Evaluating the Impact of Prophylactic Administration of Ivermectin on the Fecal Microbiome of Healthy C57BL/6J Mice (Mus musculus)

Abstract

Upon importation, laboratory mice may undergo prophylactic antiparasitic treatment during quarantine to prevent the introduction of parasites into established colonies. While quarantine protocols vary across institutions, ivermectin is commonly used, administered either orally or topically. However, the impact of these practices on the fecal microbiome remains poorly understood, raising concerns about unintended consequences for experimental outcomes. This study investigated the effects of ivermectin on fecal microbiome composition in naïve, healthy male and female C57BL/6J mice. Animals received either ivermectin-impregnated feed (12 ppm, ad libitum for 4 weeks), weekly topical ivermectin solution (2.0 mg/kg for 4 weeks), or no treatment (controls). Fecal samples were collected for 16S rRNA-based microbiome analysis before ivermectin treatment, immediately posttreatment, and 4 weeks after treatment cessation. Weekly body weights were recorded, and histopathologic evaluation of the small intestine and colon was performed at study completion. Both oral and topical ivermectin treatments resulted in significant alterations in microbiome α and β diversity at the end of treatment, with more pronounced effects observed in female mice. Some of these changes persisted for up to 4 weeks after treatment cessation. Furthermore, the findings indicate a sex-specific effect of ivermectin on specific bacterial orders, with Bacillales predominantly affected in male mice, whereas Coriobacteriales and Bacteriodales were primarily impacted in female mice. During treatment, males receiving topical ivermectin weighed significantly less than controls, while females receiving dietary ivermectin weighed significantly more. Histopathological analysis revealed no abnormalities in intestinal tissues across all groups at 4 weeks posttreatment. These findings demonstrate that ivermectin administration induces measurable and persistent changes in the fecal microbiome of healthy mice. Researchers should consider these effects when designing experiments, and institutions must weigh the benefits of colony protection against potential microbiome-related confounding variables.

Introduction

The Guide for the Care and Use of Laboratory Animals considers animal quarantine a component of adequate veterinary care,¹ and research institutions commonly require a quarantine period for rodents purchased from noncommercial vendors and may provide prophylactic antiparasitic treatment to prevent the introduction of parasites that impact colony health status and research. The drug, route, and duration of treatment vary among institutions, but ivermectin is a common drug of choice. Despite its widespread use, the potential downstream effects of ivermectin on host physiology and experimental outcomes remain underexplored.

Ivermectin, a member of the avermectin class of compounds, exerts its antiparasitic effects by binding to glutamate-gated chloride channels, leading to paralysis and death in invertebrates.² Beyond its well-known antiparasitic properties, ivermectin has been reported to exhibit antimicrobial, immunomodulatory, and even behavioral effects.^3–6 Toxicity remains a concern with ivermectin administration, as secondary neurologic effects have been well-documented across multiple species, including humans, particularly in cases of high dosing or compromised blood-brain-barrier integrity.^7–11 Finally, ivermectin may influence certain animal models. For instance, one study¹² reported that ivermectin can alter the tamoxifen-independent activity of Cre^ER fusion proteins in the offspring of treated dams, which has important implications for the use of this drug class in transgenic mouse studies.

Understanding how prophylactic treatments influence the microbiome is critical, given the growing recognition of its role in modulating immune responses and disease susceptibility. In mice, clinically reported doses range from 0.2 to 4.0 mg/kg.¹³ In one study,¹⁴ daily oral gavage of ivermectin at a high experimental dose resulted in significant alterations to gut microbiome composition and induced histopathologic changes in the cecum. Similarly, studies^15,16 in tigers and deer reported notable shifts in gut microbial communities following treatment with a combination of ivermectin and fenbendazole. However, other investigations^17–19 across various species found no significant changes in gut microbiota following ivermectin administration. These conflicting findings highlight the need for species-specific and context-dependent evaluations of ivermectin’s impact on the microbiome, particularly in laboratory animals where microbial composition can influence experimental outcomes. For example, it is unknown if ivermectin administered at clinically relevant doses to healthy male and female mice during quarantine significantly alters the fecal microbiome or microanatomy of the gastrointestinal tract. Given the association between the gut microbiome and mucosal immunity, significant changes may confound data for various studies. This is particularly important in research areas involving inflammation, metabolism, or immune modulation, where microbiome composition can influence experimental outcomes. Understanding these effects is essential for refining quarantine protocols and ensuring reproducibility in preclinical studies.

