Journal of the American Association for Laboratory Animal Science Vol 59, No 6 Copyright 2020 November 2020 by the American Association for Laboratory Animal Science Pages 712–718

Metaphylactic Antibiotic Treatment to Prevent the Transmission of Corynebacterium bovis to Immunocompromised Mouse Offspring

Emily C Pearson,1 Umarani Pugazhenthi,2 Derek L Fong,1,3 Derek E Smith,4 Andrew G Nicklawsky,4 Lauren M Habenicht,1,3 Michael K Fink,1,3 Jori K Leszczynski,1,3 Michael J Schurr,5 and Christopher A Manuel1,3,6,*

Current methods for eradicating Corynebacterium bovis, such as depopulation, embryo transfer, and cesarean rederivation followed by cross fostering, are expensive, complex, and time-consuming. We investigated a novel method to produce immunocompromised offspring free of C. bovis from infected NOD.Cg-Prkdcscid Il2rgtm1Wgl/SzJ (NSG) breeding pairs. Adult NSG mice were infected with C. bovis, paired, and randomly assigned to either a no-antibiotic control group (NAB, n = 8) or a group that received amoxicillin–clavulanic acid (0.375 mg/mL) in their drinking water for a mean duration of 7 wk (AB group, n = 7), spanning the time from pairing of breeders to weaning of litters. The AB group also underwent weekly cage changes for 3 wk after pairing to decrease intracage C. bovis contamination, whereas the NAB mice received bi-weekly cage changes. Antibiotics were withdrawn at the time of weaning. All litters (n = 7) in the AB group were culture- and qPCR-negative for C. bovis and remained negative for the duration of the study, whereas all litters in the NAB group (n = 6) remained C. bovis positive. A single adult from each breeding pair was sampled at weaning and at 5 and 10 wk after weaning to confirm the maintenance of (NAB) or to diagnose the reemergence (AB) of C. bovis infection. By the end of the study, C. bovis infection had returned in 3 of the 7 (43%) tested AB adults. Our data suggest that metaphylactic antibiotic use can decrease viable C. bovis organisms from adult breeder mice and protect offspring from infection. However, using antibiotics with frequent cage changing negatively affected breeding performance. Nevertheless, this technique can be used to produce C. bovis-free NSG offspring from infected adults and may be an option for salvaging infected immunocompromised strains of mice that are not easily replaced.

Abbreviations: AB, antibiotics group; C. NAB, no antibiotics group; NSG, NOD.Cg-Prkdcscid Il2rgtm1Wgl/SzJ

DOI: 10.30802/AALAS-JAALAS-20-000005

Corynebacterium bovis is a common bacterial pathogen among Rederivation methods that are likely to be effective in the immunocompromised mouse colonies. Clinical signs associated elimination of C. bovis include embryo transfer and cesarean with C. bovis infection in athymic nude mice (Foxn1, nu/nu) rederivation followed by cross fostering. These measures can be include an asymptomatic persistent carrier state and 2 to 7 d expensive and require a high degree of technical skill and con- of dermal hyperkeratosis, dehydration, lethargy, and decreased siderable time. Conversely, antibiotic administration through body condition.5,9,11,24 Haired immunocompromised mouse feed or drinking water requires minimal labor, treats multiple strains such as NOD.Cg-Prkdcscid Il2rgtm1Wgl/SzJ (NSG) mice animals concurrently, provides no additional stress to the ani- also can develop clinical C. bovis infection and present with mals, and requires no specialized surgical skills.17 rough hair coat, decreased body condition, scaly skin, alopecia, In one study, antibiotic administration prevented the detec- conjunctivitis, and erythematous pinnae.5,9,25 tion of C. bovis from infected mouse skin tested by culture during C. bovis has a negative effect on cancer research due to the treatment.5 However, after discontinuing long-term antibiotic induced changes to the immune response and some institu- administration, C. bovis can again be cultured, with definitive tions choose to exclude this pathogen from their facilities.20,29 clearance of only 13% of infected adult mice.5 We recently Depopulation, environmental and equipment sanitation, and demonstrated that prophylactic antibiotic therapy can prevent repopulation are often elected, given that the majority of suscep- the infection of immunodeficient mice after acute exposure.16 tible strains can easily be purchased from C. bovis-free vendors. As opposed to prophylaxis, metaphylaxis is the treatment of However, when unique noncommercial immunocompro- an animal population that has been infected with a microbial mised strains are infected, depopulation may not be an option. agent but is not currently experiencing any level of clinical disease.3 Metaphylaxis differs from prophylaxis and therapeu- tic antimicrobial use because the expectation is to control an Received: 16 Jan 2020. Revision requested: 05 Mar 2020. Accepted: 04 May 2020. infection that is already present, rather than to prevent or cure 1Office of Laboratory Animal Resources,2 Division of Endocrinology, Metabolism, and Diabetes, School of Medicine, and Departments of 3Pathology and Immunology and infection. Using antibiotics in a metaphylactic manner requires Microbiology, Anschutz Medical Campus, University of Colorado–Denver, Aurora, a population-management mindset. Colorado; 4Biostatics Core, 5Department of Immunology and Microbiology, University of The goal of our study was to investigate the metaphylactic use Colorado Cancer Center, Aurora, Colorado; and 6University of Colorado Cancer Center, Aurora, Colorado of amoxicillin–clavulanic acid for C. bovis-infected NSG breed- *Corresponding author. Email: [email protected] ing pairs in order to prevent post-parturition transmission of

