction of the dust
and debris on the prefilter (by adding the lysis buffer directly
to the dust and debris sample) allowed for better detection of
pinworms and mites on the prefilter, DNA was extracted from
5 samples of dust and debris by adding the lysis buffer directly
to the sample. All 5 direct-extraction samples were positive for
Helicobacter spp., parvovirus, M. pulmonis, S. obvelata, and Myo-
coptes musculinus DNAbut not MAV DNA, indicating that direct
extractionallowedforbetterdetectionofseveralinfectiousagents.
Sentinel testing data. PCR assays of feces and ceca collected
from the 32 sentinel mice exposed to cages washed at either 110
or 180 °F (31 d after exposure) were all negative for Helicobacter
spp., pinworm, parvovirus, and MAV DNAand for MHV RNA
(Table 5). Similarly, sera from the 32 sentinel mice exposed to
washed cages (31 d after exposure) were all negative for MPV,
MVM, MAV, and MHV antibodies (Table 5). In contrast, approxi-
mately half of the ceca from sentinel mice exposed to unwashed
cages (31 d after exposure) were positive for parvovirus and S.
obvelata DNA (Table 5). In contrast, parvovirus and S. obvelata
DNA were detected in the feces of only one sentinel housed
in an unwashed cage, underscoring the lower sensitivity of
fecal PCR analysis to detect these 2 infectious agents (Table 5).
Helicobacter spp., MVM, and MAV DNA and MHV RNA were
not detected in the feces or ceca from sentinel mice exposed to
unwashed cages (Table 5). Serology performed on the sentinel
mice exposed to unwashed cages revealed a low level of infec-
tion by MVM and MHV and a higher level of infection by MPV
(Table 5). Sera from all sentinels housed in washed or unwashed
cages were negative for ectromelia virus, murine rotavirus,
lymphocytic choriomeningitis virus, pneumonia virus of mice,
reovirus, Sendai virus, Theiler encephalomyelitis virus, and M.
pulmonis. Fur mites were not detected in any of the sentinels by
fur pluck or by fur-swab PCR assay. The difference between the
percentage of infected sentinels housed in washed compared
with unwashed cages was significant (P < 0.005) only for MPV
and pinworms, primarily because of inconsistent transmission
of the other agents by unwashed (positive control) cages. No
difference in transmission was seen between cages washed at
110 and 180 °F, indicating that both temperatures were effective
at inactivating or removing MPV and pinworms.
Steam-useestimations.Steamusewasestimatedfor2representa-
tivetunnelwasherstodemonstratepotentialcostsavingsofusing
140 °F domestic hot water, compared with water at 180 to 200 °F,
the industry standard. Because the steam is not currently metered
directly at the washers, the manufacturer’s data and operating
schedule were used to estimate energy required to heat water.
Tunnel washer 1 (model TT27X22, Metalwash, Northwestern
Systems) was manufactured in 1991. Calculations were based
on washer operating time estimates of 6 h daily, 5 d each
week, for 52 wk annually, and the manufacturer’s water-use
data of 12.5 gal/min (1,170,000 gal yearly). Steam is used to
boost domestic hot water (approximately 140 °F) to a final
rinse temperature of approximately 200 °F (∆T = 60 °F). We
used 200 °F instead of 180 °F in the calculation because the
control valve for this machine does not control to a specific set
point; it controls according to steam flow, resulting in water
within the wash and rinse tanks reaching more than 200 °F.
Table 5. Testing for Helicobacter and pinworms in contact-infection cage
sentinels 31 d postexposure to cages
180 °F
wash
110 °F
wash Unwashed
MPV serology 0/16a 0/16b 5/7a,b
MVM serology 0/16 0/16 1/7
Parvovirus fecal PCR 0/16 0/16 1/7
Parvovirus cecal PCR 0/16a 0/16b 4/7a,b
MVM fecal and cecal PCR 0/16 0/16 0/7
MHV serology 0/16 0/16 1/7
MHV fecal and cecal PCR 0/16 0/16 0/7
MAV serology 0/16 0/16 0/7
MAV fecal and cecal PCR 0/16 0/16 0/7
Helicobacter fecal PCR 0/16 0/16 0/7
Helicobacter cecal PCR 0/16 0/16 0/7
Pinworm fecal PCR 0/16 0/16 1/7
Pinworm cecal PCR 0/16a 0/16b 5/7a,b
Pinworm cecal observation 0/16a 0/16b 5/7a,b
Pinworm tape test 0/16a 0/16b 5/7a,b
Data are given as the number of samples positive for virus, Helicobacter
spp., or S. obvelata / number of samples tested
aSignificant (P < 0.005) difference between unwashed and 180 °F washed
cage groups
bSignificant (P < 0.005) difference between unwashed and 110 °F washed
cage groups
no. of MMBTU yearly
= (no. gallons of water per year)
× ∆ ° × ÷( F) 8.3 BTU 1,000,000 galΤ
= (1,170,000) (60 F) 8.3 BTU 1,000,000 gal× ° × ÷
= 583 MMBTU per year for tunnel washer 1
= × ÷ ×2080 h 900 lb h 1186 BTU
÷ ÷lb of steam 1,000,000
= 2220 MMBTU annually, according
to default parameters, for tunnel washer 2.
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Vol 54, No 6
Journal of the American Association for Laboratory Animal Science
November 2015
Cost-savings estimates from reduced steam use. The potential
savings is based on the estimated reduction in steam use and
the current steam costs from the local utility ($14/MMBTU).
For tunnel washer 1, the savings are based on the assumption
that building-supplied domestic hot water would be used
with no additional local steam heating. The potential savings
is 583 MMBTU and US$8162 annually. Additional savings as-
sociated with ‘stand-by’ steam use and with energy costs to
mitigate heat gain within the washing room can be assessed
further. For tunnel washer 2, we assumed that steam use could
be reduced by at least 75% with changes to the wash and rinse
parameters. The total potential savings is 75% × 2220 MMBTU
= 1665 MMBTU × $14/MMBTU = US$23,310 annually.
Discussion
Our hypothesis that cage washing alone at either 110 or 180 °F
is sufficient to decontaminate soiled cages that had housed mice
infected with viruses was confirmed. In the experimental infec-
tion study, 97% and 100% of cages had detectable levels of MPV
and MHV, respectively, on their surfaces at the time of washing.
MHV and MPV infection occurred in most sentinels exposed to
the unwashed cages, whereas washing of cages at 110 °F or 180
°F resulted in inactivation or removal of MPV and MHV from
all soiled cages that had housed mice acutely infected (9 d after
inoculation) with these agents. In the second study, pet-store mice
naturally infected with MPV infected most of the SW mice that
were housed with them. Similar to the experimental MPV study,
cage washing alone at either 110 or 180 °F was sufficient to inacti-
vateorremoveMPVfromallsoiledcagesinthesecondstudy.The
results of the MPV studies presented here combined with those
of our previous study11 showed that none of the 46 washed cages
(14 in previous study and 32 in current studies) transmitted MPV,
whereas 8 of 11 of the unwashed cages in this study and all 14 of
the unwashed cage bottoms in the previous study11 transmitted
MPV. The ability of cage washing to remove or inactivate 2 very
different viruses— MPV (a DNA virus that is very stable in the
environment and difficult to inactivate)5,6,16,27,38,42,49 and MHV
(an enveloped RNAvirus that is less environmentally stable but
is shed in high levels during acute infection3,9,20—suggests that
cage washing is broadly effective at decontaminating cages that
have housed mice infected with murine viruses.
Pinworms eggs, like MPV, are highly stable in the environ-
ment and difficult to inactivate with most disinfectants.13,32 Cage
washing alone at either 110 or 180 °F inactivated or removed S.
obvelata from all cages, suggesting that cage washing is effective
at preventing infection with a wide range of murine infectious
agents. Although more than half of the pet-store mice were in-
fested with A. tetraptera, only 14% of SW mice became infested
with A. tetraptera, and A. tetraptera DNA was detected on only
5% of cages prior to washing. In contrast, less than 25% of the
pet-store mice were infested with S. obvelata, but more than 90%
of the SW mice became infested with S. obvelata. This result is
probably due to differences in the lifecycles of the 2 pinworm
species. The duration of exposure of the second set of SW mice
to the pet-store mice was only 21 d, to avoid the aforementioned
clinical disease, which presumably was precipitated by M. pul-
monis infection of the SW mice. This relatively short time frame
favored S. obvelata infection, because S. obvelata eggs are released
in a single burst of approximately 350 eggs, are infective 5 to 20
h after deposition on the perianal skin and hair, and can result
in retrofection (migration of hatched larvae from the anus to the
cecum of the mouse).7,41 In contrast, A. tetraptera eggs are released
intermittently over a 3- to 4-wk period and are not infective for 5
to 8 d after excretion in the feces.2,23,34,41 In addition, A. tetraptera
has a longer prepatent period (21 to 25 d) than does S. obvelata,
which has a prepatent period of only 11 to 15 d.2,7,41
During the second study, the efficacies of several methods of
pinworm detection were compared to determine the optimal
sample to use for routine monitoring for pinworms in our
facility. PCR and visual observation of cecal contents from SW
mice were significantly better (Table 5, P < 0.01) at detecting
pinworm infection (90% to 92%) than was testing of samples
collected noninvasively (PCR of cage swabs, tape tests, PCR of
fecal pools, and PCR of individual feces). These data agree with
previous studies from our lab, which indicated that PCR and
observation of cecal contents were equally effective at detecting
pinworm infections.20 In light of our data, PCR of single fecal
pellets collected directly from mice is not recommended as a
reliable method for S. obvelata detection. The low sensitivity
of the fecal PCR for Syphacia in our hands may be the result
of inefficient DNA extraction, because others have suggested
that inhibitors of pinworm PCR can be present in fecal DNA.17
Although only 22% of individual feces samples were positive
for S. obvelata DNA on day 55 (Table 5), 37% of cages contained
a mouse that was positive for S. obvelata DNA, indicating that
most cages had only a single pinworm-positive mouse. Even
though the pinworm PCR primers used were designed to am-
plify both S. obvelata and A. tetraptera, PCR assay of the cecal
contents did not detect A. tetraptera, whereas observation of
cecal contents detected A. tetraptera in 14% of the index mice.
