What do we know (Proceedings)

The mammalian intestinal tract contains a complex, dynamic, and diverse society of pathogenic and nonpathogenic bacteria. There has been a plethora of research focusing on the mechanisms by which pathogenic bacteria influence intestinal function and induce disease; however, recent attention has focused on the indigenous non-pathogenic microorganisms and the ways in which they may benefit the host. Documentation of the health benefits of bacteria in food dates back to as early as the Persian version of the Old Testament (Genesis), and Plinius, a Roman historian in 76 BC, recommended the use of fermented milk products for the treatment of gastroenteritis (Bottazi).

One-hundred years ago, the Nobel Prize-winning Russian scientist, Elie Metchnikoff, suggested that the ingestion of lactobacillus-containing yogurt decreased the number of toxin-producing bacteria in the intestine, contributing to the longevity of Bulgarian peasants (Metchnikoff 1907). 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 which stimulates the growth of another” and was thus contrasted with the term antibiotic (Lilly, 1965). The meaning of the word has subsequently evolved to apply to those bacteria that “contribute to intestinal balance.” The current and more complete definition of a probiotic refers to “a preparation of or a product containing viable, defined microorganisms in sufficient numbers, which alter the microflora in a compartment of the host and by that exert beneficial health effects on the host.”.

Different strains of probiotic bacteria may exert different effects based on specific capabilities and enzymatic activities, even within one species. Different microorganisms express habitat preferences that may differ in various host species. Four microhabitats in the gastrointestinal tract were outlined by Freter (1992) as follows: 1) the surface of epitheliums cells; 2) the crypts of the ileum, cecum, and colon; 3) the mucus gel that overlays the epithelium; and 4) the lumen of the intestine. The luminal content of bacteria depends greatly on bowel transit, resulting in a relatively low microbial density in the small bowel. It should be emphasized that a proven probiotic effect found in one strain or species cannot be transferred to other strains or species because of differences in strain characteristics and habitat preferences.

Because of these multiple mechanisms of action, many different probiotics have potential applications to various diseases. Those in most widespread use, which have undergone the most clinical testing in humans and animals, include Lactobacillus species (such as L acidophilus, L rhamnosus, L bulgaricus, L reuteri, and L casei); Bifidobacterium species; and Saccharomyces boulardii, which is a nonpathogenic yeast. In dogs and cats, Enterococcus faecium has also received a lot of attention in clinical use in Europe and the US. Nevertheless, despite the explosion of interest and publications on probiotics in recent years, the clinical application of probiotics has been limited by the paucity of well-designed and mechanistically based laboratory, translational, and clinical studies.

Probiotics: regulatory aspects and safety

The guidelines for what is required for a product to be called a probiotic were published by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), and require that strains be designated individually, speciated appropriately, and retain a viable count at the end of their shelf life in the designated product formulation that confers a proven clinical end-point (FAO/WHO 2002). The fact that some products continue to be of dubious quality and carry unsupported health claims, complicates the process. This problem is compounded by the diverse categories that encompass probiotic products, including: food, functional food, novel food, natural remedy (Denmark, Sweden and Finland), natural health product (Canada), dietetic food (Italy), dietary supplement (USA), and biotherapeutic and pharmaceuticals (probiotic pharmaceuticals are available in Canada, China, and a variety of European countries).

The definition of a probiotic requires that the term only be applied to live microbes having a substantiated beneficial effect. Thus, microbes administered alive are considered probiotics regardless of their ability to survive intestinal transit. Although a preparation of non-viable bacteria may mediate a physiologic benefit, they are not considered to be “probiotics” under the present definition. Furthermore, any strains that do not confer clinically established physiological effects should not be referred to as probiotics.  In vitro testing to establish mechanisms of action are insufficient substantiation for the use of the term, “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 (FAO/WHO 2002).

Despite the prolonged marketing of “probiotic” products, there is little or no enforced worldwide regulation regarding labeling for quality or efficacy. A relatively large number of products are mislabeled based on inaccurate use of nomenclature for genus and species, inaccurate cell count or unsubstantiated structure/function statements continue to be sold worldwide (Weese 2001).  