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C. bovis prior to weaning. We hypothesized that the metaphy- was removed from frozen stock, and cultured on tripticase soy lactic antibiotics would either eliminate all viable C. bovis or agar with 5% sheep blood (catalog no. 221261, Becton Dicken- decrease skin burden to undetectable levels on adult breeders. son, Franklin Lakes, NJ) at 37 °C for 72 h. At 24 hours prior to This situation would facilitate a C. bovis-free window dur- mouse inoculation, CUAMC1 was propagated in heart infusion ing which transmission to neonates and weanlings would be broth (catalog no. 238400, Becton Dickenson) containing 1% prevented, thus providing a novel and noninvasive method to Tween 80 for 24 h. Bacterial concentration was determined by eliminate C. bovis from unique immunocompromised mouse comparing the culture’s absorbance at OD600 with a standard strains that are not easily replaced. curve generated by serial dilutions. Mice were anesthetized with isoflurane, and 50 µL of culture broth containing 2× 107 Materials and Methods cfu of C. bovis was applied to the dorsal neck of each mouse. Animals and facility. Thirty male and 26 female, 7 wk old, A sterile swab (BactiSwab Dry, Remel, Lenexa, KS) was used NOD.Cg-Prkdcscid Il2rgtm1Wgl/SzJ mice (NSG) were purchased to distribute the inoculum on the skin, outer and inner pinnae, from Jackson Laboratories, Bar Harbor, ME. The mice were muzzle, lip commissures, and eyelids bilaterally. At 2 wk after documented to be free of ectromelia virus, Theiler mouse experimental inoculation, mice in each cage were swabbed, to encephalomyelitis virus, Hantaan virus, K virus, lactic dehydro- confirm infection by qPCR analysis. Cages that failed to gener- genase elevating virus, lymphocytic choriomeningitis, mouse ate an established infection were supplemented with 50 mL of adenovirus, mouse cytomegalovirus, mouse hepatitis virus, soiled bedding from an experimentally positive cage. At 2 wk mouse minute virus, mouse parvovirus, mouse thymic virus, after exposure to C. bovis-soiled bedding, all remaining cages pneumonia virus of mice, polyoma virus, reovirus 3, rotavirus, were confirmed positive by qPCR analysis. Animals were posi- Sendai virus, Bordetella spp., CAR bacillus, Citrobacter rodentium, tive via qPCR analysis for a minimum of 3 wk (21.6 ± 1.4 d) prior Clostridium piliforme, Corynebacterium kutscheri, Corynebacterium to the start of the experiment. bovis, Mycoplasma pulmonis, Salmonella spp., Streptobacillus mon- Experimental design. After qPCR confirmation of C. bovis oliformis, Encephalitozoon cuniculi, fur mites, lice, follicle mites, infection, male and female NSG mice were randomly grouped pinworms, roundworms, and tapeworms. On arrival, mice into breeding pairs, which then were assigned to receive either were tested by skin swab and confirmed negative forC. bovis treatment with antibiotic-containing drinking water (AB, n = by qPCR analysis. All animal manipulations were approved 8) or standard facility water (NAB, n = 8). Because a low birth by the IACUC of University of Colorado–Denver Anschutz rate occurred in AB, 10 additional breeding pairs were added to Medical Campus, an AAALAC-accredited institution. Personal reach a statistically significant sample size. On the day after pair- protective equipment required to enter the facility include a ing, the AB group began receiving drinking water containing hair bonnet and disposable gown worn over personal clothing; amoxicillin–clavulanic acid (amoxicillin trihydrate–clavulanate additional equipment was required in the quarantine animal potassium, 0.375 mg/mL, Zoetis, Parsippany-Troy Hills, NJ). housing room and included an additional disposable gown Medicated water was prepared by mixing 0.37 g of amoxicillin and shoe covers.15 