The majority of the SW contact mice infested with A. tetraptera
(80%) were coinfested with S. obvelata, R. nana, Hymenolepis
diminuta, Entamoeba muris, or trichomonads (or multiple para-
sites), whereas only 55% of mice infested with S. obvelata were
also infested with these other parasites. Perhaps coinfection
with these other parasites interfered with the amplification of
A. tetraptera DNAbut not S. obvelata DNA. Several studies have
compared the various methods of pinworm detection, with
divergent results.14,17,22 Further optimization of DNAextraction
methods and amplification conditions is required before the
sensitivities of the various pinworm-detection methods can be
accurately compared in our laboratory.
Given that all 14 of the index mice housed in the unwashed
cages were infected with Helicobacter spp. (Table 5) it is inter-
esting to note that none of the sentinels placed in these cages
became infected. This finding suggests that Helicobacter spp.
are not readily transmitted by the residual waste present on
unwashed cages. Several reports have shown efficient transmis-
sion of Helicobacter spp. to sentinel mice via exposure to soiled
bedding,29,48 although the frequency of exposure and dose of
soiled bedding were higher than those in our current study. Our
results are more in line with 2 other studies, which reported that
H. hepaticus, H. typhlonius, H. mastomyrinus, and H. muridarium
were not transmitted to soiled-bedding sentinels.10,22
Although the contact-infection study was not intended to
measure transmission of M. pulmonis, more than half of contact-
exposed mice seroconverted to M. pulmonis, and lungs from
almost half of the contact-exposed mice were positive for M.
pulmonis DNA. We therefore assessed the transmission of M.
pulmonis by soiled unwashed cages. Several mycoplasmal spe-
cies, when dried on paper discs, have been stable for several
weeks,33 and because lungs from 8 of the 14 contact-exposed
mice in the unwashed cages were positive for M. pulmonis DNA,
it would have been reasonable to expect that sentinels placed in
unwashed cages would have become infected with M. pulmonis.
But our results showed that M. pulmonis was not transmitted
by unwashed cages. Our data are consistent with information
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753
Effect of cage-wash temperature on pathogen removal
In the contact-infection study, an initial PCR-based attempt
to detect pinworms and fur mites in samples of debris from the
dump station prefilter after the disposal of soiled bedding was
unsuccessful when the DNA was extracted indirectly from the
sample, even though the majority of mice housed in the repre-
sented cages were infested with pinworms and fur mites. We
initially thought that the larger size of these parasites (pinworms
eggs are 30 to 40 μm × 90 to 140 μm)41 led to inefficient aerosoliza-
tion of these agents or parts of these agents during soiled bedding
disposal. But a recent study showed that fur-mite DNA can be
detected on swabs from the shelf of a horizontal manifold of an
IVC rack housing a single cage of fur mite infested mice one week
after placement of the cage on the rack indicating that aerosoliza-
tion of detectable levels of debris containing fur mite components
occurred.26 Follow-up testing of samples of prefilter dust and
debris by using direct DNA extraction revealed that pinworms
andfurmiteswerepresentinallofthesamplesofdustanddebris.
It seems that smaller agents such as viruses and Helicobacter spp.
are readily washed from the debris; therefore indirect extraction
resulted in a sufficient quantity of nucleic acids for detection by
PCR analysis. In comparison, the larger infectious agents such
as pinworms and fur mites were not readily transferred from
the debris to the PBS during the indirect extraction procedure,
possibly because these agents were too large to be easily drawn
into the pipettor tip or because they were tightly associated with
the debris (for example, fur mites attached to fur would not be
transferred). Because debris samples collected prior to soiled-
bedding dumping were extracted by using the indirect method,
we perhaps would not have detected any pinworms or fur mites
present on the prefilter because the predumping samples were
extracted by using the indirect method. Because pinworms and
fur mites have not been detected in the facilities served by this
ABDC for over 9 mo, we concluded that the S. obvelata and Myo-
coptes musculinus DNA detected in the postdumping samples
was deposited during dumping of the cages in this experiment.
PCR-based testing of the prefilter of the dump station, by
using direct nucleic acid extraction, shows promise for murine
infectious agents screening at the facility level when more than
10 cages housing mice infected with a virus, bacteria, or parasite
are dumped. However, MHV and MAV, which were detected on
only a few cages in the second study, were not detected in the
debris present on the prefilter of theABDC after soiled-bedding
disposal, indicating that a threshold level of contaminated bed-
ding needs to be present on the ABDC prefilter. Further studies
to determine the sensitivity of this testing paradigm are needed.
Taken together, our studies demonstrate that cage decontam-
ination does not require washing at 180 °F and that autoclaving
of washed cages is unnecessary. Rather, cage washing at 110
°F with appropriate detergents and cycle times was sufficient
to decontaminate cages that had housed mice infected or
infested with common murine infectious agents— thereby
representing a significant advancement to the ‘greening’ of
vivarium operation. The effectiveness of washing at 110 °F is
an important finding in the context of sustainability and the
need for steam infrastructure. For example, the energy usage
when 140 °F domestic hot water is used to wash caging rather
than water steam boosted to 200 °F was calculated to result
in a savings of 583 MMBTU when a 1991 Metalwash Tunnel
Washer was operated for 1560 h annually, for a cost savings
of approximately $8000 annually. Similarly, 1665 MMBTU
can be saved by operating a newer but larger Getinge Tunnel
Washer at 140 °F for 2080 h yearly, yielding a cost savings of
approximately $23,000 each year. Additional energy savings
might be achieved by scheduling cage washing to minimize
presented in a recent study, which showed that M. pulmonis
was not transmitted to soiled bedding or contact sentinels.22 It
is important to note that although M. pulmonis was restricted to
the lungs of infected mice and although M. pulmonis DNA was
not detected on cage surfaces, at least one mycoplasmal species,
which had 98% homology with M. moatsii19 and 99% homology
with several uncultured bacterial species found in the jejunum
of mice, was detected in the feces of most pet-store and SW mice
and on cage surfaces. These findings underscore that testing for
M. pulmonis in the environment by PCR assay should not be
performed by using generic mycoplasmal primers.
Although Myocoptes musculinus was detected by PCR in 91% of
the contact exposed pet-store and SW mice, it was not transmitted
to sentinels housed in unwashed cages. These data are consistent
with several previous studies, which have shown that soiled bed-
dingtransmissionoffurmitestosentinelsoccursinfrequently.20,22,28
But2otherstudies37,43 haveshownefficienttransmissionbysoiled
bedding to sentinel mice in 8 to 19 wk. The mice in the first study
wereinfestedwithbothMyobiamusculiandMyocoptesmusculinus,
and transmission occurred to all cages, but the fur-mite species
that was transmitted is unclear.37 In the second study, mice were
housed in open-top cages that received soiled bedding from other
open-top cages within the room that housed mice infested with
Myobia musculi, and transmission occurred in 3 of 4 cages.43 To our
knowledge, our current study is the first to evaluate fomite-based
transmission of fur mites from mice infested with Myocoptes mus-
culinus in the absence of Myobia musculi infestation.
The number and type of infectious agents present in pet-store
mice and the potential for some of these agents to be spread
by fomites prompt biosecurity concerns when animal care and
laboratory personnel have pet-store mice as pets at home. Of
particular concern is that these ‘clinically normal’ pet-store mice
transmitted fatal disease to naive SW mice, albeit by direct con-
tact. Nevertheless, these findings underscore the risk of pet-store
mice and supports having a facility rodent-adoption program
so that animal care and laboratory personnel wishing to acquire
pet mice can officially do so through a pathogen-free source.
Early detection of an infectious agent can often lessen its
overall effect on an animal program. Unfortunately, many
routine sentinel-exposure protocols rely on periodic sampling
of and exposure to soiled bedding within a housing rack,
and therefore positive sentinel findings may represent an
infection that began weeks to months previously. Environ-
mental sampling is a promising adjunct to sentinel-exposure
programs for early pathogen detection. Realizing that testing
of individual ventilated cage rack filters is one environmental
screening approach, we tested a complementary approach:
the ability to reliably detect pathogens from an ABDC, a
site where soiled bedding is concentrated and aerosolized.
Because the ABDC serves large portions of a facility, the abil-
ity to reliably detect ‘excluded’ pathogens from the ABDC
would allow for screening of large portions of a facility by
using a few filter samples. Therefore, we assessed whether
PCR-based testing of the accumulated dust-debris present on
the prefilter of the ABDC after disposal of the soiled bedding
from cages housing experimentally infected sentinel mice
detected both MHV and MPV particles (diameter, 0.028 to
0.160 μm).1 Helicobacter DNA was detected in all samples of
dust and debris present on the prefilter of the ABDC (both
pre- and postdisposal samples from cages housing infected
sentinel mice in both studies), indicating the accumulation
of Helicobacter spp. (0.2 to 0.3 μm × 1.5 to 5 μm)18 during the
routine dumping of cages from endemically infected cages
within this animal facility.
jaalas14000172.indd 753 11/23/2015 1:20:00 PM
754
Vol 54, No 6
Journal of the American Association for Laboratory Animal Science
November 2015
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Assoc Lab Anim Sci 51:648.
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Infect 73:64–70.
	17.	Feldman SH, Bowman SG. 2007. Molecular phylogeny of the
pinworms of mice, rats, and rabbits, and its use to develop
molecular-beacon assays for the detection of pinworms in mice.
Lab Anim (NY) 36:43–50.
	18.	Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS,
Collins MJ Jr, Gorelick PL, Ward JM. 1994. Helicobacter hepati-
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the amount of time during which the tunnel washer is run-
ning but not actively washing caging and by factoring in the
decrease in energy needed to mitigate the heat gain within
the room so that room temperature and humidity are accept-
able to personnel working in the wash facilities. Apart from
the cost of the energy required to boost domestic hot water
to 180 °F, the boiler required to supply the steam necessary to
heat the volume of the water necessary may not be available
in the building or nearby, resulting in significant increased
construction or renovation costs. However, detailed manu-
facturer steam requirements are critical to understand the full
range of savings associated with washer operating parameters,
particularly requirements for stand-by modes maintaining
temperature settings both during and after active use. Direct
steam metering is not trivial in these settings but is required
for more precise energy cost-savings estimates.
