GI flora: Understanding the role of probiotics in veterinary medicine (Sponsored by Nestle Purina)

The mammalian intestinal tract contains a complex, dynamic, and diverse society of pathogenic and nonpathogenic bacteria. Researchers have estimated that the human body contains 1 014 cells—only 1 0 percent of which are not bacteria and belong to the human body proper.1 A large body of research has focused on the mechanisms by which pathogenic bacteria influence intestinal function and induce disease; however, recent attention has been focused on the indigenous nonpathogenic microorganisms and how they may benefit the host. Initial colonization of the sterile newborn intestine occurs with maternal vaginal and fecal bacterial flora. The first colonizers can include species of enterobacteria, streptococci, and staphylococci. These bacteria metabolize oxygen and favor the growth of anaerobic bacteria, including lactobacilli and bifi-dobacteria. This overview will introduce the history of probiotics, their safety and regulatory background, provide information on their mechanisms of action, and introduce possible ways probiotics may be used to treat clinical diseases.

History of probiotic health claims

Documentation of the healthy consumption of bacteria in food dates back to the Old Testament (Gen. 18:8). Plinius, a Roman historian in 76 B.C., recommended the use of fermented milk products for the treatment of gastroenteritis.2 In the early 20th century, Elie Metchnikoff, a Nobel Prize-winning Russian scientist, suggested that the ingestion of Lactobacillus-containing yogurt contributed to the longevity of Bulgarian peasants by decreasing the pathogenic bacteria in the intestine.3 These observations led to the concept of a "probiotic," derived from the Greek, meaning "for life." The term probiotic was first used in 1965 to define substances secreted by one microorganism that stimulates the growth of another. The meaning of the word probiotics subsequently evolved to apply to those bacteria that contribute to intestinal balance. The World Health Organization defines probiotics as "live microorganisms, which when administered in adequate amounts, confer a heath benefit on the host."4

Different strains of probiotic bacteria may exert different effects based on specific capabilities and enzymatic activities—even within one species.5, 6 Different microorganisms express habitat preferences that may differ in various host species. The four habitats in the gastrointestinal tract are: the surface of epithelial cells; the crypts of the ileum, cecum, and colon; the mucus gel that overlays the epithelium; and the lumen of the intestine.7 The luminal content of bacteria depends greatly on bowel transit, resulting in a relatively low microbial density in the small bowel.

Because probiotics have multiple mechanisms of action, many have potential applications for managing various diseases. Those probiotics that have undergone the most clinical testing in people and livestock and are used widely include Lactobacillus species (such as L. acidophilus, L. rhamnosus, L. delbrueckii ssp. bulgaricus, L. reuteri, and L. casei); Bifidobacterium species; Enterococcus faecium and Saccharomyces boulardii, which is a nonpathogenic yeast. In dogs and cats, lactobacilli and bifidobacte-ria have also been used clinically. However, Enterococcus faecium has also garnered attention in clinical use in Europe and in the United States (see Figure 1). Despite the interest in probiotics for clinical use, understanding their clinical application in veterinary medicine has been limited by the paucity of well-designed laboratory, translational, and clinical studies.

Figure 1. Fortiflora for dogs and cats

Safety and regulation of probiotics

The Food and Agriculture Organization of the United Nations and the World Health Organization have published recommended guidelines for what constitutes a probiotics product intended for use in humans. Requirements allow that the included strains be designated individually and speciated appropriately. The formulations must retain viable strain counts at the end of their shelf lives and must confer a proven clinical end-point.8 Some human products continue to be of dubious quality and carry unsupported health claims, which suggests that some manufacturers are not enforcing the recommended guidelines. This problem is compounded by the diverse categories that encompass probiotic products for humans, including food, functional food, novel food, natural remedy (Denmark, Sweden, and Finland), natural health product (Canada), dietetic food (Italy), direct fed microbial (animal feed), dietary supplement (human food) (USA), and biotherapeutic and pharmaceuticals (probiotic pharmaceuticals are available in Canada, China, and a variety of European countries).

A true probiotic contains live microbes having a substantiated beneficial effect. Although a preparation of nonviable bacteria may mediate a physiologic benefit, they are not considered to be probiotics under the present definition. Furthermore, any strains that do not confer benefits should not be referred to as probiotics. In vitro testing to establish mechanisms of action are insufficient substantiation for classifying a microbial strain as a probiotic. The basis for a microbe being termed a probiotic should be proven efficacy and safety under the recommended conditions of use, with considerations given to target population, route of administration, and dose applied.8

Despite prolonged marketing of probiotic products, a relatively large number of human products are mislabeled with inaccurate use of nomenclature for genus and species, inaccurate cell count, or unsubstantiated structure and function statements.9 From a scientific perspective, the suitable description of a probiotics reflected on the label should include the following information:

• genus and species identification, with nomenclature consistent with current scientifically recognized names

• strain designation

• viable count of each strain at the end of shelf life

• recommended storage conditions

• recommended dose

• an accurate description of the physiological effect (as far as is allowable by law)

• contact information for post-market surveillance.