Probiotic preparations labeled for use in dogs or cats are classified as nutritional supplements, not pharmaceutical products. As a result, they are not highly regulated, and specific product labeling and demonstration of efficacy are not required. This important point is best illustrated by a recent study by Weese et al. in which nineteen commercially available canine and feline diets purporting to contain probiotics were evaluated bacteriologically (Weese 2003). 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 contained all of the claimed organisms, while 1 or more of the listed contents were isolated from 10 of 19 (53%) products, and five (26%) diets did not contain any relevant growth. The diets that were tested contained between 0 and 1.8 × 105 CFU/g. Of equal concern, the question of what constitutes a minimal effective dose of a probiotic has yet to be defined.

Antibiotic resistance and probiotic

Antibiotic resistance screening has shown that the spontaneous mutation rate to antibiotic resistance among Lactobacilli can be quite high in the order of 2 × 105, depending on the strain. Several animal isolates of Lactobacillus acidophilus and Lactobacillus reuteri were tested for antibiotic resistance and all 16 L. reuteri strains were resistant to vancomycin and polymyxin B irrespective of their source, while only four of thirty L. acidophilus strains were vancomycin resistant and seven were chloramphenicol resistant. 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 E. faecalis has been demonstrated in germ-free mice. In most cases, antibiotic resistance to LAB is not of the transmissible type, 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. Although the enterococcal transmissible vancomycin resistance poses an important issue, to date there is no evidence of this occurring in clinical cases.

Mechanisms of probiotic action (from august, consultations in feline internal medicine, 2010, marks and zoran)

Probiotics block intestinal bacterial effects

Probiotics have been identified to mediate maintenance of the gastrointestinal microbial balance via two mechanisms: production of antibacterial substances, such as bacteriocins (e.g. lantibiotics) and acids (e.g. acetic, lactic, and proprionic), and competitive inhibition of pathogen and toxin adherence to the intestinal epithelium. Several strains of Lactobacilli and Bifidobacteria are able to decrease adhesion of both pathogens and their toxins to the intestinal epithelium, and they can displace pathogenic bacteria even if the pathogens have attached to intestinal epithelial cells prior to probiotic treatment (Collado 2007, Candera 2005).  One of the mechanisms underlying pathogenic bacteria binding to intestinal epithelial cells is through the interaction between bacterial lectins and carbohydrate moieties of glycoconjugate receptor molecules on the cell surface (Mukai 2004, Sun 2007).

Probiotics regulate mucosal immune responses

Both in vitro and in vivo studies show effects of probiotics on host immune functions, including upregulation of immune function that may improve the ability to fight infections or inhibit tumor formation; downregulation of immune function that may prevent the onset of allergy or intestinal inflammation. The following is a brief overview of the many possible ways individual probiotic species may have an effect on the animal's mucosal (and ultimately, systemic immune response)

Enhancing Host Innate Immunity

Probiotics have the potential to stimulate innate immune responses against microorganisms and dietary antigens newly encountered by the host through several mechanisms. Intestinal dendritic cells can retain commensal bacteria by selectively activating B lymphocytes to produce IgA to reduce mucosal penetration by bacteria. The dendritic cells carrying commensals are restricted to the intestinal mucosal lymphoid tissues, and thus avoid potential systemic immune responses (MacPherson 2004).

Modulation of Pathogen-induced Inflammatory Responses

The host innate defenses must modulate responses appropriate to the level of threat provided by a given pathogen. If the response is too weak, the infection may not be cleared, leaving the host susceptible to systemic infection. However, if it is too strong the result may be excess tissue damage. A mechanism of probiotic protection from pathogen induced injury and inflammation is modulating the balance of pro- and antiinflammatory cytokine production.

Increasing Antiinflammatory Cytokine Production

Probiotics can induce dendritic cells to produce antiinflammatory cytokines, including IL-10, which suppress the Th1 response (Hart AL, 2004). However, the role of IL-10 production in probiotic prevention of Th1 responses by probiotics is controversial, and may be through both IL-10- dependent and –independent mechanisms.

