Animal Diseases, Applications of Metagenomics
KeywordsHuman Microbiome Project Cellulolytic Bacterium Irritable Bowel Disease Lactobacillus Amylovorus Antibiotic Growth Promoter
This entry describes current knowledge about the microbiome of the gastrointestinal tract of animals and its relationship to infectious diseases. It also describes how the microbiome changes during infections.
Rene Dubos’ pioneering work on microbial ecology led to the hypothesis that the microbes of mammals living in intimate contact with each other coevolved with animals (Dubos et al. 1965; Yolton and Savage 1976). Dubos stated, “It is to be expected, therefore, that anatomical structures and physiological needs have been determined in part by the microbiota (microbiome) which prevailed during evolutionary development, and that many manifestations of the body at any given time are influenced by the microbiota now present.” Thus, during the coevolution of the microflora and the host, a set of mutualistic or even symbiotic relationships developed between the host and microbes. This hypothesis is one of the forces driving work to understand the interactions between microbes and their mammalian hosts.
The term microbiome, which was coined by Joshua Lederberg, is used to describe the entire microbial content of an environment including bacteria, viruses, protozoa, and fungi. It has long been recognized that the microbiome of the mammalian gastrointestinal tract is important for the health and development of animals. It has been hypothesized that one way that the microbiome in the gastrointestinal tract contributes to the health of animals is by excluding pathogenic bacteria and viruses from the host by occupying physical space within the gastrointestinal tract and/or by producing inhibitory substances or by competing for nutrients that result in the inhibition of pathogenic microbes. The recognition that microbiomes of animals make important contributions to the health and well-being of animals and humans and the limited detail we have of the microbial populations in mammals led to the initiation of the human microbiome project. A similar animal microbiome project has not been initiated. Nevertheless, data is emerging from numerous laboratories describing the composition and functions of the animal microbiomes. The aims of the human microbiome project are tightly focused on obtaining baseline data to “characterize the microbial communities found at several different sites on the human body, including nasal passages, oral cavities, skin, gastrointestinal tract, and urogenital tract, and to analyze the role of these microbes play in human health and disease” (Peterson et al. 2009). In particular there is a specific goal to understand the relationships between changes in the composition of the microbiome and its bearing on health and disease. The same holds for animals.
Many studies have been performed that demonstrated important metabolic functions that microbiomes contribute. For example, the rumen of cattle is known to contain large numbers of cellulolytic bacteria that are essential for the breakdown of cellulose. This is essential for ruminants because mammals lack enzymes to degrade plant cellulose. Since cellulose is a major component of their diets, the absence of cellulolytic bacteria to assist in metabolism would result in the animal’s wasting of this abundant energy source. Bacteria are known to provide other metabolic activities for the host including the stimulation of water transport in the colon (stimulated by volatile fatty acids produced by bacteria) (Yolton and Savage 1976), recycling of bile salts (Shimada et al. 1969), production of vitamin K (Ramotar et al. 1984), and providing exogenous alkaline phosphatases (Yolton and Savage 1976). The gastrointestinal microflora also is an essential stimulus in the development of the animal’s immune system (Rakoff-Nahoum et al. 2004; Mazmanian et al. 2005). Work with germ-free animals has shown that the indigenous microflora stimulates the immune system by promoting the development and expansion of the lamina propria in the intestines (Savage 1977). However, fewer studies have focused on the role that microbiomes play in prevention or resistance to diseases in animals. Those studies that have been performed have mainly been based on studies pertinent to humans using rodent models and mainly relate to chronic diseases. For example, a recent study showed that the gut microbiota is responsible for the selective loss of invariant natural killer T cell (iNKT) subsets and that, in their absence, the host can become more prone to autoimmune diseases including colitis and asthma (Leslie 2012). In another study, the composition of the microbiome in mice has been closely correlated with obesity (Turnbaugh et al. 2006).
Microbiome Changes in Mice Due to Infectious Diseases
One of the important functions ascribed to the microbiome is resistance to infection. It has been suggested that the normal microbiota play important roles in excluding pathogens either by occupying space or by directly interfering with specific pathogens (Berg 1996). A well-known example of how the human gastrointestinal microbiome occupies space thereby preventing infections is related to diarrhea caused by Clostridium difficile. When the gastrointestinal microbiome is perturbed by therapeutic use of antibiotics, C. difficile, if already present in the gastrointestinal tract, can increase in concentration resulting in a severe diarrheal disease called pseudomembranous colitis. However, few examples of similar animal diseases are known. Those studies that have been performed mainly relate to human infectious diseases employing mouse models. For example, in one study by Reeves et al. (2011), the antibiotic cefoperazone was added to drinking water of mice. Mice were then challenged orally with C. difficile and followed the mice clinically and microbiologically to determine the composition of the gut microbiome. Mice that became clinically ill were colonized mainly by members of the phylum Proteobacteria, while mice that did not become ill or normal nonchallenged mice were mainly colonized by members of the phylum Firmicutes. In another mouse-based study, Schmitz et al. (2011) compared mice exposed to “altered Schaedler flora” to specific pathogen-free mice and their susceptibility to infection with Helicobacter felis. Mice that received the altered Schaedler flora became ill and subsequently were unable to clear H. felis, while the specific pathogen-free mice, while susceptible to disease, were able to clear the H. felis challenge strain. Correlated with this result was the observation that mice that cleared H. felis acquired several species of Lactobacillus in the stomach.