trihydrate–clavulanate potassium, previously determined to Animal housing and manipulations. On arrival, all mice were be equivalent to 1/4 teaspoon, into 375 mL of facility water acclimated for 1 wk and housed 4 per cage according to sex. Mice for an anticipated dose of 75 mg/kg based on an estimated 5 were moved to a quarantine room, where they were housed in mL of water consumption daily.17 Water bottles were refreshed autoclaved JAG 75 cages (Allentown Caging, Allentown, NJ) on weekly,19,23,28 and were provided to the AB group throughout a 70-cage, single-sided, IVC rack (MicroVent, Allentown Caging) gestation and until the date of weaning. Antibiotic administra- providing 40 air changes per hour. The intracage environment tion averaged approximately 7 wk (49.8 ± 2.1 d). Breeding pairs included aspen chip bedding, a compressed cotton square, ster- that failed to produce a litter or demonstrated infanticide did ile cage furniture or cardboard roll, ad libitum irradiated rodent not progress further in the study. diet (2920X, Teklad Extruded Diet, Envigo, Indianapolis, IN), Viable offspring from breeding pairs (AB group, 7 litters; NAB and autoclaved reverse-osmosis–purified, hyperchlorinated (3 group, 6 litters) were weaned at 28 d of age. Litters were weaned to 5 ppm) water in 375-mL water bottles (Allentown Caging). into sex-segregated cages, with a maximum of 1 cage per sex The macroenvironment was maintained at 22.2 ± 1 °C (72 °F) with 5 mice per cage. All additional weanlings were euthanized and 30% to 40% humidity with at least 12 complete air changes by using CO2 asphyxiation followed by cervical dislocation. per hour and a controlled 14:10-h light:dark cycle. All animal Litters born before the 28-d weaning of the first litter were eu- manipulations were conducted in an animal transfer station thanized by decapitation after anesthesia (approximately 5 to 10 (ATS2, Allentown Cages) with work surfaces moistened with min of hypothermia). At the time of weaning, offspring in the disinfectant (1:18:1 Clidox S, Pharmacal, Naugatuck, CT). Cages AB group were transferred to an adjacent housing room to mini- were changed on a 2-wk cycle for the duration of the experiment, mize the risk of contamination from the positive control (NAB) with the exception that the AB group (but not the NAB group) group. For 10 wk after weaning, weanlings and one parent were received weekly changes during the first 3 wk after breeder monitored by culture and qPCR analysis for reemergence of C. pairing. A clean glove-changing practice was used to prevent bovis (Figure 1). At the conclusion of the monitoring period, all contamination of C. bovis infection between cages. Because this animals were euthanized as described for weanling mice. For bacterium is environmentally stable, the animal housing room the AB group, the experiment used 2 cohorts of mice; the first and all contents were considered to be C. bovis-positive. A C. cohort was bred in December, the second in April. bovis-free glove box, containing standard nitrile exam gloves, Sample collection. Pelt swabs were collected for C. bovis qPCR was stored inside a zip-lock plastic bag within the housing room. analysis and bacterial culture by passing a swab (BD BBL Cul- Once the cage lid was removed, these gloves were donned to tureSwab EZ, Becton Dickinson, Sparks, MD) over the animal’s manipulate all aspects of the intracage environment without right lateral thorax, inner and outer pinnae, periocular region, touching anything else in the room. muzzle, and oral cavity in a consistent pattern. When both par- Bacterial inoculation. C. bovis CUAMC1, isolated in 2014 from ents were present, the female mouse was used to obtain qPCR an infected nude mouse on the Anschutz Medical Campus, samples. Swabs were submitted to the University of Colorado