Suppressing Proinflammatory Cytokine Production

Probiotics such as LGG have been shown to inhibit lipopolysaccharide (LPS) and Helicobacter pylori-stimulated TNF production by murine macrophages (Penna 2003). In addition, LGG-conditioned cell culture media decreases TNF production in macrophages, indicating that soluble molecules derived from LGG exert this immunoregulatory role (Penna 2003).

Upregulation of Host Immune Responses to Defend Against Infection

Probiotics and commensal microflora may regulate a balance between pro- and antiinflammatory mucosal responses leading to intestinal homeostasis. Probiotics facilitate this important function by stimulation of host immunological functions, including Th1 responses through dendritic cell-directed T cell activation. During colonization of mice with B. fragilis, dendritic cells take up and retain a bacterial polysaccharide which promotes maturation of dendritic cells, Th1-type cytokine production including IL-4, IL-12, and IFN-γ, and subsequent CD4 + T cell expansion (Mazmanian 2005).

Regulation of Immune Responses by Probiotic DNA

There have been a number of intriguing studies documenting the beneficial immunomodulatory properties of probiotic DNA in people and murine models. DNA isolated from the probiotic VSL#3 mixture decreases LPS-activated IL-8 production and TNF and IFN- release in vivo and in vitro (Jijon 2004).

Differential Activation of TLRs by Probiotics in Immune Cells

Different probiotic bacteria stimulate distinct TLRs on host cells, an essential consideration in designing any therapeutic trials. Probiotic bacteria possess molecular recognition patterns similar to pathogenic bacteria; however, the probiotic organisms do not normally initiate pathogenic inflammatory responses. It appears that probiotics exert both up- and downregulatory effects on immune responses, and TLR-regulated signaling pathways appear to be one of the mechanisms for these immunoregulatory actions. The probiotic E. coli Nissle 1917 express increased levels of both TLR2 and TLR4 (Grabig 2006) whereas the probiotic VSL#3 mixture mediates its immunostimulatory response via TLR9 signaling (Rachmilewitz 2004). There is clearly a need to define the mechanism(s) for observed differences among the signals induced by probiotics and pathogens, which use similar receptors to induce divergent responses.

Probiotics regulate intestinal epithelial cell functions

Substantial evidence indicates that probiotic bacteria stimulate intestinal epithelial cell responses, including restitution of damaged epithelial barrier (Zyrek 2007), production of antibacterial substances and cell-protective proteins (Tao 2006), and prevention of cytokine-induced intestinal epithelial cell apoptosis (Yan 2002). Many of these responses result from probiotic stimulation of specific intracellular signaling pathways in the intestinal epithelial cells.

Current applications for probiotics in humans

One important characteristic of probiotics is their ability to suppress the proliferation and virulence of pathogenic organisms, and this is an increasingly well-documented role of probiotic bacteria in the gastrointestinal tract and genitourinary tract. However, it is becoming increasingly clear that probiotic microbiota 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) (Spinler and Verslovic, 2008).