In the U.S., probiotic preparations labeled for use in dogs or cats are classified by AAFCO (Association of American Feed Control Officials) and FDA's Center of Veterinary Medicine (CVM) as direct fed microorganisms or microbials, not pharmaceutical products. CVM has listed bacterial species that are considered safe when used in direct fed microbial (DFM) products. These include multiple species of Lactobacillus, Bifidobacterium and Enterococcus. In addition to using approved species and defined fermentation product ingredients, AAFCO has established labeling requirements for DFM products such as probiotics.

Criteria for probiotics

All products that are sources of DFM are required to provide potency guarantees for each microorganism in colony forming units per gram (CFU/g), along with a distinct listing of the microorganism species. Additionally, AAFCO requires the statement "contains a source of live (viable) naturally occurring microorganisms" as an indication of safety and efficacy.

Although these requirements apply to all such products, there is not universal compliance in the industry. This important point is best illustrated by a recent study in which nineteen commercially available canine and feline diets purporting to contain probiotics were evaluated bacteriologi-cally.9 Quantitative bacterial cultures were performed on all products and the labeling claim of each product was compared to the qualitative and quantitative culture results. None of the products were found to contain all of the claimed organisms, while one or more of the listed contents were isolated from 1 0 of 1 9 (53 percent) products. Eleven (58 percent) diets contained additional, related products, and five (26 percent) diets did not contain any relevant growth. The diets tested contained between 0 and 1.8 x 105 CFU/g , with the exception of Enterococcus faecium in dogs and cats. No dose-response trials appear to have been carried out in people or animals, and the question of what constitutes an effective dose of a probiotic has yet to be defined for most probiotic strains.

Antibiotic resistance

Antibiotic resistance screening has shown that the spontaneous mutation rate to antibiotic resistance among lactobacilli can be as high as 2 x 105, depending on the strain.10 Several animal isolates of L. acidophilus and L. reuteri were tested for antibiotic resistance and all 1 6 L. reuteri strains were resistant to vancomycin and polymyxin B irrespective of their source. Only four of 30 L. acidophilus strains were vancomycin-resistant and seven were chloramphenicol-resistant.11 Antibiotic resistance plasmids from lactobacilli have been detected in a number of studies. Although enterococci are normal inhabitants of the gastrointestinal tract and are widely used as both human and animal probiotics, in vivo conjugative transfer of antibiotic resistance plasmids from L. reuteri to Enterococcus faecalis has been demonstrated in germ-free mice.1 2 In most cases, antibiotic resistance in lactic acid bacteria is not transmissible, but represents an intrinsic species or genus-specific characteristic of the organism. Knowledge of the ability of a proposed probiotic strain to act as a donor of conjugative antibiotic resistance genes is a prudent precaution. Safety of some probiotic strains has been extensively studied. For example, it has been proven that Enterococcus faecium SF68 (NCIMB 1 0415) does not acquire or transmit antibiotic resistance.

Probiotics therapy

One important characteristic of probiotics is their ability to suppress the proliferation and virulence of pathogenic organisms. This characteristic is increasingly well documented in probiotic bacteria in the gastrointestinal tract and genitourinary tract. However, probiotics are also thought to have direct effects on human physiology and immunity, including allergic disease (e.g., asthma, hay fever), autoimmune diseases (e.g., multiple sclerosis and type I diabetes), diseases of the oral cavity (e.g., periodontal disease and caries), and the nervous system (e.g., autism and depression).13 Table 1 gives an overview of the mechanisms of action of probiotics.