In this study, we investigated whether ivermectin administration, either topically or via the diet, over a 4-week period, significantly alters the fecal microbiome composition of healthy male and female mice. We also assessed potential changes in body weight and histopathology of the small intestine and colon following treatment.

Materials and Methods

Ethical review.

This study protocol was approved by the IACUC of Emory University, an institution accredited by AAALAC International. The study adhered to the principles of the Guide for the Care and Use of Laboratory Animals.

Experimental animals.

The study subjects included 6-week-old male and female C57BL/6J mice purchased from The Jackson Laboratory (Strain No. 000664; RRID:IMSR_JAX:000664). The animals were confirmed to be free of specific pathogens upon arrival from the vendor and were subsequently housed in a facility that excludes the following infectious agents: K virus, lactic dehydrogenase elevating virus, lymphocytic choriomeningitis virus, mouse adenovirus, mouse cytomegalovirus, mouse hepatitis virus, mouse minute virus, mouse norovirus, mouse parvovirus, mouse poxvirus (Ectromelia), mouse rotavirus-A, mouse thymic virus, pneumonia virus of mice, polyoma virus, reovirus, Theiler murine encephalomyelitis virus, Bordetella bronchiseptica, Citrobacter rodentium, Corynebacterium bovis, Corynebacterium kutscheri, Filobacterium rodentium, Helicobacter spp., Klebsiella oxytoca, Klebsiella pneumoniae, Mycoplasma pulmonis, Pseudomonas aeruginosa, Rodentibacter heylii, Rodentibacter pneumotropicus, Salmonella spp., Staphylococcus aureus, Streptobacillus moniliformis, Streptococcus pneumoniae, beta-hemolytic Streptococcus spp. (A, B, C, G), Encephalitozoon cuniculi, Pneumocystis murina, Tritrichomonas spp., murine fur mites (Myobia, Myocoptes, and Radfordia), and pinworms (Aspiculuris and Syphacia).

Husbandry.

Same-sex groups were housed at a density of 5 mice per cage in autoclaved static microisolation cages (Lab Products, Aberdeen, MD) with filter tops, 1/8-inch corncob bedding (Bed-o’Cobs; The Andersons, Maumee, OH), and one cotton nestlet (Ancare, Bellmore, NY). Cage change occurred weekly by animal care staff for the duration of the study. All cages were accessed only under a HEPA-filtered, class II, type A2, biologic safety cabinet (NuAire, Plymouth, MN). When opening cages and handling animals, disposable personal protective equipment (ie, gown and gloves) was changed between each treatment group. Irradiated PicoLab Rodent Diet 20 (5053; LabDiet, Richmond, IN) was offered ad libitum. Autoclaved reverse osmosis water was provided in a water bottle with no restrictions. The parameters of the macroenvironment included a temperature range of 70-74 °F, a relative humidity range of 30%-70%, and a 12:12 light/dark photoperiod.

Study design.

Animals were individually identified with tail marks drawn in permanent marker ink and assigned to one of 3 groups by cage. One group received an ivermectin-impregnated diet for 4 weeks. One group received weekly topical applications of ivermectin solution for 4 weeks, and the last group received no treatment. Fecal pellets were collected under aseptic conditions according to a previously published protocol²⁰ for each individual animal at 3 time points: pretreatment, end of treatment, and 4 weeks posttreatment. Body weights were obtained weekly in grams by placing the animal in a container on a tared digital scale (CB 501; Adam Equipment, Oxford, CT). Each animal was visually examined for overall health twice weekly by study staff.

Sample size.

This study consisted of 30 animals total, with each group containing 10 animals, 5 males and 5 females. Power analysis for animal numbers was performed based on the research described in Pal et al²¹ and in consultation with a contracted biostatistician at Emory University.

Experimental procedures.