713 Vol 59, No 6 Journal of the American Association for Laboratory Animal Science November 2020

Denver Quantitative PCR Core for DNA isolation and analysis as previously described.15 Time points of sample collection for C. bovis qPCR analysis included at arrival, after C. bovis inoculation, 24 h after pairing for breeding, 3 d after birth, 28 d after birth (weaning), and 5 and 10 wk after weaning. qPCR was used as the primary assay for C. bovis status determination prior to and after the withdrawal of antibiotics. We anticipated that during antibiotic administration, mice would be culture negative, yet positive by qPCR due to residual C. bovis DNA in their fur. An increase in copy number after antibiotic withdrawal would suggest an active C. bovis infection. Pelt swabs for bacterial culture of C. bovis were obtained by using the same swabs, sampling procedure, and interval as for qPCR analysis. To reduce bacterial load on the swab, we did not sample the oral cavity. When both parents were available, culture samples were taken from the male. The swabs were shipped to IDEXX BioAnalytics for C. bovis isolation and iden- tification. Culture swab tips were vortexed in sterile PBS, and a 100-μL aliquot was plated on BBL Trypticase Soy Agar with 5% sheep blood (TSA II, Becton Dickinson). Plates were incubated

at 35 °C with 7% CO2 and monitored for colony growth for 72 h. Individual bacterial colonies displaying morphology consistent with Corynebacterium spp. were analyzed by MALDI-TOF mass spectrometry for species-level identification, as described previ- ously.25 C. bovis culture was the primary test used to determine C. bovis status during the administration of antibiotics. Data analysis. Both qPCR and culture results were used to evaluate the presence of C. bovis. Any qPCR value greater than 0 was considered to be C. bovis-positive. Statistical significance was assessed using the Fisher exact test for differences between the AB and NAB groups in the proportion of mice with C. bovis at each time period. The numbers of viable and nonviable lit- ters were compared between AB and NAB groups by using the Fisher exact test. Treatment group characteristics were summa- rized according to bacterial counts and percentages to describe and visually compare each categorical value. The McNemar test was used to assess changes in C. bovis-positive status of mice be- tween the withdrawal of antibiotics and the end of the study, for both qPCR and culture results. To compare agreement between qPCR and culture results, we applied intraclass correlation with a 95% confidence interval based on agreement by using a 2-way mixed-effects model. To aid in visualization due to the extreme range in qPCR values, copy numbers were transformed by using

log10(qPCR + 1) and graphed in correlation with culture results by using R version 3.5.0 (The R Foundation, www.r-project.org). Significance was defined as a P value of 0.05 or lower and based on a 2-sided alternative. All analyses were performed by using R version 3.5.0 (The R Foundation, www.r-project.org) and SAS version 9.4 (SAS Institute, Cary, NC).

Results C. bovis Infection. Mice were qPCR-positive for C. bovis for a minimum of 3 wk prior to the start of the experiment. During the study, 2 C. bovis-positive adult breeding pairs and 1 set of weaned offspring, all from the NAB group, developed charac- teristic clinical symptoms of C. bovis infection. All other mice, Figure 1. Illustration of the experimental design from C. bovis inocula- independent of C. bovis status, remained asymptomatic for the tion to the end of the study. Male mice, blue; female mice, pink; either duration of study. sex, white. After inoculation, mice were housed in sex-segregated cag- qPCR and culture diagnostics. Adult breeding pairs in the AB es for a minimum of 3 wk prior to randomization into breeding pairs group showed a decrease in qPCR copy number from pairing and treatment groups. Treatment provided to the AB group involved weekly cage changing from pairing to birth (*) and antibiotics in the to weaning, which was statistically significant (P < 0.05, Figure 2) drinking water from pairing to weaning. C. bovis was monitored by at the time of weaning. When the C. bovis culture status was qPCR analysis and culture at each time point from pairing to 10 wk assessed for the AB group, all breeding pairs were positive at after weaning. pairing. At 3 wk after antibiotic administration, corresponding

