Use Of Germ-free Animals Research Articles

Comparative Efficacy of Two Types of Antibiotic Mixtures in Gut Flora Depletion in Female C57BL/6 Mice.

Over the last decade, interest in the role of the microbiome in health and disease has increased. The use of germ-free animals and depletion of the microbial flora using antimicrobials are 2 methods commonly used to study the microbiome in laboratory mice. Germ-free mice are born, raised, and studied in isolators in the absence of any known microbes; however, the equipment, supplies, and training required for the use of these mice can be costly and time-consuming. The use of antibiotics to decrease the microbial flora does not require special equipment, can be used for any mouse strain, and is relatively inexpensive; however, mice treated in this manner still retain microbes and they do not live in a germ-free environment. One commonly used antibiotic cocktail regimen uses ampicillin, neomycin, metronidazole, and vancomycin in the drinking water for 2 to 4 wk. We found that the palatability of this mixture is low, resulting in weight loss and leading to removal of mice from the study. The addition of sucralose to the medicated water and making wet food (mash) with the medicated water improved intake; however, the low palatability still resulted in a high number of mice requiring removal. The current study evaluated a new combination of antibiotics designed to reduce the gut microbiota while maintaining body weights. C57BL/6NCrl mice were placed on one of the following drinking water regimens: ampicillin/neomycin/metronidazole/vancomycin water (n = 16), enrofloxacin/ampicillin water ( n = 12), or standard reverse osmosis deionized water (RODI) ( n = 11). During an 8 day regimen, mice were weighed and water consumption was measured. Feces were collected before and after 8 d of treatment. Quantitative real-time PCR (real-time qPCR) for 16S bacterial ribosome was performed on each sample, and values were compared among groups. The combination of enrofloxacin and ampicillin improved water intake, together with a greater reduction in gut flora.

Gut microbes contribute to variation in solid organ transplant outcomes in mice

BackgroundSolid organ transplant recipients show heterogeneity in the occurrence and timing of acute rejection episodes. Understanding the factors responsible for such variability in patient outcomes may lead to improved diagnostic and therapeutic approaches. Rejection kinetics of transplanted organs mainly depends on the extent of genetic disparities between donor and recipient, but a role for environmental factors is emerging. We have recently shown that major alterations of the microbiota following broad-spectrum antibiotics, or use of germ-free animals, promoted longer skin graft survival in mice. Here, we tested whether spontaneous differences in microbial colonization between genetically similar individuals can contribute to variability in graft rejection kinetics.ResultsWe compared rejection kinetics of minor mismatched skin grafts in C57BL/6 mice from Jackson Laboratory (Jax) and Taconic Farms (Tac), genetically similar animals colonized by different commensal microbes. Female Tac mice rejected skin grafts from vendor-matched males more quickly than Jax mice. We observed prolonged graft survival in Tac mice when they were exposed to Jax mice microbiome through co-housing or fecal microbiota transplantation (FMT) by gastric gavage. In contrast, exposure to Tac mice did not change graft rejection kinetics in Jax mice, suggesting a dominant suppressive effect of Jax microbiota. High-throughput sequencing of 16S rRNA gene amplicons from Jax and Tac mice fecal samples confirmed a convergence of microbiota composition after cohousing or fecal transfer. Our analysis of amplicon data associated members of a single bacterial genus, Alistipes, with prolonged graft survival. Consistent with this finding, members of the genus Alistipes were absent in a separate Tac cohort, in which fecal transfer from Jax mice failed to prolong graft survival.ConclusionsThese results demonstrate that differences in resident microbiome in healthy individuals may translate into distinct kinetics of graft rejection, and contribute to interpersonal variability in graft outcomes. The association between Alistipes and prolonged skin graft survival in mice suggests that members of this genus might affect host physiology, including at sites distal to the gastrointestinal tract. Overall, these findings allude to a potential therapeutic role for specific gut microbes to promote graft survival through the administration of probiotics, or FMT.

Gut Microbiome and Obesity. How to Prove Causality?

In recent years, the gut microbiota (the microorganisms that live in our digestive tract) has become an area of great interest. Indeed, this intestinal microbial community performs essential functions in maintaining our health, and has been proven to influence host physiology and metabolism. Thereby, dysregulation of this gut microbiota may be implicated in the development of various diseases, including obesity. However, studies rarely assess causality, which requires the use of germ-free animals and microbiota transplant. Using these strategies, some gut microbiota were shown to confer obesity and associated metabolic disorders to mice, suggesting a causative link between gut bacteria and metabolic diseases.

Indigenous microbiota and Leishmaniasis.

Animals are colonized by their indigenous microbiota from the early days of life. The estimated number of associated bacterial cells in humans is around of 10(14) per individual, most of them in the gut. Several studies have investigated the microbiota-host relationship, and the use of germfree animals has been an important tool in these studies. These animals, when infected with a pathogen, have shown to be sometimes more resistant and other times more susceptible than conventional animals. Leishmaniasis is a worldwide public health problem and presents a spectrum of clinical manifestations. However, very few studies have addressed the role of the indigenous microbiota on the outcome of this disease. In this review, we will highlight and discuss the data available on the ways by which the microbiota can influence the outcome of the disease in murine experimental models of cutaneous infection with Leishmania.

Gut microbiota and obesity.

