Germ-Free Animals

Germ-free, or GF, animals are laboratory animals that completely lack microbes, making them useful tools for microbiome research. Researchers create GF animals in laboratories by delivering the newborn animals in a way that protects them from microbes, which are microscopic organisms such as bacteria and viruses. They then house the GF animals in sterile conditions to ensure that the animals stay germ free. The creation of GF animals began in the late nineteenth century. Prior to that, scientists had no way to study the effects of the microbiome on overall health. The creation of GF animals allowed researchers to examine the microbiome under controlled conditions. They could colonize the animal with specific microbes and study their effects on the animal’s health without the confounding presence of other microbes. Researchers have used GF animals as a living model to study the microbiome, which has provided evidence for a relationship between the microbiome and health, including a role for the microbiome in shaping the development of multiple body systems.

Researchers use GF animals to study the microbiome and its effects on health. The microbiome is a community of microbes living on and in the human body. Those microbes often colonize mucosal membranes, which are surfaces in the body that line organs and cavities, such as the gastrointestinal tract, respiratory tract, and reproductive tract. Microbes can be harmful or commensal, which means that they do not hurt the host, though most microbial colonization is commensal. Daily environmental factors, such as diet and germ exposure, contribute to the microbiome composition, making each individual’s microbiome unique. The diversity of microbial colonization during infancy affects development of multiple body systems, such as the immune system and nervous system. Without a microbiome, humans would not develop into adulthood normally. Purposely creating GF humans would therefore be unethical.

Research to develop GF animals began in the late nineteenth century. In 1885, Louis Pasteur, who studied chemistry and microbiology in France, was one of the first to conceptualize the idea of animals living without microorganisms. However, he also noted that their development would likely be impossible due to the vital role microbes play in the host’s health. Then in 1895, George H. F. Nuttall and Hans Thierfelder, who at the time conducted research together in Germany, disproved Pasteur’s theory and created the first GF animals. The pair carried out experiments to determine whether having a microbiome was necessary for normal life. They delivered guinea pigs via sterile Cesarean section, which is a procedure where a surgeon cuts into the mother’s abdomen to deliver the neonate. Delivery by Cesarean section ensured that the neonates did not encounter the mother’s vaginal microbes as they would have during vaginal delivery. Nuttall and Thierfelder sterilized a glass chamber using high temperature steam to kill all bacteria, and then raised the guinea pigs in the sterile chamber. However, since the researchers stored the sterile food for the germ-free animal in the chamber, when the food ran out, the researchers moved the animals out, and the animals were no longer GF animals.

In 1915, Ernst Küster, a professor and surgeon who worked in Germany, designed a sterile chamber where he housed a GF goat for thirty-four days. For sterile food delivery, Küster devised a smaller compartment that led into the main compartment. Materials were sterilized with steam in the small compartment before being passed into the main compartment, so they did not contain any bacteria. Even after Küster found a way to feed GF animals without exposing them to bacteria, the goat only lived for thirty-four days. Researchers attempted similar experiments but could ensure that their animals remained germ-free for only a short period of time.

In 1932, James Reyniers, who taught bacteriology at the University of Notre Dame, in Notre Dame, Indiana, and his colleagues generated one of the first colonies of GF animals. Reyniers’s group researched microbes and therefore investigated creating GF animals so that they could introduce a single microbe species in the absence of all other potentially confounding microbes. The isolator they used to house the GF animals was similar to the one Küster designed. The isolator was a stainless-steel drum that could undergo steam sterilization and could also be attached to other isolators. The apparatus also included long rubber gloves to handle the animals and materials without introducing bacteria. In the laboratory of the Notre Dame group, a pair of GF rats mated inside the sterilized chamber, which was one of the first times a GF animal reproduced and gave birth to GF newborns. That occurrence demonstrated that GF animals could mate, though not as readily as animals with microbes.

Researchers used stainless steel chambers to house GF animals until 1957, when the researchers at Notre Dame conducting germ-free research developed a chamber with walls that consisted of transparent, flexible vinyl film. Switching to flexible film allowed researchers to introduce food and other materials more conveniently into the isolator. In previous steel chambers, researchers had to wait for the steam to sterilize the chamber before adding materials. The new chambers allowed researchers to use heat to quickly seal packages containing materials like food into the flexible-film chamber and then use a hot wire inside the chamber to cut into the package and release the contents. Researchers at that time also began using peracetic acid, which is an acid that kills most bacteria within minutes, for sterilization, as peracetic acid could sterilize surfaces more quickly than steam.

During the mid-1900s, researchers in the farming industry also began using GF animals to aid in livestock production. Initially, they found that administering antibiotics to farm animals grown for human consumption aided in physical growth, while also reducing the amount of food the animals needed. That growth effect was due to the antibiotic’s killing of intestinal bacteria that hindered growth. However, at the time, researchers’ use of antibiotics in agriculture incited public debate because many people feared that the antibiotics would cause bacteria to become resistant to it and could ultimately harm humans. Therefore, using animals raised in completely sterile conditions to ensure the absence of the growth inhibiting bacteria was an alternative option for creating farm animals that had increased physical growth.

In the 1970s, researchers also began to extend the sterile isolators used to house GF animals towards applications in humans. In 1971, a boy named David Vetter in Houston, Texas, was born with severe combined immune deficiency, which is a genetic disorder that affects the immune system, making the body highly susceptible to infection, and exposure to microbes unsafe. Because his sister was not a match for a bone marrow transplant, which is a treatment that could have replaced Vetter’s dysfunctional immune cells with healthy cells, instead researchers helped create a sterile, germ-free environment for him to grow up in. From birth, Vetter stayed in a sterile isolator, and NASA even provided him with a custom-built space suit so he could potentially travel around outside of his bubble, without encountering microbes. Ultimately, he did receive a bone marrow transplant but died in 1984. Vetter became known as Houston’s bubble boy, and was one of the first GF humans to grow up in a GF environment.