714 Metaphylaxis for C. bovis infected breeders

Table 1. Numbers of breeders and offspring culture-positive for C. bovis Treatment group AB (n = 7) NAB (n = 6) Adults At breeder pairinga 7e 2 At birth of pups 2e 6 At weaning of pupsb 0e 6 5 wk after weaningc 3 6 10 wk after weaningc 3 6 Offspring 5 wk after weaning 0e 6 10 wk after weaningd 0e 6 aqPCR data used at breeder pairing to designate positive C. bovis status bAdults and pups cultured immediately prior to weaning by using the same sample cOne adult of the breeding pair was retained for sampling at 5 and 10 wk after weaning of pups dBoth male and female offspring were cultured by using the same sample Figure 2. Longitudinal overview of qPCR results by mouse breeding eP ≤ 0.05 (Fisher exact test) between treatment groups pair. Left panel, breeding pairs in the nonantibiotic-treated (NAB) group; right panel, breeding pairs in the antibiotic-treated (AB) group. Lines indicate trajectories for individual breeding pair, and points cor- Discussion responding to a culture result at a given measurement time are color- Techniques to produce SPF offspring are common in coded. qPCR results are shown on the log (qPCR+1) scale. 10 laboratory rodent colonies. Viruses and bacteria have been eradicated successfully during gestation through weaning to the time of pup birth, 2 of the 7 (29%) breeding pairs in the in mice by embryo transfer, cesarean section, cross fostering, AB group remained culture-positive (Table 1). All of the 7 AB and antimicrobial administration.6,7,26,27 In the current study, breeding pairs were culture-negative at weaning. At 5 and 10 we demonstrate the use of metaphylactic antibiotic treatment wk after antibiotics were discontinued, 3 of the 7 (43%) adults to control C. bovis infections in adult breeding pairs of NSG returned to culture-positive status. In comparing litter culture mice to produce C. bovis-free offspring. The C. bovis-negative status at 10 wk after weaning, the AB group differed significantly status of offspring was confirmed at 10 wk and, in a subset of (P < 0.05) from the NAB group: the AB group had a lower pro- offspring, at 31 wk after weaning. The success of this method portion of C. bovis-positive litters. The weaned offspring from depends on antibiotic-induced elimination of viable C. bovis the AB group were qPCR- and culture-negative at both 5 and from the skin of infected mice after appropriate antibiotic 10 wk after weaning (Table 1). One pair of weaned males in the treatment.5 We hypothesized that if the skin of breeding pairs AB group was retained beyond the end of the study, monitored are culture-negative for C. bovis, the risk of pups acquiring an at 31 wk after weaning, and shown to be negative for C. bovis infectious dose would be diminished or eliminated. In addi- via qPCR and culture analysis (data not shown). tion, eliminating bacterial shedding from infected mice would As anticipated, adult breeding pairs in the NAB group eliminate intracage contamination. Here we demonstrated that showed an increase in mean qPCR copy number from pair- metaphylactic administration of amoxicillin–clavulanic acid ing to weaning (Figure 2). Culture results for breeders in the decreased C. bovis skin copy numbers based on qPCR analysis, NAB group at the time of pairing did not agree with qPCR and that mice became culture-negative while on antibiotics results, with only 2 of 6 breeder pairs positive according to (Figure 2 and Table 1). When combined with weekly cage culture and all positive by PCR analysis. Nevertheless, NAB changes during gestation and changing gloves prior to handling group showed both qPCR- and culture-positive status among anything within each cage, transmission of infection to neonates all pup cages and adult cages from birth through 10 wk after and weanlings was prevented. In combination, metaphylactic weaning (Table 1). antibiotic treatment and specific management practices can be The qPCR and culture test results did not agree across the used as a tool to facilitate the propagation of C. bovis-free NSG entire experiment. However, they displayed intraclass cor- mice from breeders infected with C. bovis. relation (95% CI) of 0.74 (0.64–0.82), which indicates moderate C. bovis is a facultatively anaerobic, gram-positive bacterium agreement between the 2 methods. that is susceptible to a wide range of antibiotics.30 For this study, Breeding performance. Breeding success was judged by visu- we chose amoxicillin–clavulanic acid (amoxicillin trihydrate– ally assessing viable litters at 4 wk after parturition. In the AB clavulanate potassium) due to its broad spectrum of activity, group, 16 of the 18 (89%) pairs gave birth to live pups, compared ease of administration through drinking water, efficacy against with all 8 (100%) pairs in the NAB group (Table 2). Of the 16 C. bovis,30 ability to reach the minimal inhibitory concentration litters produced in AB group, 9 litters (56%) were cannibalized. of amoxicillin for C. bovis at a reasonable oral dose,17 and rou- For NAB, 8 litters were born, and 2 (25%) were cannibalized. tine use at our facility with a history of reliable consumption The pairing-to-parturition interval did not differ significantly by research mice. Our treatment is regarded as metaphylactic between groups and ranged from 19 to 31 d. Litter size was because we do not expect the antibiotic treatment to cure the similar between groups (pups per litter: AB 6.2 +/- 3.7, NAB adult mice. As previously demonstrated, infected mice placed 6.8+/-1.5). The number of viable litters surviving to weaning on antibiotics for more than 8 wk became culture-positive 0 to age was 7 of 18 (39%) in the AB group and 6 of 8 (75%) in the 55 d after treatment was discontinued.5 However, experimen- NAB group (Table 2). tal infection with C. bovis can be prevented by prophylactic