Acknowledgments
This work was supported by Grants for Laboratory Animal Science
(GLAS) from AALAS. We thank Alison Faruolo for assistance with
animal care and viral diagnostics and Fu-Chen Yang for assistance with
parasitology. We thank Marco Garcia, Eric Georgelos, and Julie Paquette
for assistance with cage washing and cage-wash energy calculations.
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jaalas14000172.indd 755 11/23/2015 1:20:00 PM

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Effect of Cage-Wash Temperature on the Removal of Infectious Agents

  • 1. Vol 54, No 6 November 2015 Pages 745–755 Journal of the American Association for Laboratory Animal Science Copyright 2015 by the American Association for Laboratory Animal Science 745 The use of evidence-based standard operating procedures in animal resource centers is crucial to cost containment and sustainable energy use. Understandably, biosecurity is a major driver of procedures and processes in rodent facilities and permeates virtually all aspects of animal resource operations, making it necessary to balance the cost: benefit of detection, prevention, and control of infection. As an example, recent publications that suggested that mouse parvovirus (MPV) infection can be caused by MPV-contaminated grains that were not inactivated during their processing into pelleted ro- dent feed25,36,47 have led to changes in husbandry procedures including the use of autoclaved or irradiated food. Although the successful control and prevention of MPV infections in our facilities has been attributed to the practice of autoclaving mouse cages preassembled with bedding and food prior to their use in the facility, this procedure was not definitively proven to be the sole factor in MPV eradication.30 Because wash centers are historically the largest utilities consumer in animal facilities,15 this practice of autoclaving cages prior to use is not only labor- intensive but also energy-intensive, and the labor and energy consumption is amplified in facilities that lack a bulk autoclave and in which the cages must be transported to be autoclaved. In addition, during outbreaks with MPV and other infectious agents, our long-standing standard operating procedure for soiled cages is to autoclave them to inactivate infectious agents prior to removing the soiled bedding and cage washing. This labor- and energy-intensive practice of using autoclaving to decontaminate cages has historically been justified because MPV is a nonenveloped virus that is highly stable in the environment and difficult to inactivate.5,6,16,27,38,42,49 Despite the environmen- tal stability of the virus, infections with MPV are difficult to detect because the amount of virus shed is low; transmission can be inefficient, resulting in inconsistent seroconversion of mice housed in the same cage; and PCR analysis can detect low levels of MPV DNA in the feces of mice that are unable to transmit virus to contact sentinels.4,30,31 We recently demon- strated that cage washing alone removed or inactivated MPV from 14 cages that had housed outbred mice acutely infected with MPV; these findings are not surprising when the inefficien- cies of MPV transmission are considered.11 These initial results challenge the cost:benefit ratio of autoclaving cages as a means to decontaminate them prior to cage washing during an MPV outbreak. Our findings with MPV led us to question whether cage washing alone might be effective for decontaminating cages after exposure to other infectious agents that are stable Effect of Cage-Wash Temperature on the Removal of Infectious Agents from Caging and the Detection of Infectious Agents on the Filters of Animal Bedding-Disposal Cabinets by PCR Analysis Susan R Compton1,* and James D Macy1,2 Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission. Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180 °F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening. Abbreviations: ABDC, animal bedding disposal cabinet; MAV, murine adenovirus K87; MHV, mouse hepatitis virus; MNV, mu- rine norovirus; MPV, mouse parvovirus; MVM, minute virus of mice; MMBTU, million British thermal units; SW, Swiss Webster Received: 19 Nov 2014. Revision requested: 24 Dec 2014. Accepted: 30 Jan 2015. 1Section of Comparative Medicine, Yale University School of Medicine, and 2Animal Resources Center, Yale University, New Haven, Connecticut *Corresponding author. Email: susan.compton@yale.edu jaalas14000172.indd 745 11/23/2015 1:19:58 PM
  • 2. 746 Vol 54, No 6 Journal of the American Association for Laboratory Animal Science November 2015 virus, MHV, MPV, minute virus of mice (MVM), murine noro- virus (MNV), pneumonia virus of mice, reovirus, Sendai virus, Theiler encephalomyelitis virus, and Mycoplasma pulmonis and were free of bacterial and parasitic infections at the time of shipment. Random-bred female mice were obtained from 3 local pet stores (4 mice per store) to serve as sources of Helicobacter spp. and pinworms. Mice were housed in IVC (70 cages per rack) under positive pressure (ACE MicroVent,Allentown, NJ). Cages containing corncob bedding (Harlan Teklad, Indianapolis, IN), rodent chow (Global 2018S, Harlan Teklad) and nesting material (Cotton squares,Ancare, Bellmore, NY) were preassembled and autoclaved. Mice had unrestricted access to hyperchlorinated (4 to 6 ppm) water delivered by water bottle. The mice were housed and husbanded according to standard biocontainment procedures. The animal room had a negative pressure differen- tial relative to the corridor, a 12:12-h light:dark cycle, 10 to 15 air changes hourly, room temperature of 22.2 ± 1.1 °C, and room humidity of 50% ± 10% and was used exclusively for this study. All animal care and experimental procedures were performed in an AAALAC-accredited animal facility, were approved by the Yale IACUC, and adverse events (described later) were reported to the IACUC.All animal care followed the Guide for the Care and Use of Laboratory Animals,24 and experimental procedure were in accordance with federal policies and guidelines governing the use of vertebrate animals. PCR analysis. Individual fecal pellets were collected from the anus of each mouse, and pools of 6 to 10 pellets were collected from soiled bedding. The nape, abdomen, and rump of mice were swabbed by using sterile Hydraflock swabs (Puritan, Guil- ford, ME) for detection of fur mites. Cage swabs were collected by swabbing the perimeter of the cage just above the bedding and then passing the swab through the soiled bedding in an X pattern from cage corner to cage corner to detect viruses, Heli- cobacter spp., and pinworms. Cecal samples were collected from mice after carbon dioxide euthanasia. All samples were frozen in 1.5-mL tubes at –20 °C prior to PCR analysis. DNAand RNA were extracted from samples by using DNeasy or RNeasy kits (Qiagen, Valencia, CA) according to the manufacturer’s instruc- tions. PCR assays were performed by using iTaq Universal SYBR Green kit (BioRad, Hercules, CA), and RT–PCR assays were per- formed by using iScript One-Step RT–PCR kit with SYBR Green (BioRad) and a thermocycler (CFX Connect, Biorad). Primers specific for cilia-associated respiratory bacillus, Helicobacter spp., MHV, parvovirus (MPV and MVM), murine adenovirus K87 (MAV), MNV, murine rotavirus, Myobia musculi, Myocoptes musculinus, Pasteurella pneumotropica, pinworms (Aspiculuris tetraptera and Syphacia obvelata), Pneumocystis murina, reovirus, and Theiler encephalomyelitis virus were used as previously described.12,20,21,44 RT–PCR analysis for lymphocytic choriomen- ingitis virus was performed by using the primers LCMVS710 (5′ AGG CTC AGA TGG CAA GAC C 3′) and LCMVS1641 (5′ GCC CAA ATG TTG TGA CAC TCT 3′). RT–PCR analysis for pneumonia virus of mice was performed by using PVM1616 (5′ CCCAACATG GAG GTCAAG CAG 3′) and PVM1987 (5′ GCA TTG CCAAGCACAACACTG 3′). RT–PCR analysis for Sendai virus was performed by using SEN399 (5′ GGAGTAAAC GCC GAT GTC AAA 3′) and SEN1274 (5′ CCC TTG GCT GTA TCC GTC ACT 3′). Mycobacteria spp. PCR analysis used MYB404 (5′ TTT CTC GGA TTG ACG GTA GG 3′) and MYB1235 (5′ TGA GAC CGG CTT TAA AAG GA 3′). Mycoplasma pulmonis PCR analysis used MYPUL180 (5′ TTA GAT CGC ATG ATT TAG AT 3′) and MYPUL875 (5′ TGC GAG CAT ACT ACT CAG 3′). Serology. Cardiocentesis was performed after carbon dioxide overdose. Sera were tested in immunofluorescent antibody in the environment, even if they are shed for longer periods of time or at higher levels than is MPV. In our previous study,11 we showed that cage washing was effective at preventing fomite-based transmission of MPV by cage components, and we postulated that water temperature and detergent type contribute to MPV decontamination either directly through inactivation of the virus and/or indirectly through effective mechanical removal of organic waste and residual virus from the cage. The Guide for the Care and Use of Laboratory Animals24 states that “disinfection from the use of hot water alone is the result of the combined effect of temperature and the length of time at a given temperature” and that “effec- tive disinfection can be achieved with wash and rinse water at 143–180 °F or more.” Theoretically, contact times to disinfect equipment at these temperatures would need to be 1800 s at 143 °F (61.7 °C) and 0.1 s at 180 °F (82.2 °C).46 Contact times of 6 s or more at 168 to 180 °F (75.6 to 82.2 °C) killed 3 types of bacteria (Pseudomonas aeruginosa, Salmonella cholerasuis, and Staphylococcus aureus) in one study,45 and contact times of 2 min or more at 160 °F (71.1 °C) killed 5 types of bacteria (Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Providencia rettgeri, and Staphylococcus epidermidis) in another.39 However, these 2 studies were performed by using hot water in a test tube and, therefore, detergent and mechanical spraying action that occur within rack washers was not a factor. Traditionally, the temperature used for the wash and rinse water in our facilities has been 180 °F (82.2 °C) with a belt speed of 2 to 3 ft./min (0.6 to 0.9 m/min). We postulated that if the volume and force of the wash water, combined with detergents, consistently diluted or removed infectious agents to below the level necessary for the transmission of infection, then wash temperatures high enough to inactivate the agents would be unnecessary. Given the energy usage and infrastructure neces- sary to boost ‘domestic’ hot water to 180 °F, the purpose of this study was to compare the efficacy of cage washing by using the domestic hot-water temperature (110 °F [43.3 °C]) and the traditional steam-boosted wash temperature (180 °F [82.2 °C]) on preventing the transmission from contaminated caging of 3 of the most prevalent viral agents (MNV, MPV, MHV; prevalence, 32.6%,1.8%, and 1.6%, respectively ) and bacterial and parasitic agents (Helicobacter spp. and pinworms; prevalence,15.9% and 0.3%, respectively).35 Effective and efficient decontamination of caging goes hand- in-hand with effective and efficient detection of infection. Timely detection of infection is important to biosecurity, and environmental sampling is a promising adjunct to sentinel exposure programs for the early detection of infectious agents. PCR analysis of cage and rack components, including the out- flow prefilter of ventilated racks, has been shown to be of use for several infectious agents including MPV, MHV, Helicobacter spp., and fur mites.10,26,31 The ability to reliably detect infectious agents from a site where soiled bedding debris is aerosolized and concentrated might provide an efficient adjunct for infectious agent screening. In the current study, we determined whether monitoring by PCR analysis of dust and debris collected from the animal bedding disposal cabinet (ABDC) prefilter could be used as an efficient adjunct to sentinel programs to screen for contamination by infectious agents. Materials and Methods Mice. Female Swiss Webster mice (Crl:CFW [SW]; age, 4 to 6 wk) were obtained from Charles River Laboratories (Kingston, NY). Vendor reports indicated that mice were seronegative for ectromeliavirus,murinerotavirus,lymphocyticchoriomeningitis jaalas14000172.indd 746 11/23/2015 1:19:58 PM