Table 1. Mechanisms of probiotic action

Probiotic therapy in dogs

To date, the study of probiotic efficacy in dogs is still in its infancy, and the small number of studies that have evaluated the effects of probiotics in dogs were designed to screen for potential new activity of new probiotics. In these early studies, most have been focused on the intestinal microflora in apparently healthy dogs. Specifically, probiotic strains of human or canine origin (lactobacilli, bifidobacteria, and enterococci) were used in adult dogs to assess effects on intestinal microbial populations, reduction of specific pathogens in feces, and immunomodulation.14-20 In many of these studies, the effect of probiotics added to the food in healthy dogs had an equivocal effect on fecal microflora and pathogens.18 ,21 Further, it is important to note that studies designed to determine the potential of a new probiotic were not randomized, controlled trials, and the strains of probiotic varied from study to study, therefore making generalization more difficult to make. In addition, many studies focused on fecal isolation and quantitative cultures of putative pathogenic bacteria such as Clostridium perfringens, rather than evaluating more meaningful end points such as shifts in the microbial flora, mucosal immunopathology, and alterations in intestinal integrity. Only two studies addressing the role of probiotics in management of dietary sensitivity and food-responsive diarrhea have been published to date, both with positive results.15, 17 Only one of these studies was a randomized, placebo-controlled clinical trial.15 The results of that study were clinically positive because all dogs in the study improved after being placed on the elimination diet. However, the results showed no specific changes in the inflammatory cytokine patterns or a specific benefit of the probiotic.15 The immunomodulatory effects of E. faecium SF68 have been studied in dogs. The results of one study showed that the probiotic was associated with increased fecal IgA concentrations and increased vaccine-specific circulating IgG and IgA concentrations.22 These puppies also had improved fecal quality and decreased variability in fecal quality when compared to the control puppies. An additional study with elderly dogs showed that elderly dogs fed E. faecium SF68 maintained higher IgA levels than control dogs.23 While increased immunoglobulins may suggest enhanced immune response and should signal an enhanced ability to handle new immunological challenges, proving the clinical relevance of increased IgA is difficult to prove without purposely exposing dogs to infectious diseases. Additional studies are warranted in dogs to further assess the immunomodulatory effects of probiotics. Because of the numerous published studies proving beneficial effects of probiotics in people and livestock, tremendous interest has been shown among commercial petfood companies that market probiotics for use in dogs or cats. However, most of the evidence surrounding the use of probiotics in puppies or adult dogs with stress colitis or antibiotic-responsive diarrhea is anecdotal, with no prospective, randomized, placebo-controlled studies in these disorders published to date.

Probiotic therapy in cats

There is also a paucity of published information pertaining to probiotic use in cats, and there are no clinical studies reporting a beneficial effect of probiotic therapy for any feline disease.24 One study evaluating the effect of dietary supplementation with the probiotic strain of L. acidophilus (DSM 13241) administered in 15 healthy adult cats demonstrated the recovery of the probiotic from the feces of the cats in association with a significant reduction in Clostridium spp. and E. faecalis.25 However, immunomodulatory effects were reported based on decreased lymphocyte and increased eosinophil populations, and increased activities of phagocytes in the peripheral blood. This study was not a randomized trial and the changes reported in the populations of peripheral blood cells cannot be extrapolated into evidence of systemic health benefits. Evaluation of the effect of supplementation with E. faecium SF68 on immune function responses following administration of a multivalent vaccine was evaluated in specific pathogen-free kittens.26 This prospective, randomized, placebo-controlled study resulted in the recovery of E. faecium SF68 from the feces of seven of nine cats treated with the probiotic, and a nonsignificant increase in feline herpes virus-1 -specific serum IgG levels. Concentrations of total IgG and IgA in serum were similar between the probiotic and placebo groups and the percentage of CD4+ T lymphocytes was only significantly increased in kittens at 27 weeks (and no other time). Probiotics have also been evaluated in juvenile captive cheetahs, a population with a relatively high incidence of bacterial-associated enteritis. Administration of a species-specific probiotic containing

Lactobacillus Group 2 and E. faecium to 27 juvenile cheetahs was associated with a significantly increased body weight in the treatment group, while there was no increase in the control group.27 In addition, administration of the probiotic was associated with improved fecal quality in the probiotic group. All studies were performed in healthy kittens or cats, and there are no published studies to date evaluating the use of probiotics in cats with gastrointestinal disorders such as bacterial or parasitic-associated diarrhea, food allergy, antibiotic-associated diarrhea, or inflammatory bowel disease. However, despite the paucity of studies of probiotics in cats, clinical use of probiotics in practice for prevention of diarrhea in kittens or cats receiving antibiotics, for kittens or cats undergoing diet changes, and for kittens with parasitic diarrheas is likely to be safe, and in many cases, may be effective for management of diarrhea.

Future considerations

The potential benefits and specific indications for probiotics in dogs and cats have yet to be clearly defined, and our understanding of the nature and diversity of the canine and feline intestinal micro-flora during health and disease is slowly expanding. The diverse microbial content of the intestinal tract is not adequately reflected by fecal analysis, which has been the predominant sample analyzed to date. The application of genome analysis to the study of the microbial ecology of the gastrointestinal tract should facilitate the identification of major culturable and nonculturable populations, and provide a tool for studying shifts in these populations over time and under different conditions. The completion of prospective, randomized, placebo-controlled studies in dogs and cats that rely on clinically relevant end points that relate to particular physiologic or pathologic conditions is needed to define what role probiotics will have. Probiotics do appear to have a potential role in the prevention and treatment of various gastrointestinal illnesses, but it is likely that benefits achieved are specific to the bacterial species used and to the underlying disease context. Further work will help us better define the appropriate probiotic species and the specific indications for their use.

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