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Table 2. Breeding performance Treatment group AB NAB Cohort no. 1 2 Totala 1 Number of breeding pairs 8 10 18 8 Parturition rate 6/8 (75%) 10/10 (100%) 16/18 (89%) 8/8 (100%) Litter mortality 5/8 (63%) 6/10 (60%) 11/18 (61%) 2/8 (25%) Mean interval (d) between pairing and parturition 21.67 23 23.3 20.3 Viable litters 3/8 (38%) 4/10 (40%) 7/18 (39%) 6/8 (75%) No. of C. bovis-positive litters 0 0 0 6 No. of C. bovis-negative litters 3 4 7 0 aAverage provided where appropriate administration of oral amoxicillin–clavulanic acid.16 To show ill as well as to propagate the bacteria as chronic carriers. that metaphylactic antibiotic treatment reliably creates a C. Bacterial shedding results in environmental contamination, bovis-free ‘window’ only while mice are on treatment, we dis- ultimately perpetuating the infection in other susceptible mice. continued antibiotics for breeders after weaning the offspring Giving these mice antibiotics in conjunction with facility-wide and monitored for the reemergence of C. bovis. Concurrently, measures to clear the infection will ultimately reduce overall to decrease the potential for cross-contamination to offspring, antibiotic use. weanlings in the AB group were moved into a separate room, At our institution, nude mice are most likely to be from which was entered before entry into the room containing both commercial stock and, when naïve, can easily be replaced. the AB and NAB breeder adults and NAB offspring. At 10 wk In contrast, NSG mice or mice with similar genetic defi- after the removal of antibiotic treatment from the breeders in cits have been crossed with other strains to produce new the AB group, 57% (4 of 7) were PCR-positive and 34% (3 of strains that are not commercially available. These mice are 7) were culture-positive (Figure 2 and Table 1). These results extremely valuable to researchers, eliminating depopulation continue to support that antibiotic therapy alone does not as an option for eradicating infectious agents. Therefore, we eliminate established C. bovis infections from immunodeficient chose NSG mice for this study because several valuable NSG mice. In contrast, by monitoring the pups in the AB group for crosses required C. bovis eradication at our facility. Addi- 10 wk after weaning, we show that metaphylaxis successfully tional studies must be conducted to determine whether this protected offspring in AB group from infection. technique can be applied to other strains of immunocom- In our study, we could not determine when neonates began to promised mice. Our data support the hypothesis that when ingest antibiotics. Although we did not quantify the antibiotic therapeutic levels of antibiotics are administered, viable C. dose obtained by pups while nursing, some literature suggests bovis is eliminated from the skin of infected adult breeding that antibiotics can be transported across the mammary gland pairs, suggesting that similar outcomes can be expected in and reach the nursing pups.18 Antibiotic metabolites are similar other mouse strains. to blood plasma components and, depending on factors includ- In the current study, pup mortality occurred within the first ing concentration gradients, protein affinity, lipid solubility, and 3 d of birth. Perinatal pup mortality in 0% to 50% of litters has state of ionization, they can successfully penetrate breast milk.18 been reported in previous studies.4,14,31,32 For AB cohorts 1 and When considering the safety of amoxicillin–clavulanic acid in 2, pup mortality from breeding pairs occurred in 3 of 6 (50%) regard to a human fetus, the American Academy of Pediatrics and 6 of 10 (60%) litters, respectively. In contrast, only 2 of 6 Committee on Drugs considers amoxicillin–clavulanic acid and (25%) litters in the NAB group experienced pup mortality. penicillin-like drugs safe for pregnant mothers to use.1 No harm- Little research exists to explain preweaning or perinatal pup ful effects have been reported in infants who consumed breast mortality. Some studies suggest that litter survivability in- milk that potentially contained amoxicillin or clavulanic acid.2 In creases when nest building behaviors are displayed and when addition, the milk:plasma ratio reportedly is very low when oral the dam spends increased time within the nest.33 Whereas penicillin-like drugs and clavulanic acid are prescribed orally to parity does not affect litter loss, strain appears to have some nursing human mothers.2 However, once mouse pups transition predictive value for litter survivability.32 Documented strate- from nursing to drinking the provided antibiotic-containing gies to improve litter survivability include supplying the cage water, we anticipate that they would consume a prophylactic with the appropriate type and quantity of nesting materials,12 dose prior to weaning and separation from the dam. Despite reducing the frequency of cage changing, and providing clarity regarding whether antibiotics consumed by the pups environmental enrichment like polycarbonate houses and contributed to preventing infection transmission, the offspring tissue paper.14 As a result, not only the increased cage-change of the AB group were diagnostically negative for bacteria. frequency after pairing but also the complete removal and Antimicrobial stewardship has become an area of focus replacement of nesting material might have contributed to across many professions.10,13,21,22 As the incidence of bacte- poor litter survivability of the AB group as compared with the rial resistance grows and alternative therapeutics are scant, NAB group. Nevertheless, our proposed method appears to prudent use is mandated to keep the population safe. The affect pup viability, which will require confirmation through metaphylactic manner in which this study deploys antibiotics future studies. However, in practice, once the breeding animals adheres to antimicrobial stewardship principles. By placing have received initial antibiotic treatment and cage changing, infected adults on antibiotics prior to birth, offspring are able subsequent litters can be produced by maintaining breeder to mature in a C. bovis-free environment, preventing infection. animals on antibiotics alone, thus potentially increasing lit- C. bovis can affect research and cause mice to become clinically ter survivability for subsequent litters. Although we did not