  • 3. 747 Effect of cage-wash temperature on pathogen removal removed by ‘dumping’ the soiled bedding from the contami- nated cages into the ABDC; cages were then scraped prior to loading into the tunnel washer (Better Built Metal Wash Tunnel Washer, Northwestern Systems, Delta, Canada), which had a belt speed of 2.4 ft/min (0.74 m/min). Cages were exposed to hot water in the wash and rinse cycles for a total of 4.6 min. The chemicals used in the tunnel washer were Enviro-Kleen 1500, GLPC7, and Acidulate 150 (Quip Labs, Wilmington, DE). After the soiled bedding from all 36 cages was dumped, 4 approxi- mately 1-mL samples of dust and debris were collected from the prefilter of the ABDC, and PCR analysis was performed to determine whether detectable amounts of MPV, MHV, or MNV had been deposited on the prefilter during dumping of soiled bedding. Then, the first 16 cages, with filter tops and wire bars, were washed at a wash temperature of 110 °F (43.3 °C), which was confirmed by using a chromel–alumel type K thermocouple connected to a Meter (Fluke, Everett, WA). The thermocouple was located in the rinse tank, which stores and maintains the preset water temperature for this tunnel washer. Before the second set of 16 cages was washed, the steam system was activated, and the water in the tunnel washer tank was allowed to heat to 180 °F (82.2 °C). The wash temperature was confirmed to be 180 °F by using temperature-sensitive tape (Thermolabel, Paper Thermometer, Greenfield, NH), and then the second set of 16 cages was washed. The final 4 cages were not washed and served as positive-control cages to confirm that the amount of virus present on the soiled cages was sufficient to cause infection of naive sentinel mice. After cage washing at either 110 or 180 °F, cages were reassembled, transported back to the animal room, and placed on the IVC rack to dry for 2 h. The efficacy of cage sanitation was assessed by ATPase testing (Accupoint 2 Sanitation Monitoring System with surface sam- plers, Neogen, Lansing, MI). Autoclaved food, autoclaved bedding, autoclaved nesting material, and an autoclaved water bottle filled with hyper- chlorinated water were added to each washed cage, and then a 4-wk-old female SW sentinel mouse was placed into each cage. One week after the addition of sentinel mice to the cages (that is, 1 wk after exposure to the washed cages), feces were collected from sentinel mice for PCR analysis. At 2 wk after exposure to the washed cages, sentinel mice were moved to clean cages. At 3 wk after exposure to the washed cages, mice were euthanized, and feces and blood were collected for PCR analysis and serology, respectively. Helicobacter spp. and pinworm contact-infection study. The study design included a 2-stage approach to produce SW mice infected with Helicobacter spp. and pinworms because stocks of these infectious agents were not readily available. Initially, 12 random-bred female mice were obtained from 3 local pet stores (4 mice per store); 9 of these mice were housed individually. We added 4-wk-old female SW mice (n = 4 per cage; 36 SW mice total) to each of the 9 cages to initiate infection with Helicobacter spp. and pinworms, understanding that other infectious agents harbored by the pet-store mice would also be transmitted to the SW contact mice. The extra 3 pet-store mice were housed separately as replacements for pet-store mice that might become ill during the study. Feces from pet-store mice were tested for Helicobacter spp., pinworms, lymphocytic choriomeningitis vi- rus, MAV, MHV, MNV, parvovirus, murine rotavirus, reovirus, and Theiler encephalomyelitis virus by PCR analysis on their arrival to our facility. Pet-store mice were rotated among the 9 cages of contact SW mice on days 5, 10, 15 and 20 to produce a more uniform infection profile than would be obtained if each cage of contact SW mice were exposed to only a single pet-store assays as previously described40 for antibodies to one or more of the following agents: ectromelia virus, lymphocytic chori- omeningitis virus, MAV, MHV, MPV, MVM, murine rotavirus, pneumonia virus of mice, reovirus, Sendai virus, Theiler en- cephalomyelitis virus, and M. pulmonis. Parasitology. To detect S. obvelata, cellophane tape tests were performed by applying tape to the perianal region of the mouse. The adherent surface of the tape was placed onto a glass microscope slide, and slides were observed by using a microscope at 10× and 40× magnification. Slurries were made in 0.9% saline from pools of 6 to 10 feces collected from soiled bedding. A small amount of the slurry was placed on a glass microscope slide, a cover slip was placed on the sample, and the sample was examined for evidence of ova or adult endoparasites by microscopy at 10× and 40× magnification. Portions of the cecum and colon from each mouse were submitted for direct examination for intestinal parasites. Immediately after collection of the cecum and colon, approximately 0.5 mL of 0.9% saline was added to the sample in a culture dish and mixed with a wooden applicator. A small amount of the sample was placed on a glass microscope slide, cover-slipped, and examined for evidence of ova or adult endoparasites by microscopy at 10× and 40× magnification. Parasites were identified and speciated morphologically. The cecocolonic contents in the culture dish were examined again, at 24 h after the initial exam, under low- power (10×) microscopy for evidence of adult endoparasites. Fur plucked from the nape and rump of each mouse was placed in a drop of mineral oil on a microscope slide; a cover slip was placed on the sample; and samples were examined by using a microscope under low power (10× magnification) for evidence of fur-mite ova, larvae, and adults, which were identified and speciated morphologically. Statistical analysis. Two-tailed Fisher exact probability tests were performed by using an online statistics application (vas- sarstats.net). A P value of less than 0.05 denoted statistical significance. Experimental viral infection study. Unanesthetized 4-wk-old female SW index mice (n = 36) were inoculated orally with 300 ID50 of MPV1d (20 μL of a 10% spleen stock), 36 unanesthetized 4-wk-old female SW index mice were inoculated orally with 3000 ID50 of MHV-Y (20 μL of a 10% intestinal stock), and 36 unanesthetized 4-wk-old female SW index mice were inoculated orally with 100 ID50 of MNV-G8 (20 μL of a 10% colon stock). Mice were housed in 36 cages, with each cage containing one MPV-inoculated mouse, one MHV-inoculated mouse, and one MNV-inoculated mouse. On day 9 after inoculation, all inocu- lated (index) mice were removed from their respective soiled cages and were placed in clean cages (3 mice per cage) for an additional 2 wk to allow for seroconversion. Feces were collected from all index mice at 10 d after inoculation, and feces and blood was collected from index mice at 23 d to test for MPV-, MHV-, and MNV-specific nucleic acids in feces and antibodies in serum. On day 9 after inoculation, soiled cage tops and bottoms were marked with heat-resistant ink. Each cage was swabbed, and PCR analysis was performed to confirm that all cages had detectable levels of viral nucleic acids on their surfaces prior to cage washing. Cages were immediately transported to the wash center. Prior to removing the soiled bedding from the cages, approximately 1-mL samples (n = 4) of dust and debris were collected from the prefilter of theABDC (model NU607400, Labgard Class 1 Animal Bedding Disposal Cabinet, NuAire, Plymouth, MN) by using forceps. PCR analysis was performed on the 4 samples to determine whether infectious agents had previously been deposited on the prefilter. Soiled bedding was jaalas14000172.indd 747 11/23/2015 1:19:58 PM
  • 4. 748 Vol 54, No 6 Journal of the American Association for Laboratory Animal Science November 2015 the prefilter of the ABDC. PBS was added to 5 samples of dust and debris, and RNA and DNA were extracted from the debris–PBS mixture (indirect extraction). DNA was extracted from 5 samples of dust and debris directly after the addition of lysis buffer (direct extraction). PCR analysis to determine whether detectable amounts of infectious agents had been deposited on the prefilter during soiled bedding dumping was performed on all dust and debris samples. Cages were washed in a Better Built Metal Wash Tunnel Washer with a belt speed of 2.4 ft./min (0.74 m/min) by using EnviroKleen 1500, GLPC7, and Acidulate 150. Cages were exposed to hot water in the wash and rinse cycles for a total of 4.6 min. The first 16 cages were washing by using a wash temperature of 110 °F, after which the steam system was activated and the water in the tank was heated to 180 °F. The wash temperature was confirmed to be 180 °F by using Thermolabel temperature-sensitive tape (Paper Thermom- eter), and then the second 16 cages were washed. The final 7 cages were not washed and served as positive-control cages to confirm that the amount of infectious agents present on the soiled cages was sufficient to infect the sentinel mice. Cages were reassembled and transported back to the animal room, where they were placed on the IVC rack to dry for 2 h. Autoclaved food, autoclaved bedding, autoclaved nesting material, and a clean water bottle were added to each cage, and a single 4-wk-old female SW sentinel mouse was placed in each cage. On days 55 and 56, fecal pellets and fur swabs were collected from the 78 infected mice for PCR analysis, tape