The human intestine harbors a complex bacterial community called the gut microbiota. This microbiota is specific to each individual despite the existence of several bacterial species shared by the majority of adults. The influence of the gut microbiota in human health and disease has been revealed in the recent years. Particularly, the use of germ-free animals and microbiota transplant showed that the gut microbiota may play a causal role in the development of obesity and associated metabolic disorders, and lead to identification of several mechanisms. In humans, differences in microbiota composition, functional genes and metabolic activities are observed between obese and lean individuals suggesting a contribution of the gut microbiota to these phenotypes. Finally, the evidence linking gut bacteria to host metabolism could allow the development of new therapeutic strategies based on gut microbiota modulation to treat or prevent obesity.

Control of commensal microbiota by the adaptive immune system

The symbiotic relationship between the mammalian host and gut microbes has fascinated many researchers in recent years. Use of germ-free animals has contributed to our understanding of how commensal microbes affect the host. Immunodeficiency animals lacking specific components of the mammalian immune system, on the other hand, enable studying of the reciprocal function—how the host controls which microbes to allow for symbiosis. Here we review the recent advances and discuss our perspectives of how to better understand the latter, with an emphasis on the effects of adaptive immunity on the composition and diversity of gut commensal bacteria.

Irritable bowel syndrome: a microbiome-gut-brain axis disorder?

Irritable bowel syndrome (IBS) is an extremely prevalent but poorly understood gastrointestinal disorder. Consequently, there are no clear diagnostic markers to help diagnose the disorder and treatment options are limited to management of the symptoms. The concept of a dysregulated gut-brain axis has been adopted as a suitable model for the disorder. The gut microbiome may play an important role in the onset and exacerbation of symptoms in the disorder and has been extensively studied in this context. Although a causal role cannot yet be inferred from the clinical studies which have attempted to characterise the gut microbiota in IBS, they do confirm alterations in both community stability and diversity. Moreover, it has been reliably demonstrated that manipulation of the microbiota can influence the key symptoms, including abdominal pain and bowel habit, and other prominent features of IBS. A variety of strategies have been taken to study these interactions, including probiotics, antibiotics, faecal transplantations and the use of germ-free animals. There are clear mechanisms through which the microbiota can produce these effects, both humoral and neural. Taken together, these findings firmly establish the microbiota as a critical node in the gut-brain axis and one which is amenable to therapeutic interventions.

Of Microbes and Cancer

Recently, due to the use of germ-free animals in cancer research, many facts have been accumulated indicating that bacteria may produce different effects (enhancing or inhibiting) on carcinogenesis, depending on the features of the carcinogenic substance metabolism. At the same time, epidemiological investigations have shown that in human populations with a high or a low risk of cancer development (mostly cancer of the colon and mammary gland) the composition of the intestinal bacterial flora and its metabolic properties have peculiar features. In both instances, certain bacterial species are presumed to take part in the formation of final carcinogenic metabolites or in the synthesis of endogenic carcinogens (and/or cocarcinogens).

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion ( t 1 2 ) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t 1 2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t 1 2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [ 14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.

Gnotobiotic animals in research: their uses and limitations.

The gnotobiotic laboratory animal is potentially a very valuable tool for investigating any suspected interaction between the host and its associated microflora or between different components of that flora. However, like many other good ideas, the production of gnotobiotes is simple in concept but complicated in execution. In the early stages the greatest obstacles to the general use of germ-free animals were the expense and the restricted amount of space that could be maintained free from contaminants. Nowadays, with isolators made of plastic film and improved methods of chemical sterilization, gnotobiotic equipment has become available, even for large animals, at a relatively modest price. Further, colonies of germ-free rats and mice have been established in a number of breeding centres and their offspring can be easily transported to the research laboratory. Already the use of germ-free animals has contributed much to our knowledge of the host-microbe relationship but it has also focused attention on their limitations and on some characteristics that must be taken into account when interpreting the results of experiments with gnotobiotes.

Experimental induction of caries by the use of streptococcal strain isolated from the carious dentin.

Since 1954, ORLAND et al. [1, 2] have carried out a series of experiments by the use of germ-free animals [3] and reported the facts that the caries in a rodent family was bacterially caused and that enterococcus was one of the bacterial strains responsible. In their wake, FITZGERALD et al. succeeded in inducing the caries by a slightly aerobic and non-proteolytic streptococcal strain which was isolated from the agnotobiotic rat [3].However, both ORLAND and FITZGERALD were cognizant of the fact that this animal existed in somewhat unnatural conditions.In 1960, therefore, FITZGERALD et al. [4] removed 5 streptococcal strains from the carious cavity of hamsters fed on a carious-inducing diet and, by planting the strains either singly or in combination in caries-inactive rats, they were successful in this experimental induction of the caries. At the same time, they inoculated lactobacillus and diphtheroid bacillus which were isolated from the carious cavity singly into caries-free animals. However, this attempt proved a failure.The authors had isolated about 330 streptococcal strains from the oral cavities or carious dentin of Wister rats and of them, they selected Str. bovis which was consiered to be closely linked to the cariogenecity.Interestingly enough, there was found none of these strains that corresponded to 5 streptococcal strains used by FITZGERALD or to those which ORLAND and FITZGERALD employed to induce successfully the caries in germ-free animals.On the strength of this fact, the authors were inclined to think that streptococcus responsible for the caries in a rodent was not of a single kind but rather there must be streptococcal strains responsible for the caries which were proper to the species of animals and their growth environment.The present experimental study was conducted so as to substantiate this inference above.

The use of germfree animals and techniques in the search for unknown etiological agents.

The use of germfree animals and techniques in the search for unknown etiological agents.

Flexible Film Apparatus for the Rearing and Use of Germfree Animals

Flexible Film Apparatus for the Rearing and Use of Germfree Animals