Animals were allowed to acclimate to the animal facility for 2 weeks before the start of the study. Fecal collection occurred in the morning (between the hours of 9:30 am and 12:00 pm) every 4 weeks for a total of 3 fecal collections per animal. The animals were placed in individual autoclaved cages with no bedding for 10-15 minutes to produce 3-8 fecal pellets before being returned to their home cage. The fecal pellets were collected with an autoclaved toothpick and placed into a sterile microfuge tube (Lot No. K197262L; Eppendorf North America, Enfield, CT). The fecal pellets were immediately placed on ice, then stored at −80 °C until the end of study.²⁰ The fecal samples were processed and analyzed with the Microbiome Sequencing Service: 16S/ITS Amplicon Sequencing (Zymo Research, Irvine, CA).

Ivermectin treatment.

After initial fecal collection, treatment was started for each ivermectin group. In place of a standard diet, the ivermectin diet treatment group received irradiated ivermectin-impregnated diet (5053/12 ppm ivermectin Red 1/2 IRR; Lot No. 24Jan18RTD1; TestDiet, Richmond, IN) to be consumed ad libitum for 4 weeks per our internal quarantine standard operating procedure. This diet delivers an estimated dose of 1.3 mg/kg ivermectin as previously described.¹³ The animals in the topical ivermectin treatment groups received ivermectin solution (ProMectin Injection for Cattle and Swine 1% Sterile Solution; Lot No. 220180; Saint Joseph, MO) applied with a pipette between the shoulder blades at an estimated dose of 2.0 mg/kg. The application occurred for the topical groups while obtaining body weights for all groups, so as not to increase handling time for any group. The topical treatment was repeated weekly for a total of 4 treatments.

Tissue collection and histopathology.

At the end of the study, the animals were euthanized via inhalation of CO₂ in their home cages for necropsy. Small intestine and colon were collected from all animals and immersion fixed in 10% neutral-buffered formalin (VWR International; Radnor, PA) for 48 hours at room temperature. Tissues were embedded in paraffin, sectioned at a 4-µm thickness, and stained with hematoxylin and eosin for routine histopathologic analysis. Histologic sections were evaluated under a light microscope by a board-certified veterinary pathologist (TN).

Inclusion and exclusion criteria.

There were no excluded animals or data sets.

Randomization.

Cages were randomized by the animal care technician on arrival to the animal facility. All cages were housed on the same rack level for the duration of the study. The animals were handled in the following order to prevent cross-contamination of treatment groups: control groups, diet groups, and then topical groups.

Blinding.

The primary study administrator could not be blinded due to the red color of the ivermectin diet. The pathologist and microbiome technicians were blinded to treatment groups for analysis.

Outcome measures.

Alpha diversity (species diversity within samples), β diversity (species composition between samples), and abundance of bacterial orders were the primary outcome measures assessed in this study. In addition, body weight and histopathologic changes to the gastrointestinal tract were evaluated.

Statistical methods.

Grouping was conducted by sex and time point in the study due to sex differences²² and expected microbiome drift over time.²³ Alpha diversity, β diversity, and composition abundance analysis were performed by Zymo Research using Qiime v.1.9.1.²⁴ Body weight and bacterial abundance at the order level were tested between control and experimental groups (diet or topical) at each time point using Wilcoxon ranked sum testing performed in SAS 9.4. Alpha diversity was analyzed using the Chao1 index with linear mixed model regressions to analyze differences between control and experimental groups at each time point. Modeling used the restricted/residual maximum likelihood (REML) method. Beta diversity was analyzed using Bray-Curtis dissimilarity (BCD). The BCD matrix was provided to and analyzed using principal coordinate analysis (PCoA). The results of PCoA were then statistically analyzed using ANOVA F statistics. The subsequent P value was derived using F statistics. Analysis of α and β diversity was conducted in Python 3.4. All P values were reported at α = 0.05.

Results

Evaluation of ivermectin administration on body weight.

Weekly body weight measurements revealed sex-specific responses to ivermectin treatment.

Male mice fed an ivermectin-impregnated diet showed no significant differences in body weight throughout the study period (Figure 1A), while female mice in the same group weighed significantly more than control animals at weeks 1, 3, 4, 5, and 6 (P < 0.05; Figure 1B).

Figure 1.