tests were performed and fur plucks were collected and analyzed. Then the mice were euthanized by CO2 asphyxiation, and blood was collected for serology and ceca were collected for direct observation and PCR. On days 65 and 78 (days 14 and 27 after exposure to contami- nated cages) sentinels were moved to clean cages. One month after exposure to the washed cages (on day 82), sentinel mice were euthanized, and feces and blood were collected for PCR and serology, respectively. Results Experimental viral infection study. To determine the effect of water temperature during cage washing on the removal or inac- tivation of viruses, cages were washed when peak levels of virus were expected to be present in the cages. Specifically, 9 d after experimental inoculation of mice with MPV, MHV, and MNV, by which time a substantial amount of each virus should have accumulated in the soiled bedding and on the cage surfaces, 16 soiled cages were washed at 110 °F, and 16 soiled cages were washed at 180 °F. In addition, 4 cages from which the bedding was removed, the surfaces scraped to remove adhered bedding, but not washed were used to confirm that the amount of virus present on the soiled cage surface was sufficient to infect the sentinel mice (positive control). Experimental viral infections of mice. PCR analysis of feces collected individually from the 108 index mice, 10 d after ex- perimental inoculation, detected MPV in 94% and MHV in 98% of the mice, indicating that most of the mice were actively shedding MPV and MHV when cages were washed (Table 1). In addition, at 23 d after inoculation, 97% of the index mice were seropositive for MPV (all 3 mice in one cage did not seroconvert to MPV), and all index mice were seropositive for MHV (Table 1). These data indicate that all mice inoculated with MHV became infected, and all but one mouse inoculated with MPV became infected; thereafter the inoculated mice infected their mouse. Cages were changed on days 10, 18, 30, 42, 51, 65, and 78. Two SW mice were found dead on day 7 in a cage in which a water bottle accident had occurred, and 2 of the ‘extra’ pet- store mice were added as replacements to that cage to restore the cage census to 5 mice (45 mice total: 11 pet-store mice and 34 SW contact mice). The remaining pet-store mouse was eu- thanized on day 20, when it was determined that it would not be needed as a replacement. On days 22 and 23, feces were collected individually from pet-store and SW contact mice for PCR analysis, fecal pools were collected for direct examination, fur swabs were collected for PCR analysis and tape tests, and fur plucks were performed. Unexpected adverse events occurred between days 22 and 36. A total of 5 SW contact mice were found dead on days 23 and 29, despite careful monitoring, and 6 SW contact mice were euthanized because they were observed to be acutely dyspneic, dehydrated, and hunched or circling on days 20, 23, 25, and 26. On day 30, pinworm- and Helicobacter-infected mice (11 pet- store mice and the 23 remaining SW contact mice) were placed individually in 34 cages, and a second set of 68 naïve 4-wk-old female SW mice were distributed (2 per cage) among the 34 cages, resulting in 102 mice total at a density of 3 mice per cage. Ten more of the original SW contact mice were found dead on days 30 to 36, despite careful monitoring, and 3 more of the origi- nal SW contact mice were euthanized because they were noted to be acutely dyspneic, and dehydrated or hunched on day 35. Therefore, on day 37, the remaining 12 original contact SW mice were euthanized because of concerns that they also would develop similar acute clinical disease. The euthanasia or death of all 36 of the original SW contact mice left 23 of the 34 cages to be used to generate soiled bedding containing Helicobacter spp. and pinworms without an infected mouse, and the second set of 68 SW mice had not yet had sufficient time to acquire and shed both Helicobacter spp. and pinworms by day 37 (7 d after expo- sure to infected mice). Given concerns that exposing additional naive SW mice would result in clinical disease or mortality, we decided that no additional SW mice would be used. One of the 68 SW mice appeared runted and was euthanized on day 42. Therefore on day 42, the remaining 78 (11 pet-store and 67 SW mice from the second set of SW mice) animals were distributed among 39 clean cages (2 mice per cage) to generate soiled cages to be washed at 110 and 180 °F on day 51. Prior to cage washing (day 50), pools of feces were collected from each cage for PCR analysis to determine whether all of the cages contained mice that were infected with Helicobacter spp., were infested with pinworms, and were infected with other agents. On day 51, 9 d after the last cage change and 21 d after exposure of the second set of 67 SW contact mice to infected mice, this cohort of contact-infected mice (11 pet-store mice and the second set of 67 SW mice) were moved to clean cages, and soiled cage tops and bottoms were marked with heat- resistant ink. Each cage was swabbed, and PCR analysis was performed to confirm that all cages had detectable levels of Helicobacter spp. and pinworm DNA on their surfaces prior to cage washing. Cages were transported to the wash center. Prior to removal of the soiled bedding, 3 approximately 1-mL samples of dust and debris and were collected from the pre- filter of the ABDC and were submitted for PCR analysis to determine whether MAV, MHV, MNV, parvovirus, Helicobacter spp., pinworms or fur mites had previously been deposited on the prefilter. Soiled bedding was dumped into the ABDC, and cages were scraped prior to loading into the tunnel washer. After the bedding from all 39 cages was dumped, 10 approxi- mately 1-mL samples of dust and debris were collected from jaalas14000172.indd 748 11/23/2015 1:19:58 PM
  • 5. 749 Effect of cage-wash temperature on pathogen removal much higher ATP levels (7614; 42,358; greater than 99,999; and greater than 99,999) were detected on the 4 unwashed cages, which were visibly ‘dirty.’ Testing of SW sentinels exposed to washed and unwashed cages. PCR analysis of feces collected from the 32 sentinel mice, 7 d after exposure to washed cages, were all negative for MPV DNAand MHV RNA, whereas PCR of feces collected from 3 of the 4 sentinel mice, 7 d after exposure to unwashed cages, were positive for MPV DNA and MHV RNA (Table 1). Similarly, all serologic samples from the 32 sentinel mice, 21 d after exposure to washed cages, were negative for MPV and MHV, whereas 3 of 4 sentinel mice, 21 d after exposure to unwashed cages, were MPV- and MHV-seropositive (Table 1). One of the unwashed cages did not transmit MPV or MHV, indicating that the viral load in this cage was below the transmission threshold. These results indicate that both 110 and 180 °F were effective at de- contaminating soiled cages in which mice with active infections of MPV and MHV were housed. Helicobacter spp. and pinworm contact-infection study. The effect of water temperature during cage washing on decontami- nation of cages harboring common murine bacteria (Helicobacter spp.) and parasites (A. tetraptera and S. obvelata) of laboratory mice was determined. To generate the soiled cages to be washed, SW mice were infected with Helicobacter spp. and pinwormsby contact with pet-store mice. Then 16 soiled cages were washed at 180 °F, and 16 soiled cages were washed at 110 °F. The final 7 cages were dumped and scraped clean but not washed and therefore served as positive control cages to confirm that the amount of infectious agents present on the soiled cages was sufficient to infect the sentinel mice. Infection and testing of mice used as sources for cage con- tamination. Initial testing of pet-store mice. Pet-store mice (n = 9) were tested, within 1 wk of arrival, by fecal and fur-swab PCR analysis and by visual observation of pools of feces. Infection with Helicobacter spp. was detected in all mice (H. bilis [n = 5 mice], H. typhlonius [n = 2], H. ganmani [n = 1], H. mastomyrinus [n = 1]). Coinfection with A. tetraptera and S. obvelata was detected in 2 mice, and infection with A. tetraptera alone was detected in 3 mice. In addition, pet-store mice also were infected with Myocoptes musculinus (n = 5 mice), Roden- tolepis nana (n = 9), Hymenolepis diminuta (n = 5), lice (n = 2), MPV or MVM (n = 4), MHV (n = 5), and MAV (n = 3). Fecal PCR did not detect MNV, lymphocytic choriomeningitis vi- rus, murine rotavirus, reovirus, or Theiler encephalomyelitis virus in the pet-store mice. Antemortem testing of SW and pet-store mice. PCR analysis of feces from individual mice (33 SW and 11 pet-store mice) on day 22 detected Helicobacter spp. DNA in 98% of mice, S. obvelata DNA in 93% of mice, and A. tetraptera DNA in 4.5% of mice. Fur-swab PCR analysis on day 23 detected Myocoptes musculinus DNA in 93% of mice and Myobia musculi DNA in 73% of mice. Fur-pluck observation on day 23 detected mite eggs on 36% of the mice and detected lice eggs on 41% of the mice. Postmortem testing of SW mice exhibiting clinical disease. PCR evaluation of lungs from the 21 SW mice that were euthanized between days 22 and 37 detected parvovirus in 100%, M. pul- monis in 82%, and Pasteurella pneumotropica in 23% of mice but not Pneumocystis murina, Mycobacteria spp., cilia-associated respiratory bacillus, MHV, Sendai virus, or pneumonia virus of mice. Visual observation of cecal contents from the 12 SW mice euthanized on day 37 revealed all 12 of mice had S. obvelata, 5 had A. tetraptera, 5 had R. nana, 2 had Hymenolepis diminuta, and 2 had Entamoeba muris. PCR assessment of feces and cecal cage mates. Experimental infection with MNV was attempted but was unsuccessful. Testing of cages for viral contamination. On day 9, 36 cages were swabbed, just prior to removal of the soiled bedding from the cages. MPV DNA was detected on all but one cage, and MHV RNA was detected on all cages (Table 1). ABDC testing. The presence of MPV and MHV in soiled bedding was confirmed by testing of dust and debris that was aerosolized during soiled bedding disposal and that was deposited on the ABDC prefilter. PCR-based testing of 4 samples of the dust and debris collected from the prefilter of the ABDC prior to soiled bedding disposal from the cages housing MPV- and MHV-infected mice, indicated the pres- ence of MNV RNA and Helicobacter spp. DNA in all 4 samples but not MPV DNA or MHV RNA. This result was expected, because both MNV and Helicobacter spp. are endemic in this facility, whereas MPV and MHV are not. In contrast, PCR- based testing of 4 samples of the dust and debris present on the prefilter of the ABDC after soiled-bedding disposal from the cages housing MPV- and MHV-infected mice detected MHV and MNV RNA as well as MPV and Helicobacter spp. DNA. The detection of MHV RNA and MPV DNA in all postdumping