716 Metaphylaxis for C. bovis infected breeders test this method on subsequent litters from antibiotic-treated References breeding pairs, we have no reason to suspect that antibiotics 1. American Academy of Pediatrics Committee on Drugs. 2001. The would become ineffective for subsequent litters from the same transfer of drugs and other chemicals into human milk. Pediatrics breeders. 108:776–789. https://doi.org/10.1542/peds.108.3.776. We found discrepancies among the results from culture 2. Bar-Oz B, Bulkowstein M, Benyamini L, Greenberg R, Soriano I, and qPCR diagnostics throughout the study. When results Zimmerman D, Bortnik O, Berkovitch M. 2003. Use of antibiotic and analgesic drugs during lactation. Drug Saf 26:925–935. https:// were analyzed at pairing of breeding animals, 4 of 13 (32%) doi.org/10.2165/00002018-200326130-00002. breeding pairs were qPCR-positive but culture-negative. 3. Berman J, Francoz D, Dubuc J, Buczinski S. 2017. A randomised For facility-wide monitoring measures, qPCR analysis is the clinical trial of a metaphylactic treatment with tildipirosin for test of choice.8 However, qPCR testing has little reliability bovine respiratory disease in veal calves. BMC Vet Res 13:176. in definitively determining C. bovis status (positive or nega- https://doi.org/10.1186/s12917-017-1097-1. tive) during antibiotic administration. Statistical evaluation 4. Bond TLY, Neumann PE, Mathieson WB, Brown RE. 2002. Nest revealed that these 2 diagnostic tests have moderate to good building in nulligravid, primigravid and primiparous C57BL/6J reliability, according to intraclass correlation assessment. and DBA/2J mice (Mus musculus). Physiol Behav 75:551–555. https://doi.org/10.1016/S0031-9384(02)00659-5. Potential limitations of this study include the experimental 5. Burr HN, Lipman NS, White JR, Zheng J, Wolf FR. 2011. Strate- nature of the infection tested in mice. For nonexperimental gies to prevent, treat, and provoke Corynebacterium-associated applications of this method, animals are likely to be infected hyperkeratosis in athymic nude mice. J Am Assoc Lab Anim Sci chronically, whereas our mice were infected for a minimum 50:378–388. of 3 wk. Even with this relatively short duration of infection, 6. Buxbaum LU, DeRitis PC, Chu N, Conti PA. 2011. Eliminating our data mirror natural C. bovis infection, because the trend murine norovirus by cross-fostering. J Am Assoc Lab Anim Sci for C. bovis to return after the withdrawal of antibiotics was 50:495–499. preserved.5 Although mice were culture- and qPCR-positive 7. Clark SE, Purcell JE, Bi X, Fortman JD. 2017. Cross-Foster red- erivation compared with antibiotic administration in the drinking for C. bovis, the majority did not display clinical signs of in- water to eradicate Bordetella pseudohinzii. J Am Assoc Lab Anim fection and appeared healthy. This situation may have had a Sci 56:47–51. positive influence on breeding success, given that clinically 8. Dole VS, Henderson KS, Fister RD, Pietrowski MT, Maldonado affected NSG mice exhibit rough hair coat, decreased body G, Clifford CB. 2013. Pathogenicity and genetic variation of 3 condition, dehydration, scaly skin, alopecia, conjunctivitis, strains of Corynebacterium bovis in immunodeficient mice. J Am and erythematous pinnae.5,9,25 These signs contribute to Assoc Lab Anim Sci 52:458–466. the animal’s overall dull demeanor, which may lead to low 9. Duga S, Gobbi A, Asselta R, Crippa L, Tenchini ML, Simonic T, fecundity. The AB group required 2 cohorts of breeding to Scanziani E. 1998. Analysis of the 16S rRNA gene sequence of the coryneform bacterium associated with hyperkeratotic dermatitis reach statistically significant sample size. Even though the of athymic nude mice and development of a PCR-based detec- cohorts did not differ significantly, seasonality could be a tion assay. Mol Cell Probes 12:191–199. https://doi.org/10.1006/ factor. Another variable between the AB and NAB groups mcpr.1998.0168. was the initial 3 wk of increased cage changing after pair- 10. Dyar OJ, Huttner B, Schouten J, Pulcini C, ESGAP (ESCMID ing the breeders in the AB groups. We hypothesize that the Study Group for Antimicrobial stewardship). 