Researchers in 2023 typically follow a general protocol to create GF animals, but the details of the protocol differ across species. The general protocol usually involves delivering GF animals in a sterile manner, raising them in a sterile environment, and feeding them a sterile diet. However, because animals can have different ways of producing offspring, the methods of generating the GF animal differ slightly. For example, chickens lay eggs and thus procedures such as Cesarean sections are more complex and uncommon for them. To generate GF chickens, researchers usually collect eggs and sterilize them using a solution of peracetic acid. The eggs then stay in a sterile hatching isolator that maintains a warm temperature optimal for hatching. Researchers use an egg candler, which is a light that allows for visualization of the inside of the egg, to verify embryo development in fertilized eggs. Those chicks then hatch in a separate sterile hatching isolators. One day after the chicks hatch, researchers confirm that they are germ free by analyzing stool samples and seeing if any bacterial cultures arise from them. The GF chicks grow up in sterile isolators and consume sterile food and water until the end of the experiment.

Unlike for chickens, when generating GF mice, researchers usually utilize sterile Cesarean section. Sterile Cesarean section typically entails removing the uterus of the animal which contains the fetus, which is then placed in a sterile isolator where the fetus is delivered.  Researcher often use mice for GF research due to their biological similarity to humans, as well as ease in handling. Humans and mice share approximately ninety-nine percent similarity in their genes, and most of their internal organ systems also operate similarly. As the primary method to create GF mice, the sterile Cesarean delivery method has generally remained the same since the late nineteenth century. However, as of 2023, techniques to create GF animals have become more cost-effective with improvements in technology such as isolators and feeding systems.

Another method that researchers have devised to create GF mice is through embryo transfer. In 1999, Masanori Okamoto and Tsuneya Matsumoto, who both conducted research in Japan, published a paper that detailed the embryo transfer method. In the paper, they note that sterile Cesarean section presented challenges such as improper delivery timing. As an alternative, they propose the embryo transfer method, which involves harvesting embryos from female mice and then aseptically transferring them to the uterus of a GF female. Because the GF female does not possess any vaginal microbes, the birth would then be sterile. The researchers state the embryo transfer method was effective as the researchers did not have to intervene in the birthing process, though it required a GF animal to already be present to carry the fetus to term.

Researchers use GF animals to study health effects when the animal either does not have any microbes or possesses a known set of microbes. In the latter case, researchers introduce specific microbes they are interested in studying into the GF animal. The microbes introduced may either consist of a single strain or a mixture of different microbes. Using GF animals to study the microbiome allows researchers to look at the colonization effect of the specific, known set of microbes they introduce without worrying about the impact of other microbes. Additionally, researchers can use GF animals to examine the role of the microbiome at different time points in development. Colonizing the GF animal at various critical stages of development allows for research on the temporal relationship between microbial colonization and development.

However, one limitation of GF animals is that their germ-free nature begins from birth. Therefore, they cannot model certain cases of microbiome status, such as when the microbiome composition becomes altered later in life. For example, such alterations include infections by harmful bacteria that cause irregular changes in the microbiome composition. An alternative that researchers use in those cases is antibiotic treatment, which kills specific microbes in the animal. Using broad-spectrum antibiotics, which act on most bacterial groups, can effectively deplete an animal’s microbiome. Researchers can administer antibiotics to the animal at any time, which allows for flexibility in experimental design. A pitfall of antibiotic treatment, however, is the possibility that the drug may not kill the microbes consistently, as well as possibly create harmful side effects.

Researchers have specifically used GF animals to establish the effect of the microbiome in immune system responses. In a 2010 study, Siegfried Hapfelmeier and colleagues, a group of researchers from Switzerland, Canada, and the US, demonstrated the link between microbial colonization and immune response in the gut. They looked at B cells, which are a type of immune cell that produces antibodies. Antibodies are molecules that recognize and destroy foreign proteins. In the gut, certain B cells produce specific antibodies called IgA that target microbes on mucosal surfaces. Hapfelmeier introduced microbes into GF animals and found a dose-dependent relationship where higher loads of bacterial colonization led to higher levels of IgA in response to the introduced bacteria. Therefore, using GF animals as an experimental model, Hapfelmeier concluded that there is a link between gut microbiome composition and B cell production of IgA.

The use of GF animals also contributed to confirming the presence of the gut-brain axis, which is the connection between the nervous system and the gut. A 2010 study by Karen-Anne M. Neufeld and colleagues, a group of researchers from McMaster University in Ontario, Canada, studying medicine, psychiatry, and neuroscience, found that GF mice presented with reduced anxiety when compared to mice with some form of a microbiome. The researchers observed brain-derived neurotrophic factor, or BDNF, a molecule in the brain that promotes the survival of nerve cells and influences stress-related behaviors. The authors observed that the absence of microbes in GF mice correlated with high levels of BDNF in certain areas of the brain, indicating that the lack of microbiome had some sort of impact on the reduced anxiety in the GF mice. Heather Hulme and colleagues, a group of researchers from the United Kingdom primarily studying immunology, published an article in 2022 examining the use of mass spectrometry imaging, or MSI, which can picture the distribution of molecules, to track molecular changes in relation to the gut-brain axis. The researchers used GF mice and examined the molecular changes through MSI and promoted further research on gut-brain axis using MSI.

The development of GF animals in microbiome research has allowed for more experimental possibilities when studying the microbiome. Studies that utilize GF animals contributed to establishing the link between the microbiome and the development of various body systems such as the immune system and nervous system. The technology that GF animal creation employs also applies to other areas such as industrial farming and even human treatments.