samples indicates that the accumulation of MHV and MPV particles in aerosolized dust and debris from the 16 cages’ worth of soiled bedding was sufficient to be detectable by PCR. Cage cleanliness and decontamination assessment. The cleanliness of cages after washing at 110 or 180 °F was as- sessed objectively by measuring residual ATP levels on the cage bottom to determine whether washing at the lower temperature was less effective at removing organic material. The cage-wash temperature (110 °F compared with 180 °F) did not affect cage sanitation. Specifically, the ATP levels detected in the 2 groups of washed cages did not differ sig- nificantly, given that ATP levels of 1 to 64 (mean, 14) were detected in the 16 cages washed at 180 °F, and ATP levels of 1 to 99 (mean,16) were detected in 16 cages washed at 110 °F and was consistent with visual assessments in which no differences in cage cleanliness could be detected. In contrast, Table 1. PCR and serologic analysis for experimental virus infection: cages, index mice, and sentinel mice 180 °F wash 110 °F wash Unwashed 9 DPI MPV cage swab 15/16 16/16 4/4 10 DPI MPV index feces 44/48 45/47 12/12 23 DPI MPV index sera 45/48 48/48 12/12 7 DPE MPV sentinel PCR 0/16a 0/16b 3/4a,b 21 DPE MPV sentinel sera 0/16a 0/16b 3/4a,b 9 DPI MHV cage swab 16/16 16/16 4/4 10 DPI MHV index feces 47/48 46/47 12/12 23 DPI MHV index sera 45/48 48/48 12/12 7 DPE MHV sentinel PCR 0/16a 0/16b 3/4a,b 21 DPE MHV sentinel sera 0/16a 0/16b 3/4a,b DPE, days postexposure to washed or unwashed cages; DPI, days postinoculation Data are given as the number of samples positive for viral nucleic acids or antibodies / the number of samples tested. aSignificant (P < 0.005) difference between unwashed and 180 °F washed cage groups. bSignificant (P < 0.005) difference between unwashed and 110 °F washed cage groups jaalas14000172.indd 749 11/23/2015 1:19:58 PM
  • 6. 750 Vol 54, No 6 Journal of the American Association for Laboratory Animal Science November 2015 contents from the 12 SW mice euthanized on day 37 detected parvovirus and Helicobacter spp. in 100%, S. obvelata in 85%, MAV in 50%, and MHV in 41% of the mice. Antemortem testing of SW and pet-store mice just prior to cage washing. PCR analysis of fecal pools collected from each of the 39 soiled cages 1 d prior to cage washing on day 50, when 7 d of fecal accumulation was present in the cage, detected Helicobacter spp. DNA in 97% of cages, pinworm DNA in 46%, parvovirus DNA in 100%, MAV DNA in 5%, and MHV RNA in 77% (Table 2). Postmortem testing of contact-exposed SW and pet-store mice. The 78 SW and pet-store mice were euthanized 4 to 5 d after cage washing on days 55 to 56, and serology, PCR, cecal observation, and tape tests were performed. Serology detected antibodies specific for MPV in 82%, MVM in 13%, MHV in 99%, MAV in 4%, and M. pulmonis in 61% of the mice (Table 3). PCR analysis per- formed on feces, ceca, or lungs detected nucleic acids specific for Helicobacter spp. in 97%, parvovirus in 26%, MHV in 47%, MAV in 7%, M. pulmonis in 44%, and S. obvelata in 92% of mice (Tables 3 and4).SequencingofHelicobacterPCRproductsrevealedthepres- ence of H. bilis, H. ganmani, and H. typhlonius. In addition, direct observation of cecal contents detected pinworms in 90% of mice, with S. obvelata detected in 76% of mice, A. tetraptera detected in 16% of mice, and pinworm larva of unknown species in 8% of mice (Table 4). In addition, cecal observation detected several other intestinal parasites (Entamoeba muris, 27%; trichomonads, 13%; R. nana, 13%; and Hymenolepis diminuta, 5%). PCR analyses of individual fecal pellets and tape tests were less sensitive than cecal methods (PCR analysis and observation) at detecting S. ob- velata (P < 0.005; Table 4). Fur-swab PCR assay was more sensitive than fur pluck analysis for the detection of Myocoptes musculinus (P < 0.005; Table 4). Testing of cages for bacterial, parasitic, and viral con- tamination. We swabbed 39 cages on day 51, just prior to removal of soiled bedding from the cages. Helicobacter spp. DNA was detected on 85% of cages, pinworm DNA on 69%, and parvovirus DNA on 92% of cages (Table 2). MAV DNA was detected on a single cage and MHV RNA was detected on 2 cages in the 110 °F wash group but not on any of the cages in the 180 °F or unwashed cage groups (Table 2). The efficacy of fecal-pool PCR assay on day 50 (Table 2) and of cage-swab PCR analysis (Table 2) on day 51 was significantly different only for MHV (P < 0.005), with MHV detected less frequently on cages. ABDC testing. The presence of infectious agents in soiled bedding was confirmed by testing of dust and debris gener- ated during the dumping of soiled bedding. Regarding indirect sample extraction, PCR-based testing of 3 samples of the dust and debris present on the prefilter of the ABDC prior to soiled bedding disposal from the cages housing infected mice indicated the presence of MNV RNA in 2 samples and Helicobacter spp. DNA in 3 samples but not MHV RNA or MAV, parvovirus, pinworm, or fur mite DNA. This result was expected, because both MNV and Helicobacter spp. are endemic in and are not excluded from the mouse colonies in this facility. PCR-based testing of 5 samples of the dust and debris present on the pre- filter of the ABDC after dumping of soiled bedding from cages housing infected mice was performed by using the indirect extraction method to extract nucleic acids. Parvovirus DNA, MNV RNA, and Helicobacter spp. DNA were detected in all 5 samples, indicating that sufficient parvoviral particle accumula- tion on the prefilter occurred during soiled bedding dumping. MHV RNA and MAV DNA were not detected in any of the 5 samples collected from the ABDC after dumping of soiled bed- Table 2. PCR analysis of pools of feces or swabs collected on the day prior to cage washing from cages housing Helicobacter spp. and pinworm contact-infection mice 180 °F wash 110 °F wash Unwashed Helicobacter fecal pool 16/16 16/16 6/7 Helicobacter cage swab 14/16 14/16 5/7 Pinworm fecal pool 7/16 (7S/0A) 10/16 (8S/2A) 1/7 (1S/0A) Pinworm cage swab 11/16 (11S/0A) 11/16 (11S/1A) 5/7 (4S/1A) Parvovirus fecal pool 16/16 16/16 7/7 Parvovirus cage swab 15/16 14/16 7/7 MAV fecal pool 0/16 2/16 0/7 MAV cage swab 0/16 1/16 0/7 MHV fecal pool 16/16a,b 11/16a 3/7b MHV cage swab 0/16 2/16 0/7 Data are given as the number of samples positive for infectious agent nucleic acids / the number of samples tested (the number of. cages positive for Syphacia /the number of cages positive for Aspiculuris). aSignificant (P < 0.05) difference between 110 °F and 180 °F washed- cage groups. bSignificant difference between unwashed and 180F washed cage groups (P < 0.005). Table 3. Serologic and PCR analysis of Helicobacter spp. and pinworm contact-infection mice 4 to 5 d after cage washing 180 °F wash 110 °F wash Unwashed MPV serology 25/31 26/29 10/14 MVM serology 6/31 3/30 1/14 Parvovirus fecal PCR 9/31 7/28 3/14 MHV serology 30/30 30/30 13/14 MHV fecal PCR 16/31 15/28 3/14 MAV serology 0/31 3/30 0/14 MAV fecal PCR 1/31 4/28 0/14 M. pulmonis serology 19/30 18/30 8/14 M. pulmonis lung PCR 13/32 14/32 7/14 Helicobacter cecal PCR 31/32 31/32 14/14 Helicobacter fecal PCR 24/31 24/28 9/14 Data are given as the number of samples positive for infectious agent nucleic acids or antibodies / the number of samples tested No significant difference was detected between groups for any agent Table 4. Testing for parasites in Helicobacter spp. and pinworm contact- infection mice 4 to 5 d after cage washing 180 °F wash 110 °F wash Unwashed Pinworm fecal PCR (Syphacia) 8/31 7/28 1/14 Pinworm cecal PCR (Syphacia) 31/32 28/32 13/14 Pinworm cecal observation 20/24 (19S, 3A,1L) 24/25 (18S, 6A,3L) 12/13 (10S, 1A,1L) Pinworm tape test (Syphacia) 14/25 15/25 11/13 Fur pluck observation (Myocoptes) 13/24 15/25 7/13 Fur swab PCR (Myocoptes) 30/32 26/30 13/14 Data are given as the number of samples positive for parasites / the number of samples tested (the number of. mice positive for S. obvelata /the number of mice positive for A. tetraptera/the number of mice positive for pinworm larvae) No significant difference was detected between groups for any agent jaalas14000172.indd 750 11/23/2015 1:19:58 PM
  • 7. 751 Effect of cage-wash temperature on pathogen removal The equation used to determine the steam energy (in 1 mil- lion British thermal units [MMBTU]) per year required to heat domestic hot water is: Note that additional steam is used to maintain approximately 50 gal of water within the wash tank and rinse tank at approxi- mately 200 °F continuously (8760 h per year.) Although several reasonable engineering assumptions can be made to estimate the amount of steam use associated with maintaining the wash- and rinse-tanktemperatures,itismoreappropriatetometerthissteam or solicit more detailed information from the manufacturer. No metering was performed in this analysis, so this additional steam use was not included in the savings estimates. Tunnel washer 2 (model 3236LS, Getinge, Rochester NY) was manufactured in 2009. Calculations were based on washer operating-time estimates of 8 h daily, 5 d each week, 52 wk yearly (2080hannually).Steamisusedinthewash,initialrinse,finalrinse, and dryer cycles at an average rate of 900 lb/h, according to the operatingmanual.Unlikethosefortunnelwasher1,themanufac- turer’s data for this newer tunnel washer provided steam-supply specifications for life-cycle cost analysis. Therefore, steam is used at ding. Even though the majority of mice housed in cages were infested with pinworms and fur mites, DNA from these agents was not PCR-amplified from these prefilter samples. The 8 dust and debris samples (3 before and 5 after bedding disposal) had been extracted by adding 1 mL PBS to the 1.5-mL tube contain- ing the debris, followed by adding 100 μL of the buffer–debris mixture to the lysis buffer (indirect extraction). In an effort to determine whether direct extraction of the dust and debris on the prefilter (by adding the lysis buffer directly to the dust and debris sample) allowed for better detection of pinworms and mites on the prefilter, DNA was extracted from 5 samples of dust and debris by adding the lysis buffer directly to the sample. All 5 direct-extraction samples were positive for Helicobacter spp., parvovirus, M. pulmonis, S. obvelata, and Myo- coptes musculinus DNAbut not MAV DNA, indicating that direct extractionallowedforbetterdetectionofseveralinfectiousagents. Sentinel testing data. PCR assays of feces and ceca collected from the 32 sentinel mice exposed to cages washed at either 110 or 180 °F (31 d after exposure) were all negative for Helicobacter spp., pinworm, parvovirus, and MAV DNAand for MHV RNA (Table 5). Similarly, sera from the 32 sentinel mice exposed to washed cages (31 d after exposure) were all negative for MPV, MVM, MAV, and