2017. What is anti- period of increased cage-changing decreased litter viability microbial stewardship? Clin Microbiol Infect 23:793–798. https:// in the AB group; a sham cage manipulation was not applied doi.org/10.1016/j.cmi.2017.08.026. to the NAB group. Despite these limitations, the ability to 11. Field K, Greenstein G, Smith M, Herrman S, Grizzi J. 1995. P62 produce C. bovis-free offspring with management practices Hyperkeratosis-associated coryneform in athymic nude mice. Contemp Top Lab Anim Sci 34:69. and antibiotics is valuable when more invasive eradication 12. Gaskill BN, Pritchett-Corning KR, Gordon CJ, Pajor EA, Lucas methods are not feasible. JR, Davis JK, Garner JP. 2013. Energy reallocation to breeding C. bovis affects many studies involving immunocompromised performance through improved nest building in laboratory mice. mice.29 Eradication is a difficult and expensive process, but may PLoS One 8:1–9. https://doi.org/10.1371/journal.pone.0074153. ultimately result in the use of fewer animals in research. Our 13. Gyssens IC. 2018. Role of education in antimicrobial stewardship. method aligns antimicrobial stewardship and reduction of ani- Med Clin North Am 102:855–871. https://doi.org/10.1016/j. mal numbers to achieve immunocompromised mouse offspring mcna.2018.05.011. LeidingerCS,Thöne-Reineke C, Baumgart N, Baumgart J. 2018. En- free of C. bovis. Our study demonstrates a novel way to eradicate 14. vironmental enrichment prevents pup mortality in laboratory mice. C. bovis from immunocompromised mouse strains by using Lab Anim 53:53–62. https://doi.org/10.1177/0023677218777536. metaphylactic antibiotics in infected immunocompromised 15. Manuel CA, Pugazhenthi U, Spiegel SP, Leszczynski JK. 2017. breeding pairs. The ability to maintain unique noncommercial Detection and elimination of Corynebacterium bovis from barrier immunocompromised strains during facility wide eradication rooms by using an environmental sampling surveillance program. of C. bovis is essential to research groups that would otherwise J Am Assoc Lab Anim Sci 56:202–209. be unable or unwilling to use expensive, time-consuming, and 16. Manuel CA, Schurr MJ, Henry CJ. 2016. Prophylactic antibiotics invasive eradication methods. prevent Corynebacterium bovis infection following acute exposure. PS90 abstract presented at the AALAS National Meeting, Charlotte, North Carolina, October 30–3 November 2016. J Am Assoc Lab Anim Sci 5:702. Acknowledgments 17. Marx JO, Vudathala D, Murphy L, Rankin S, Hankenson FC. We thank David Eckhoff at IDEXX BioAnalytics for his expertise and 2014. Antibiotic administration in the drinking water of mice. J assistance with the bacterial skin cultures performed for this study. We Am Assoc Lab Anim Sci 53:301–306. also thank Brandie Trotter for her contribution to the original artwork 18. Mathew JL. 2004. Effect of maternal antibiotics on breast feeding and graphic design and Chris Nestel and Kirsten Soules for providing infants. Postgrad Med J 80:196–200. https://doi.org/10.1136/ technical expertise. Statistical support was received from the University pgmj.2003.011973. of Colorado Cancer Center Support Grant (P30CA046934). The Office of 19. McIntyre AR, Lipman NS. 2007. Amoxicillin-clavulanic acid and Laboratory Animal Resources provided funding for this project as part trimethoprim-sulfamethoxazole in rodent feed and water: effects of the University of Colorado Anschutz Medical Campus Internship in of compounding on antibiotic stability. J Am Assoc Lab Anim Sci Laboratory Animal Medicine. 46:26–32.

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