Microbiome Changes in Companion Animals Due to Infectious Diseases
Suchodolski and his colleagues have studied the microbiome of dogs and cats with particular reference to irritable bowel disease. Comparing the gastrointestinal tracts of both dogs and cats with IBD, they found increases in bacteria in the family Proteobacteria decreases in the phylum Firmicutes (Suchodolski 2011). In dogs with IBD when duodenal biopsy samples were available for analysis, they found that dogs with IBD were enriched for Pseudomonas, Acinetobacter, Conchiformibious, Achromobacter, Brucella, and Brevundimonas (Suchodolski et al. 2010).
Microbiome Changes in Livestock Animals Due to Infectious Diseases
Leser et al. (2000) compared the microbiomes of healthy pigs to those with infections with Brachyspira hyodysenteriae using T-RFLP analysis of 16S rRNA gene products. They found numerous changes and suggested that this was evidence that B. hyodysenteriae destabilized the microbiome. However, another interpretation is that a destabilized microbiome resulted in susceptibility to the infection. Isaacson et al. (2011) obtained preliminary data that demonstrated that experimental challenges of pigs with Salmonella Typhimurium or Lawsonia intracellularis or both caused specific and consistent changes in the colonic and cecal microbiome measured by sequencing of 16S rRNA genes. Furthermore, coinfection of pigs with both pathogens resulted in increased shedding of S. Typhimurium over time and at much higher concentrations. This could be the result of increased inflammation in the gastrointestinal tract caused by L. intracellularis allowing S. enterica to more readily colonize and proliferate in these sites.
Recently an analysis of the pig virome was undertaken using a metagenomic approach (i.e., sequencing of the total extracted DNA rather than just the 16S rRNA gene) (Shan et al. 2011). Feces from 24 healthy pigs and 12 pigs with diarrhea were examined. Viruses were collected by differential centrifugation followed by membrane filtration. Viral nucleic acids were extracted, and the total viral community nucleic acid was sequenced using high-throughput pyrosequencing. On average 4.2 different mammalian viruses were identified in the fecal samples of healthy pigs and 5.4 unique viruses in the pigs with diarrhea. Most of the viruses identified (99 %) were RNA viruses in the families Picornaviridae, Astroviridae, Coronaviridae, and Caliciviridae. The remaining viruses were DNA viruses in the families Circoviridae and Parvoviridae.
Studies of the effects of antibiotic growth promoters on the health and growth of livestock animals have been performed to determine how they alter the gastrointestinal microbiomes. Rettedal et al. (2009) measured the effects of chlortetracycline on the ileal microbiome. They found that chlortetracycline resulted in decreases in Lactobacillus johnsonii and Turicibacter and an increase in Lactobacillus amylovorus. Collier et al. (2003) compared the microbiomes of pig feces treated with the antibiotic tylosin in comparison to nontreated controls. They used denaturing gradient gel electrophoresis and made taxonomic assignments to specific electrophoretic bands by cutting them from the gels and directly sequencing them. They found a decrease in three species of Lactobacillus, one species of Streptococcus, and one species of Bacillus and an increase of Lactobacillus gasseri in response to tylosin. Looft et al. used a study design that employed six pigs: three pigs were treated with a combination of chlortetracycline, sulfamethazine, and penicillin and three served as untreated controls (Looft et al. 2012). Pigs were treated with antibiotics at 18 weeks of age and sampled at 18, 20, and 21 weeks of age. At 20 weeks of age (2 weeks of treatment), there were decreases in bacteria in the phylum Bacteroidetes. Specific changes were decreases in Anaerobacter, Barnesiella, Papillibacter, Sporacetigenium, and Sarcina. Members of the phylum Proteobacteria were increased.
The information presented in this article provides good evidence that the gastrointestinal microbiome is important in infectious diseases. The examples of perturbations to the microbiome result in infections (C. difficile and H. felis) are examples of this phenomenon. As well, there is sufficient data to demonstrate that other infections (S. enterica and Brachyspira) contribute to alterations in the gut microbiome. Whether these changes are directly involved in disease pathogenesis is not known but further investigations certainly will establish this relationship. Also there is mounting evidence that certain chronic diseases (obesity and some autoimmune diseases) also are mediated through and interaction with the gut microbiome. This observation suggests that many chronic diseases have links to microbial agents. Future research will likely establish better and more specific relationships between these chronic diseases and the microbiomes and are likely to uncover additional diseases with microbial contributions or etiologies.
- Isaacson R, Borewicz K, Kim HB, Vannucci F, Gebhart C, Singer R, Sreevatsan S, Johnson T. Lawsonia interacellularis increases Salmonella enterica levels in the intestines of pigs. Conference of Research Workers in Animal Diseases, 2011:103.Google Scholar