MHV antibodies (Table 5). In contrast, approxi- mately half of the ceca from sentinel mice exposed to unwashed cages (31 d after exposure) were positive for parvovirus and S. obvelata DNA (Table 5). In contrast, parvovirus and S. obvelata DNA were detected in the feces of only one sentinel housed in an unwashed cage, underscoring the lower sensitivity of fecal PCR analysis to detect these 2 infectious agents (Table 5). Helicobacter spp., MVM, and MAV DNA and MHV RNA were not detected in the feces or ceca from sentinel mice exposed to unwashed cages (Table 5). Serology performed on the sentinel mice exposed to unwashed cages revealed a low level of infec- tion by MVM and MHV and a higher level of infection by MPV (Table 5). Sera from all sentinels housed in washed or unwashed cages were negative for ectromelia virus, murine rotavirus, lymphocytic choriomeningitis virus, pneumonia virus of mice, reovirus, Sendai virus, Theiler encephalomyelitis virus, and M. pulmonis. Fur mites were not detected in any of the sentinels by fur pluck or by fur-swab PCR assay. The difference between the percentage of infected sentinels housed in washed compared with unwashed cages was significant (P < 0.005) only for MPV and pinworms, primarily because of inconsistent transmission of the other agents by unwashed (positive control) cages. No difference in transmission was seen between cages washed at 110 and 180 °F, indicating that both temperatures were effective at inactivating or removing MPV and pinworms. Steam-useestimations.Steamusewasestimatedfor2representa- tivetunnelwasherstodemonstratepotentialcostsavingsofusing 140 °F domestic hot water, compared with water at 180 to 200 °F, the industry standard. Because the steam is not currently metered directly at the washers, the manufacturer’s data and operating schedule were used to estimate energy required to heat water. Tunnel washer 1 (model TT27X22, Metalwash, Northwestern Systems) was manufactured in 1991. Calculations were based on washer operating time estimates of 6 h daily, 5 d each week, for 52 wk annually, and the manufacturer’s water-use data of 12.5 gal/min (1,170,000 gal yearly). Steam is used to boost domestic hot water (approximately 140 °F) to a final rinse temperature of approximately 200 °F (∆T = 60 °F). We used 200 °F instead of 180 °F in the calculation because the control valve for this machine does not control to a specific set point; it controls according to steam flow, resulting in water within the wash and rinse tanks reaching more than 200 °F. Table 5. Testing for Helicobacter and pinworms in contact-infection cage sentinels 31 d postexposure to cages 180 °F wash 110 °F wash Unwashed MPV serology 0/16a 0/16b 5/7a,b MVM serology 0/16 0/16 1/7 Parvovirus fecal PCR 0/16 0/16 1/7 Parvovirus cecal PCR 0/16a 0/16b 4/7a,b MVM fecal and cecal PCR 0/16 0/16 0/7 MHV serology 0/16 0/16 1/7 MHV fecal and cecal PCR 0/16 0/16 0/7 MAV serology 0/16 0/16 0/7 MAV fecal and cecal PCR 0/16 0/16 0/7 Helicobacter fecal PCR 0/16 0/16 0/7 Helicobacter cecal PCR 0/16 0/16 0/7 Pinworm fecal PCR 0/16 0/16 1/7 Pinworm cecal PCR 0/16a 0/16b 5/7a,b Pinworm cecal observation 0/16a 0/16b 5/7a,b Pinworm tape test 0/16a 0/16b 5/7a,b Data are given as the number of samples positive for virus, Helicobacter spp., or S. obvelata / number of samples tested aSignificant (P < 0.005) difference between unwashed and 180 °F washed cage groups bSignificant (P < 0.005) difference between unwashed and 110 °F washed cage groups no. of MMBTU yearly = (no. gallons of water per year) × ∆ ° × ÷( F) 8.3 BTU 1,000,000 galΤ = (1,170,000) (60 F) 8.3 BTU 1,000,000 gal× ° × ÷ = 583 MMBTU per year for tunnel washer 1 = × ÷ ×2080 h 900 lb h 1186 BTU ÷ ÷lb of steam 1,000,000 = 2220 MMBTU annually, according to default parameters, for tunnel washer 2. jaalas14000172.indd 751 11/23/2015 1:19:59 PM
  • 8. 752 Vol 54, No 6 Journal of the American Association for Laboratory Animal Science November 2015 Cost-savings estimates from reduced steam use. The potential savings is based on the estimated reduction in steam use and the current steam costs from the local utility ($14/MMBTU). For tunnel washer 1, the savings are based on the assumption that building-supplied domestic hot water would be used with no additional local steam heating. The potential savings is 583 MMBTU and US$8162 annually. Additional savings as- sociated with ‘stand-by’ steam use and with energy costs to mitigate heat gain within the washing room can be assessed further. For tunnel washer 2, we assumed that steam use could be reduced by at least 75% with changes to the wash and rinse parameters. The total potential savings is 75% × 2220 MMBTU = 1665 MMBTU × $14/MMBTU = US$23,310 annually. Discussion Our hypothesis that cage washing alone at either 110 or 180 °F is sufficient to decontaminate soiled cages that had housed mice infected with viruses was confirmed. In the experimental infec- tion study, 97% and 100% of cages had detectable levels of MPV and MHV, respectively, on their surfaces at the time of washing. MHV and MPV infection occurred in most sentinels exposed to the unwashed cages, whereas washing of cages at 110 °F or 180 °F resulted in inactivation or removal of MPV and MHV from all soiled cages that had housed mice acutely infected (9 d after inoculation) with these agents. In the second study, pet-store mice naturally infected with MPV infected most of the SW mice that were housed with them. Similar to the experimental MPV study, cage washing alone at either 110 or 180 °F was sufficient to inacti- vateorremoveMPVfromallsoiledcagesinthesecondstudy.The results of the MPV studies presented here combined with those of our previous study11 showed that none of the 46 washed cages (14 in previous study and 32 in current studies) transmitted MPV, whereas 8 of 11 of the unwashed cages in this study and all 14 of the unwashed cage bottoms in the previous study11 transmitted MPV. The ability of cage washing to remove or inactivate 2 very different viruses— MPV (a DNA virus that is very stable in the environment and difficult to inactivate)5,6,16,27,38,42,49 and MHV (an enveloped RNAvirus that is less environmentally stable but is shed in high levels during acute infection3,9,20—suggests that cage washing is broadly effective at decontaminating cages that have housed mice infected with murine viruses. Pinworms eggs, like MPV, are highly stable in the environ- ment and difficult to inactivate with most disinfectants.13,32 Cage washing alone at either 110 or 180 °F inactivated or removed S. obvelata from all cages, suggesting that cage washing is effective at preventing infection with a wide range of murine infectious agents. Although more than half of the pet-store mice were in- fested with A. tetraptera, only 14% of SW mice became infested with A. tetraptera, and A. tetraptera DNA was detected on only 5% of cages prior to washing. In contrast, less than 25% of the pet-store mice were infested with S. obvelata, but more than 90% of the SW mice became infested with S. obvelata. This result is probably due to differences in the lifecycles of the 2 pinworm species. The duration of exposure of the second set of SW mice to the pet-store mice was only 21 d, to avoid the aforementioned clinical disease, which presumably was precipitated by M. pul- monis infection of the SW mice. This relatively short time frame favored S. obvelata infection, because S. obvelata eggs are released in a single burst of approximately 350 eggs, are infective 5 to 20 h after deposition on the perianal skin and hair, and can result in retrofection (migration of hatched larvae from the anus to the cecum of the mouse).7,41 In contrast, A. tetraptera eggs are released intermittently over a 3- to 4-wk period and are not infective for 5 to 8 d after excretion in the feces.2,23,34,41 In addition, A. tetraptera has a longer prepatent period (21 to 25 d) than does S. obvelata, which has a prepatent period of only 11 to 15 d.2,7,41 During the second study, the efficacies of several methods of pinworm detection were compared to determine the optimal sample to use for routine monitoring for pinworms in our facility. PCR and visual observation of cecal contents from SW mice were significantly better (Table 5, P < 0.01) at detecting pinworm infection (90% to 92%) than was testing of samples collected noninvasively (PCR of cage swabs, tape tests, PCR of fecal pools, and PCR of individual feces). These data agree with previous studies from our lab, which indicated that PCR and observation of cecal contents were equally effective at detecting pinworm infections.20 In light of our data, PCR of single fecal pellets collected directly from mice is not recommended as a reliable method for S. obvelata detection. The low sensitivity of the fecal PCR for Syphacia in our hands may be the result of inefficient DNA extraction, because others have suggested that inhibitors of pinworm PCR can be present in fecal DNA.17 Although only 22% of individual feces samples were positive for S. obvelata DNA on day 55 (Table 5), 37% of cages contained a mouse that was positive for S. obvelata DNA, indicating that most cages had only a single pinworm-positive mouse. Even though the pinworm PCR primers used were designed to am- plify both S. obvelata and A. tetraptera, PCR assay of the cecal contents did not detect A. tetraptera, whereas observation of cecal contents detected A. tetraptera in 14% of the index mice. The majority of the SW contact mice infested with A. tetraptera (80%) were coinfested with S. obvelata, R. nana, Hymenolepis diminuta, Entamoeba muris, or trichomonads (or multiple para- sites), whereas only 55% of mice infested with S. obvelata were also infested with these other parasites. Perhaps coinfection with these other parasites interfered with the amplification of A. tetraptera DNAbut not S. obvelata DNA. Several studies have compared the various methods of pinworm detection, with divergent results.14,17,22 Further optimization of DNAextraction methods and amplification conditions is required before the sensitivities of the various pinworm-detection methods can be accurately compared in our laboratory. Given that all 14 of the index mice housed in the unwashed cages were infected with Helicobacter spp. (Table 5) it is inter- esting to note that none of the sentinels placed in these cages became infected. This finding suggests that Helicobacter spp. are not readily transmitted by the residual waste present on unwashed cages. Several reports have shown efficient transmis- sion of Helicobacter spp. to sentinel mice via exposure to soiled bedding,29,48 although the frequency of exposure and dose of soiled bedding were higher than those in our current study. Our results are more in line with 2 other studies, which reported that H. hepaticus, H. typhlonius, H. mastomyrinus, and H. muridarium were not transmitted to soiled-bedding sentinels.10,22 Although the contact-infection study was not intended to measure transmission of M. pulmonis, more than half of contact- exposed mice seroconverted to M. pulmonis, and lungs from almost half of the contact-exposed mice were positive for M. pulmonis DNA. We therefore assessed the transmission of M. pulmonis by soiled unwashed cages. Several mycoplasmal spe- cies, when dried on paper discs, have been stable for several weeks,33 and because lungs from 8 of the 14 contact-exposed mice in the unwashed cages were positive for M. pulmonis DNA, it would have been reasonable to expect that sentinels placed in unwashed cages would have become infected with M. pulmonis. But our results showed that M. pulmonis was not transmitted by unwashed cages. Our data are consistent with information jaalas14000172.indd 752 11/23/2015 1:19:59 PM