Evaluation of Ivermectin Administration on Body Weight. (A) Weight of 8-wk-old C57BL/6J male mice subjected to control or ivermectin-containing diet for 4 wk. (B) Weight of 8-wk-old C57BL/6J female mice subjected to control or ivermectin-containing diet for 4 wk. (C) Weight of 8-wk-old C57BL/6J male mice subjected to topical ivermectin treatment for 4 wk. (D) Weight of 8-wk-old C57BL/6J female mice subjected to topical ivermectin treatment for 4 wk. Statistics: nonparametric Wilcoxon testing; n = 5 per group; *P < 0.05; ^†P < 0.01.

In contrast, males receiving topical ivermectin weighed significantly less than controls during weeks 2, 3, and 4 (P < 0.01; Figure 1C), which resolved following cessation of treatment. Female mice treated topically showed no significant changes in body weight compared with controls (Figure 1D).

Evaluation of ivermectin administration on microbiome α diversity.

All groups exhibited significant changes to microbiome α diversity posttreatment. Male mice fed an ivermectin-impregnated diet showed no significant changes at the end of treatment but had a significant decrease in α diversity at 4 weeks posttreatment (P < 0.01; Figure 2A). Female mice in the same group had significantly decreased α diversity at the pretreatment time point (P < 0.05) that was more significant at the end of treatment and 4 weeks posttreatment (P < 0.01; Figure 2B). Males treated with topical ivermectin exhibited a significant increase in α diversity at the end of treatment (P < 0.05; Figure 2C), while females of the same group showed significant decreases in α diversity at the end of treatment (P < 0.01) and 4 weeks posttreatment (P < 0.05; Figure 2D).

Figure 2.

Evaluation of Ivermectin Administration on Microbiome Alpha Diversity. (A) Microbiome α diversity (Chao1 index) of 8-wk-old C57BL/6J male mice subjected to control or ivermectin-containing diet for 4 wk. (B) Microbiome α diversity (Chao1 index) of 8-wk-old C57BL/6J female mice subjected to control or ivermectin-containing diet for 4 wk. (C) Microbiome α diversity (Chao1 index) of 8-wk-old C57BL/6J male mice subjected to topical ivermectin treatment for 4 wk. (D) Microbiome α diversity (Chao1 index) of 8-wk-old C57BL/6J female mice subjected to topical ivermectin treatment for 4 wk. Statistics: mixed linear model regression; n = 5 per group; *P < 0.05; ^†P < 0.01.

Evaluation of ivermectin administration on microbiome β diversity.

Microbiome β-diversity analysis revealed sex-specific impacts of ivermectin administration to mice. Male mice fed an ivermectin-impregnated diet showed significant differences in species composition at the end of treatment (P < 0.05; Figure 3A), while females of the same group showed significant changes at the end of treatment and 4 weeks posttreatment (P < 0.01; Figure 3B). Male mice treated with topical ivermectin showed no significant changes to species composition at any time point (Figure 3C), but female mice of the same group exhibited significant changes to β diversity at the end of treatment (P < 0.05; Figure 3D).

Figure 3.

Evaluation of Ivermectin Administration on Microbiome Beta Diversity. (A) Microbiome β diversity of 8-wk-old C57BL/6J male mice subjected to control or ivermectin-containing diet for 4 wk. (B) Microbiome β diversity of 8-wk-old C57BL/6J female mice subjected to control or ivermectin-containing diet for 4 wk. (C) Microbiome β diversity of 8-wk-old C57BL/6J male mice subjected to topical ivermectin treatment for 4 wk. (D) Microbiome β diversity of 8-wk-old C57BL/6J female mice subjected to topical ivermectin treatment for 4 wk. Statistics: pairwise permutational multivariate analysis of variance (PERMANOVA); n = 5 per group; *P < 0.05; ^†P < 0.01.

Evaluation of ivermectin administration on abundance.

