C. parvum in germ-free mice

Summary The effect of previous sensitization to C. parvum, by cross-reacting antigens from other bacteria, on the immunostimulatory effects of C. parvum treatment were studied in germ-free and conventional mice. It was found that the development of splenomegaly and specific delayed hypersensitivity following C. parvum injection were similar in both germ-free mice and conventional mice.Supported by U.S. Public Health Service Grant No. 5S07 RR05705 and NIH Grant no. AM 18530Visiting Investigator. Present address: Department of Experimental Immunobiology, The wellcome Research Laboratories Beckenham, Kent, England.Recipient of a post-doctoral fellowship from the National Foundation for Ileitis and Colitis Inc.Recipient of Research Career Development Award No. AM 0073 from the National Institute of Arthritis, Metabolism and Digestive Diseases     

Decreased cellular immune response of germ-free mice.

Cellular immune response to intracerebral lymphocytic choriomeningitis infection was studied in mice belonging to an identical strain but different in breeding conditions. In consequence of the cellular immune reaction on the leptomeninx, lymphocytic choriomeningitis developed and caused death in 100% of conventionally bred mice, whereas 80% of germ-free and 15% of mouse-pathogen-free mice failed to display lymphocytic infiltration of the leptomeninx and survived the infection as chronic virus carriers. This finding pointed to a deficient cellular immune response of germ-free and mouse-pathogen-free mice. The under-development of the lymphoid system due to the antigen-poor breeding conditions might be responsible for the deficiency.     

The use of laminar flow for obtaining germ-free mice.

Hysterectomy in a laminar-flow cabinet affords greater economy of time and effort in obtaining germ-free mice than other methods. No contamination has occurred during the surgical procedure or in the subsequent transfer of the neonates into germ-free isolators.     

Use of germ-free mice in experimental transplantation

A comparative study of skin allografts was performed in germ-free A/Ola mice kept in boxes and in A/Ola and BALB mice raised in ordinary conditions. Skin graft (of C57B1/6 mice) in A/Ola and BALB mice raised in ordinary conditions was shown to reject 16-21 and 12-18 days after the transplantation, respectively without cyclophosphamide (CP) use. CP application in BALB mice, grown in ordinary conditions, prolonged the lifespan of grafts to 12-29 days. The use of CP in germ-free A/Ola mice prolonged the lifespan of grafts to 19-39 days. In germ-free mice kept in boxes the use of an immunodepressant was not accompanied by infectious complications, while the animals kept in the vivarium often died of infectious diseases.     

Detection of viruslike particles in germ-free mice

Kajima, Masahiro (University of Notre Dame, Notre Dame, Ind.), and Morris Pollard. Detection of viruslike particles in germ-free mice. J. Bacteriol. 90:1448-1454. 1965.-Viruslike particles similar in structure to the mouse leukemia agent were detected by electron microscopy in thymus tissues of germ-free AK mice which had developed spontaneous leukemia. In addition, unique viruslike particles (type B) were detected in tissues from spontaneous mammary adenocarcinoma of germ-free C3H mice. Leukemia virus-like particles were also observed in the thymuses of the control AK mice and of the C3H control mice as well as of those with mammary tumors. Germ-free mice are not virus-free. The routes of transmission of leukemia as well as of mammary tumor viruses may be "vertical," through the embryo or placenta.     

Dextran sodium sulfate-induced colitis in germ-free IQI/Jic mice.

This study presents a histological examination of dextran sodium sulfate (DSS)-induced colitis in germ-free (GF) mice. A comparison of the pathology between GF and conventionalized mice (CVz) was made to determine the role that intestinal microflora play in DSS-induced colitis. To induce colitis, GF and CVz IQI/Jic mice were given either 5% or 1% DSS orally. Administration of 5% DSS, a common concentration used to induce colitis in mice, caused gross rectal bleeding and a marked decrease in hematocrit as early as day one in GF mice. These mice died on day three due to massive bleeding into the intestinal lumen. In contrast, CVz mice did not die during the seven-day experimental period. Histopathological examination three days after administration of 5% DSS did not reveal any colitis lesions in GF mice, but CVz mice had developed moderate colitis in the large intestine. Administration of a low concentration of DSS (1%), which only induces mild basal crypt loss in CVz mice, caused severe colitis in the distal colon in GF mice, and they died on day 14. These data suggest that intestinal microflora are not necessary for the induction of colitis. Furthermore, DSS may be highly toxic to GF mice, and when given at a concentration of 5% it causes massive bleeding into the intestinal lumen resulting in death prior to development of colitis.     