  • 9. 753 Effect of cage-wash temperature on pathogen removal In the contact-infection study, an initial PCR-based attempt to detect pinworms and fur mites in samples of debris from the dump station prefilter after the disposal of soiled bedding was unsuccessful when the DNA was extracted indirectly from the sample, even though the majority of mice housed in the repre- sented cages were infested with pinworms and fur mites. We initially thought that the larger size of these parasites (pinworms eggs are 30 to 40 μm × 90 to 140 μm)41 led to inefficient aerosoliza- tion of these agents or parts of these agents during soiled bedding disposal. But a recent study showed that fur-mite DNA can be detected on swabs from the shelf of a horizontal manifold of an IVC rack housing a single cage of fur mite infested mice one week after placement of the cage on the rack indicating that aerosoliza- tion of detectable levels of debris containing fur mite components occurred.26 Follow-up testing of samples of prefilter dust and debris by using direct DNA extraction revealed that pinworms andfurmiteswerepresentinallofthesamplesofdustanddebris. It seems that smaller agents such as viruses and Helicobacter spp. are readily washed from the debris; therefore indirect extraction resulted in a sufficient quantity of nucleic acids for detection by PCR analysis. In comparison, the larger infectious agents such as pinworms and fur mites were not readily transferred from the debris to the PBS during the indirect extraction procedure, possibly because these agents were too large to be easily drawn into the pipettor tip or because they were tightly associated with the debris (for example, fur mites attached to fur would not be transferred). Because debris samples collected prior to soiled- bedding dumping were extracted by using the indirect method, we perhaps would not have detected any pinworms or fur mites present on the prefilter because the predumping samples were extracted by using the indirect method. Because pinworms and fur mites have not been detected in the facilities served by this ABDC for over 9 mo, we concluded that the S. obvelata and Myo- coptes musculinus DNA detected in the postdumping samples was deposited during dumping of the cages in this experiment. PCR-based testing of the prefilter of the dump station, by using direct nucleic acid extraction, shows promise for murine infectious agents screening at the facility level when more than 10 cages housing mice infected with a virus, bacteria, or parasite are dumped. However, MHV and MAV, which were detected on only a few cages in the second study, were not detected in the debris present on the prefilter of theABDC after soiled-bedding disposal, indicating that a threshold level of contaminated bed- ding needs to be present on the ABDC prefilter. Further studies to determine the sensitivity of this testing paradigm are needed. Taken together, our studies demonstrate that cage decontam- ination does not require washing at 180 °F and that autoclaving of washed cages is unnecessary. Rather, cage washing at 110 °F with appropriate detergents and cycle times was sufficient to decontaminate cages that had housed mice infected or infested with common murine infectious agents— thereby representing a significant advancement to the ‘greening’ of vivarium operation. The effectiveness of washing at 110 °F is an important finding in the context of sustainability and the need for steam infrastructure. For example, the energy usage when 140 °F domestic hot water is used to wash caging rather than water steam boosted to 200 °F was calculated to result in a savings of 583 MMBTU when a 1991 Metalwash Tunnel Washer was operated for 1560 h annually, for a cost savings of approximately $8000 annually. Similarly, 1665 MMBTU can be saved by operating a newer but larger Getinge Tunnel Washer at 140 °F for 2080 h yearly, yielding a cost savings of approximately $23,000 each year. Additional energy savings might be achieved by scheduling cage washing to minimize presented in a recent study, which showed that M. pulmonis was not transmitted to soiled bedding or contact sentinels.22 It is important to note that although M. pulmonis was restricted to the lungs of infected mice and although M. pulmonis DNA was not detected on cage surfaces, at least one mycoplasmal species, which had 98% homology with M. moatsii19 and 99% homology with several uncultured bacterial species found in the jejunum of mice, was detected in the feces of most pet-store and SW mice and on cage surfaces. These findings underscore that testing for M. pulmonis in the environment by PCR assay should not be performed by using generic mycoplasmal primers. Although Myocoptes musculinus was detected by PCR in 91% of the contact exposed pet-store and SW mice, it was not transmitted to sentinels housed in unwashed cages. These data are consistent with several previous studies, which have shown that soiled bed- dingtransmissionoffurmitestosentinelsoccursinfrequently.20,22,28 But2otherstudies37,43 haveshownefficienttransmissionbysoiled bedding to sentinel mice in 8 to 19 wk. The mice in the first study wereinfestedwithbothMyobiamusculiandMyocoptesmusculinus, and transmission occurred to all cages, but the fur-mite species that was transmitted is unclear.37 In the second study, mice were housed in open-top cages that received soiled bedding from other open-top cages within the room that housed mice infested with Myobia musculi, and transmission occurred in 3 of 4 cages.43 To our knowledge, our current study is the first to evaluate fomite-based transmission of fur mites from mice infested with Myocoptes mus- culinus in the absence of Myobia musculi infestation. The number and type of infectious agents present in pet-store mice and the potential for some of these agents to be spread by fomites prompt biosecurity concerns when animal care and laboratory personnel have pet-store mice as pets at home. Of particular concern is that these ‘clinically normal’ pet-store mice transmitted fatal disease to naive SW mice, albeit by direct con- tact. Nevertheless, these findings underscore the risk of pet-store mice and supports having a facility rodent-adoption program so that animal care and laboratory personnel wishing to acquire pet mice can officially do so through a pathogen-free source. Early detection of an infectious agent can often lessen its overall effect on an animal program. Unfortunately, many routine sentinel-exposure protocols rely on periodic sampling of and exposure to soiled bedding within a housing rack, and therefore positive sentinel findings may represent an infection that began weeks to months previously. Environ- mental sampling is a promising adjunct to sentinel-exposure programs for early pathogen detection. Realizing that testing of individual ventilated cage rack filters is one environmental screening approach, we tested a complementary approach: the ability to reliably detect pathogens from an ABDC, a site where soiled bedding is concentrated and aerosolized. Because the ABDC serves large portions of a facility, the abil- ity to reliably detect ‘excluded’ pathogens from the ABDC would allow for screening of large portions of a facility by using a few filter samples. Therefore, we assessed whether PCR-based testing of the accumulated dust-debris present on the prefilter of the ABDC after disposal of the soiled bedding from cages housing experimentally infected sentinel mice detected both MHV and MPV particles (diameter, 0.028 to 0.160 μm).1 Helicobacter DNA was detected in all samples of dust and debris present on the prefilter of the ABDC (both pre- and postdisposal samples from cages housing infected sentinel mice in both studies), indicating the accumulation of Helicobacter spp. (0.2 to 0.3 μm × 1.5 to 5 μm)18 during the routine dumping of cages from endemically infected cages within this animal facility. jaalas14000172.indd 753 11/23/2015 1:20:00 PM
  • 10. 754 Vol 54, No 6 Journal of the American Association for Laboratory Animal Science November 2015 15. Donaho JC. 2012. The green cage wash of 2012 and beyond. J Am Assoc Lab Anim Sci 51:648. 16. Eterpi M, McDonnell G, Thomas V. 2009. Disinfection efficacy against parvoviruses compared with reference viruses. J Hosp Infect 73:64–70. 17. Feldman SH, Bowman SG. 2007. Molecular phylogeny of the pinworms of mice, rats, and rabbits, and its use to develop molecular-beacon assays for the detection of pinworms in mice. Lab Anim (NY) 36:43–50. 18. Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, Collins MJ Jr, Gorelick PL, Ward JM. 1994. Helicobacter hepati- cus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice. J Clin Microbiol 32:1238–1245. 19. Giebel J, Binder A, Kirchhoff H. 1990. Isolation of Mycoplasma moatsii from the intestine of wild Norway rats (Rattus norvegicus). Vet Microbiol 22:23–29. 20. 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Lab Anim (NY) 40:145–152. the amount of time during which the tunnel washer is run- ning but not actively washing caging and by factoring in the decrease in energy needed to mitigate the heat gain within the room so that room temperature and humidity are accept- able to personnel working in the wash facilities. Apart from the cost of the energy required to boost domestic hot water to 180 °F, the boiler required to supply the steam necessary to heat the volume of the water necessary may not be available in the building or nearby, resulting in significant increased construction or renovation costs. However, detailed manu- facturer steam requirements are critical to understand the full range of savings associated with washer operating parameters, particularly requirements for stand-by modes maintaining temperature settings both during and after active use. Direct steam metering is not trivial in these settings but is required for more precise energy cost-savings estimates. 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