Potential for using a hermetically-sealed, positive-pressured isocage system for studies involving germ-free mice outside a flexible-film isolator

Abstract

Germ-free mice are used to examine questions about the role of the gut microbiota in development of diseases. Generally these animals are maintained in semi-rigid or flexible-film isolators to ensure their continued sterility or, if colonized with specific microbiota, to ensure that no new species are introduced. Here, we describe the use of a caging system in which individual cages are hermetically sealed and have their own filtered positive airflow. This isopositive caging system requires less space and reduces animal housing costs. By using strict sterile techniques, we kept mice germ-free in this caging system for 12 weeks. We also used this caging system and approach to conduct studies evaluating a) the stability of the microbiome in germ-free mice receiving a fecal transplant and b) the stability of dietary-induced microbiota changes in fecal-transplanted mice. As has been shown in fecal transfer studies in isolators, we found that the transferred microbiota stabilizes as early as 2 weeks post transfer although recipient microbiota did not completely recapitulate those of the donors. Interestingly, we also noted some sex effects in these studies indicating that the sex of recipients or donors may play a role in colonization of microbiota. However, a larger study will be needed to determine what role, if any, sex plays in colonization of microbiota. Based on our studies, an isopositive caging system may be utilized to test multiple donor samples for their effects on phenotypes of mice in both normal and disease states even with limited available space for housing.

Keywords: MSD, multi-dimensional scaling; OTU, operational taxanomic unit; SOP, standard operating procedure.; fecal transfer; germ-free; isolator; isopositive cage; method; microbiota.

Figure 1.

Procedure for working aseptically with isopositive cages. To minimize the risk of contamination when performing procedures on germ-free or colonized mice housed in isopositive cages, personnel donned sterile gowns and gloves, and cages were disinfected with Clidox solution (1:3:1). (A) The sterile person donning sterile surgical gown and gloves. (B) The sterile person working within the biosafety cabinet following aseptic technique. Only autoclaved or Clidox disinfected materials are introduced into the biosafety cabinet and handled by the sterile person. (C) Dunk tank and rubber gloves used by the assistant to completely submerge isopositive cages in Clidox. (D) Assistant submerging the hermetically sealed isopositive cage into 1:3:1 Clidox prior to placing the cage into a previously disinfected biosafety cabinet.

Figure 2.

PCR analysis of 16s rRNA from baseline fecal samples. Prior to fecal transfer, pooled fecal samples were collected from each isopositive cage and tested for the presence of bacteria by 16s rRNA PCR. (A) Samples from the first study testing fecal transfer stability. (B) Samples from the second study using samples from donors fed a control or high vitamin D diet. Each lane represents a pooled sample from each isopositive cage; H₂O, negative control; SPF cont, DNA from feces of animals housed in the SPF facility.

Figure 3.

Fecal transfer results in smaller ceca and shorter small intestines. Germ-free mice were gavaged with a fecal slurry generated from mice housed in an SPF facility. Four weeks following fecal transfer, mice were euthanized and phenotypic changes in the gastrointestinal tract were assessed. (A) Fecal transfer to germ-free mice (left) results in smaller ceca compared to mice that remained germ-free (right). (B) Fecal transfer shortened the length of the small intestine. Intestine of a germ-free mouse following fecal transfer (left); intestine of a germ free mouse (right). Arrows point to cecum.

Figure 4.

Heat map of major OTU relative abundances in the fecal transplant experiment. The Ln of the normalized reads per OTU is shown. The hierarchical clustering was performed using average linkage of samples and OTUs based on Bray–Curtis dissimilarity index of relative abundance profiles. The sample names indicate cage number (1, 2, 3), ear tag (R for right, L for left, N for none), sex (M, F), and week (1, 2, 4) preceded by an underscore. The samples cluster almost entirely by sex and then time.

Figure 5.

(A) Multidimensional scaling of OTU abundances from the fecal transplant experiment. The donor sample is shown as a black dot (upper right). Males are shown in cyan and females in magenta. The inner circle indicates time post fecal transplant in weeks as white, gray and black for one, 2 and 4 weeks, respectively. After the initial transplant, the samples diverge from the donor at week one and become more donor-like as time progresses. Males and females appear to separate into distinct groups. (B) Community complexity for fecal transplant experiment by Shannon index. The donor sample is shown at week 0. There is no significant difference by sex.

Figure 6.

Heat map of major OTU relative abundances in the fecal transplant experiment using 2 different donor samples from mice fed a control and a high vitamin D diet. The Ln of the normalized reads per OTU is shown. The hierarchical clustering was performed using average linkage of samples and OTUs based on Bray–Curtis dissimilarity index of relative abundance profiles. The sample names indicate diet of donors (C for control, D for high vitamin D), cage number (1, 2, 3), ear tag (R for right, L for left, N for none), sex (M, F), and week (1, 2, 4) preceded by an underscore. There is a strong tendency for the week 2 and 4 samples to cluster by diet.

Figure 7.

(A) Multidimensional scaling of OTU abundances from the fecal transplant experiment with donors fed a control and high vitamin D diet. The single black data points (lower right corner) represent the 2 donor samples for the control and high vitamin D diet. Triangles represent high vitamin D and circles represent control diet. The cyan and magenta indicate sex (male and female, respectively) while the inner color indicates time post fecal transplant in weeks as white, gray and black for one, 2 and 4 weeks, respectively. As with the initial fecal transplant study, the samples diverge from the donors rapidly but begin to become more donor-like by week 4. There is a strong division between the 2 diets in post-transplant. (B) Community complexity for fecal transplant with donors fed control and high vitamin-D diets. The donor samples for each diet are shown at week 0. There is no significant difference by sex, however, the microbiota from high vitamin-D diet appears to establish a more diverse bacterial community.