All groups exhibited changes in the abundance of bacterial orders within fecal pellets throughout the duration of the study. At the pretreatment time point, males in the diet group showed a significant reduction in Anaeroplasmatales (P < 0.05). Male mice treated with the ivermectin-impregnated diet showed a significant decrease (P < 0.05) in Lactobacillales and a significant increase (P < 0.05) in Verrucomicrobiales at the end of treatment and a significant increase (P < 0.05) in the bacterial order Bacillales 4 weeks posttreatment (Figure 4A). Female mice treated with ivermectin-impregnated diet exhibited significant increases (P < 0.05) in Bacteriodales, Coriobacteriales, and Lactobacillales and significant decreases (P < 0.05) in Clostridiales at the end of treatment. While most changes to bacterial orders resolved from the end of treatment, a significant decrease (P < 0.01) in Clostridiales remained 4 weeks posttreatment, and a significant increase (P < 0.05) in Erysipelotrichales was observed (Figure 4B). In male mice treated with topical ivermectin, a significant increase (P < 0.05) in Bacteroidales and a decrease (P < 0.05) in Anaeroplasmatales were identified in the pretreatment samples. The bacterial order Anaeroplasmatales remained significantly decreased (P < 0.01) at the end of treatment, and at 4 weeks posttreatment, a significant decrease (P < 0.01) in Bacillales and Coriobacteriales (P < 0.05) was identified (Figure 4C). In female mice of the same group, there were pretreatment changes in bacterial orders that consisted of a significant increase in Bacteroidales (P < 0.01) and Lactobacillales (P < 0.05) and a significant decrease in Clostridiales (P < 0.05) and Coriobacteriales (P < 0.01). At the end of treatment, there was a significant increase in Bacteroidales (P < 0.01) and Coriobacteriales (P < 0.05). No significant changes in bacterial order abundance remained at the 4-week posttreatment time point (Figure 4D).

Figure 4.

Evaluation of Ivermectin Administration on Abundance. (A) Microbiome bacterial order abundance of 8-wk-old C57BL/6J male mice subjected to control (CON) or ivermectin-containing diet (DIET) for 4 wk. (B) Microbiome bacterial order abundance of 8-wk-old C57BL/6J female mice subjected to control (CON) or ivermectin-containing diet (DIET) for 4 wk. (C) Microbiome bacterial order abundance of 8-wk-old C57BL/6J male mice subjected to topical ivermectin treatment (TOP) for 4 wk. (D) Microbiome bacterial order abundance of 8-wk-old C57BL/6J female mice subjected to topical ivermectin treatment (TOP) for 4 wk. Statistics: nonparametric Wilcoxon testing; n = 5 per group; *P < 0.05; ^†P < 0.01. Statistical symbols were omitted for Coriobacteriales because its low abundance was below the threshold for visualization on the graph.

Necropsy and histopathology.

No abnormal clinical conditions were identified throughout the experimental timeline. Gross necropsy and histopathologic examination revealed normal small intestine and colon at 4 weeks posttreatment in all groups. One female in the topical group had an absent right uterine horn and grossly enlarged right ovary on necropsy. The reproductive tract was normal histologically in this animal, which supports an incidental diagnosis of unilateral uterine horn aplasia.

Discussion

This study demonstrates that ivermectin administration affected the fecal microbiomes of both male and female mice, regardless of the treatment route, with females being more strongly affected. Alpha diversity, or species diversity within samples, was impacted in all 4 treatment groups as compared with controls. Both males and females in the diet groups and females treated with topical ivermectin had significantly decreased α diversity after treatment. These groups also experienced significant change to their species composition, as indicated by significant changes to β diversity as compared with control groups. While males treated with topical ivermectin had significantly increased species richness after treatment, the species composition was not significantly affected. Most of the changes found at the end of treatment persisted 4 weeks posttreatment; however, the histopathology of the small intestine and colon was normal at this time.

Because the gut microbiome serves many functions for the host, any variability can have extensive effects on intestinal research. For example, the gut microbiome has been shown to influence diseases such as inflammatory bowel disease,²⁵ celiac disease,²⁶ and colorectal cancer.²⁷ In addition, the gut microbiome can affect nonintestinal organs through different axes.²⁸ For example, the communications between the distinct microbial communities of the intestinal tract and the lungs, termed the gut-lung axis, may lead to changes to the gut microbiome affecting the microbiome of the lungs.²⁹ The gut microbiome therefore has been documented to play a role in asthma,³⁰ LPS-induced lung injury,³¹ and tuberculosis³² through this axis. This axis between the gut microbiome and other organs also exists for the brain, liver, kidneys, and pancreas,²⁸ so any research specific to these organ systems may also be affected by changes to the gut microbiome of their subjects.