Alterations of brain endocannabinoidome signaling in germ-free mice

We investigated the hypothesis that the endocannabinoidome (eCBome), an extension of the endocannabinoid (eCB) signaling system with important functions in the CNS, may play a role in the microbiota-gut-brain axis. Using LC-MS/MS and qPCR arrays we profiled the brain eCBome of juvenile (4 weeks) and adult (13 weeks) male and female germ-free (GF) mice, which are raised in sterile conditions and virtually devoid of microbiota, present neurophysiological deficits, and were found recently to exhibit a strongly altered gut eCBome in comparison to conventionally raised age/sex-matched controls. The causal effect of the gut microbiome on the eCBome was investigated through the re-introduction into adult male GF mice of a functional gut microbiota by fecal microbiota transfer (FMT). The concentrations of the eCB, 2-arachidonoylglycerol (2-AG), and its 2-monoacylglycerol congeners, were significantly reduced in the brain, but not in the hypothalamus, of both juvenile and adult male and adult female GF mice. FMT rendered these decreases non-statistically significant. The eCB, anandamide (AEA), and its congener N-acylethanolamines (NAEs), were instead increased in the brain of adult female GF mice. Saturated fatty acid-containing NAEs were decreased in adult male GF mouse hypothalamus in a manner not reversed by FMT. Only few changes were observed in the expression of eCBome enzymes and receptors. Our data open the possibility that altered eCBome signaling may underlie some of the brain dysfunctions typical of GF mice.     

An improved procedure for obtaining germ-free laboratory mice

The economy of time and effort accruing from the use of laminar air-flow cabinets to obtain germ-free mice has already been established. However, a further modification of the technique, utilizing small intermediate isolators, has made it possible to transfer to the large, recipient isolators only those litters which are viable and demonstrably free from contamination.     

Monocontamination of germ-free mice by a fastidious unidentified anaerobe

Over a period of several months, 5 germ-free mouse isolators became monocontaminated with seemingly the same fastidious, anaerobic bacilli which defied identification. The organism originated in rodent diet which had been exposed to gamma-irradiation. However, it was discovered retrospectively that the irradiation doses had not been monitored, and subsequent monitoring showed the desired dose of 5 Mrad was not achieved in some parts of the load. Although inadequate sterilization was the likely cause of contamination it was not possible to explain why only this fastidious anaerobe survived the treatment, and not other more common bacterial contaminants.     

beta-Aspartylglycine, a substance unique to caecal contents of germ-free and antibiotic-treated mice.

The caecal supernatants from germ-free, antibiotic-treated and control mice were compared with respect to their content of low-molecular-weight substances (less than 3500 mol. wt.). The supernatants contained about the same amount of free amino acids. After acid hydrolysis, the caecal supernatants of germ-free and antibiotic-treated mice showed a 2.9-fold increase in free amino acids, whereas a similar treatment of the supernatant from control mice resulted in a 2.6-fold increase. By gel filtration on Sephadex G-25, and high-voltage paper electrophoresis at pH 3.5 of the fractions eluted after the void volume, it was found that the caecal supernatants of germ-free and antibiotic-treated mice contained a substance more acidic than aspartic acid. Preparative high-voltage electrophoresis, dansylation, amino acid analysis and a specific colour reaction showed the substance to be beta-aspartylglycine. After a minimal 36 h of treatment with neomycin and bacitracin, a high concentration of beta-aspartylglycine was found, and no enterococci and aerobic Gram-negative rods could be cultured from the caecal contents. The possibility that in one mouse the appearance of beta-aspartylglycine was related to a decrease in Gram-negative rods was ruled out by selective elimination of aerobic Gram-negative rods by using polymyxin B. This suggests that other bacteria concomitantly eliminated with the enterococci and aerobic Gram-negative rods, directly or indirectly, could play a role in the accumulation of beta-aspartylglycine.