It has been suggested that select food dyes may result in colitis and microbiome impacts in mice.^33,34 Compounded diets routinely used in research settings are commonly manufactured with dyes to help differentiate treated diets from standard diets.³⁵ This study was designed to emulate a practical example of prophylactic treatment of healthy mice during animal quarantine. Therefore, we did not include an additional diet group with the red food dye and lacking ivermectin, as our institution in practice would not provide an ivermectin-compounded diet without the food dye. We elected not to use additional animals to discern the potential differences between the ivermectin and the dye within the diet, but it is possible that the dye may have contributed to the microbiome changes seen in the diet groups. Similarly, there was no separate topical sham group (ie, 40% glycerol) as we elected to have the single control group serve both experimental groups, reducing the overall number of animals needed. Because stress associated with animal handling may affect the gut microbiome,^36,37 the topical application of ivermectin occurred while obtaining body weights so that all groups were handled equally.

The sexual dimorphic nature of the gut microbiome in mice is well documented in the literature^22,38–43 and replicated in this study. In our study, males and females were separated at 3 weeks of age at the vendor and remained apart. Therefore, they had diverging microbiomes for 5 weeks before the first fecal samples were collected. This sex difference is believed to be influenced by the enterohepatic circulation of sex steroids that begins at puberty.⁴³ In this study, males and females also showed different responses to ivermectin administration, with females more strongly impacted. Our results suggest a sex-specific effect of ivermectin on specific bacterial orders with Bacillales being affected in male mice and Coriobacteriales and Bacteriodales being affected in female mice; however, the specific mechanism driving this difference in this study is unknown. In addition, to our knowledge, this is the first study to report sex-specific differences in healthy mice of both sexes that received ivermectin.

An unexpected finding of this study were the significant changes in weight noted during treatment for 2 of the groups. Males treated topically with ivermectin showed significantly reduced body weight during treatment that resolved once ivermectin treatment was discontinued. A published toxicity study¹³ in C57BL/6NTac mice showed transient weight loss in animals treated with 48 ppm ivermectin, while other studies^4,44 found no significant effects on body weight. Females treated with the ivermectin diet showed significantly increased body weight during treatment and 2 weeks posttreatment. This difference was resolved by the end of the study. While it appears that there is a notable increase in body weight of the diet group at day 0, this difference was not statistically significant. The ivermectin-impregnated diet is the same base diet as fed to the control group, but the treated chow is firmer and more compact due to differences in the compounding and manufacturing processes. The texture difference may account for the differences seen in body weight between the diet and control groups in females, although this difference was not seen in males.

Animals were allowed to acclimate to the facility for 2 weeks before study initiation and to allow for the collection of pretreatment fecal samples for microbiome analysis. This was done to prevent any confounds from transport stress as well as changes in husbandry practices from vendor to institution. In both topical groups, there were significant findings noted in abundance of a few bacterial orders pretreatment, although there were no significant changes to α or β diversity. There may have been unknown environmental pressures that led to the shifts in bacterial order abundance prior to the first fecal collection in these 2 groups.

There were some limitations of this study. First, in practice, there is great variability in quarantine procedures, drug choices, and treatment routes and durations among institutions. While our study included 2 treatment routes and both sexes, evaluation was dependent on the drug of choice and duration of treatment. It remains unknown how different lengths of ivermectin treatment may affect the fecal microbiome, body weight, or gut histopathology. Due to animal numbers, we were unable to necropsy a cohort of animals at the end of treatment; therefore, it is unknown whether animals experienced any histopathologic changes to the small intestine or colon that may have resolved by 4 weeks posttreatment. Finally, we did not evaluate potential cytokine changes associated with changes to the fecal microbiome. In addition to direct immunomodulatory effects of ivermectin, it is possible that changes in the microbiome impact mucosal or systemic immunity. For example, one species found to be more abundant at the end of diet treatment in both males and females, Akkermansia muciniphila, has been shown to induce weak proinflammatory activity by stimulating production of IL-8 by enterocytes.⁴⁵ Future research is needed to determine if any immune system changes may be a direct effect of ivermectin administration to healthy animals.

Overall, this study suggests that, in healthy C57BL/6J mice, there is a lasting impact of ivermectin treatment on the fecal microbiome and transient impact on body weight. In conclusion, researchers should consider the possible impact of this drug on their data, while institutions must balance the need to protect their established colonies from exposure to parasitic infections.

Conflict of Interest

The authors have no conflicts of interest to declare.

Funding

This work was internally funded.