Probiotic therapy in dogs

To date, only a relatively small number of studies have been published evaluating the effects of probiotics in dogs, and most of these have been focused on the intestinal microflora in apparently healthy dogs. Specifically, probiotic strains of human or canine origin (lactobacilli, bifidobacter and enterococcus) were used in healthy adult dogs to assess effects on intestinal microbial populations, reduction of specific pathogens in feces, and immunomodulation (Strompfova 2004, Sauter 2006, Perelmuter 2008, Pascher 2008, Vahjen and Manner 2003, Swanson 2002, Biagi 2007). 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 (Vahjen and Manner 2003, Baillon 2004). Further, it is important to note that most of these studies were not randomized, controlled trials, and the strains of probiotic varied from study to study, making interpretation of findings more challenging. In addition, many studies focused on fecal isolation and quantitative cultures of putative pathogenic bacteria such as C. 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, with overall positive results (Pascher 2008, Sauter 2006). Only one of these studies was a randomized, placebo-controlled clinical trial (Sauter), and the results of that study, while clinically positive because all of the dogs on the study improved when they were placed on the elimination diet, showed no specific changes in the inflammatory cytokine patterns of the dogs or a specific benefit of the probiotic (Sauter 2006) .  The immunomodulatory effects of Enterococcus faecium SF68 have been studied in dogs, and the probiotic was associated with increased fecal IgA concentrations and increased vaccine specific circulating IgG and IgA concentrations (Benyacoub 2003). While increased immune globulins may suggest enhanced immune response, the clinical relevance of this finding is not known.  Additional studies are warranted in dogs to further assess the immunomodulatory effects of probiotics, and to evaluate their safety. The latter issue is particularly important given the recent finding of increased intestinal adhesion of Campylobacter jejuni in an in vitro model of canine intestinal mucus following incubation with Enterococcus faecium (Rinkinen 2003). It should be noted that this E. faecium strain is different from the E. faecium SF68 strain that is commercially available and to date, there is no clinical or anecdotal evidence of Campylobacter-associated diarrhea in dogs. Despite the paucity of prospective, randomized placebo-controlled clinical trials in dogs, tremendous interest has been shown among commercial pet-food companies who are marketing probiotics for use in dogs or cats. Unfortunately, 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

Unfortunately, there is a dearth 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 (Weese 2008). One study evaluating the effect of dietary supplementation with the probiotic strain of Lactobacillus acidophilus DSM 13241 (2 × 108 CFU/d for 4.5 weeks) 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 Enterococcus faecalis (Marshall-Jones 2006). However, the immunomodulatory effects were reported based on decreased lymphocyte and increased eosinophil populations, and increased activities of peripheral blood phagocytes. The relevance of these findings is unclear as 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 Enterococcus faecium strain SF68 on immune function responses following administration of a multivalent vaccine was evaluated in specific pathogen-free kittens (Veir 2006). This prospective, randomized, placebo-controlled study resulted in the recovery of E. faecium SF68 from the feces of 7/9 cats treated with the probiotic, and a non-significant increase in FHV-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 + lymphocytes was only significantly increased in kittens at 27 weeks and not at any other time points. 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 Enterococcus 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 (Koeppel 2006). In addition, administration of the probiotic was associated with improved fecal quality in the probiotic group. It should be emphasized that 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 IBD.

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 microflora 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 non-culturable 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 a particular physiologic or pathologic condition is needed to define a role for probiotics. Probiotics do now appear to have a potential role in the prevention and treatment of various gastrointestinal and allergic 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.

References

FAO/WHO. Guidelines for the evaluation of probiotics in food. In: Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food. 2002.

Weese JS, Arroyo L. Bacteriological evaluation of dog and cat diets that claim to contain probiotics. Can Vet J. 44:212-215, 2003.

Sauter SN, Benyacoub J, Allenspach K, et al., Effects of probiotic bacteria in dogs with food responsive diarrhea treated with an elimination diet. J Anim Phys Anim Nutr 90: 269-277, 2006.

Perelmuter K, Fraga M, Zunino P. In vitro activity of potential probiotic Lactobacillus murinus isolated from the dog. J Appl Microbiol 104: 1718-1725, 2008.

Pascher M, Hellweg P, Khol-Parisini A, et al. Effects of a probiotic Lactobacillus acidophilus strain on feed tolerance in dogs with non-specific dietary sensitivity. Arch Anim Nutr 62: 107-116, 2008.

Vahjen W and Manner K. The effect of a probiotic Entercoccus faecium product in diets of healthy dogs on bacteriological counts of Salmonella, Campylobacter, and Clostridium in feces. Arch Anim Nutr 57: 229-233, 2003.

Swanson KS, Grieshop CM, Flickinger EA, et al., Fructooligosaccharides and Lactobacillus acidophilus modify gut microbial populations, total tract nutrient digestibilities and fecal protein catabolite concentrations in healthy adult dogs. J Nutr 132: 3721-3731, 2002.

Biai G, Cipollini I, Pompei A, et al. Effect of a Lactobacillus animalis strain on composition and metabolism of the intestinal microflora in adult dogs. Vet Microbiol 124: 160-165, 2007.

Baillon MLA, Marshall-Jones ZV, Butterwisk RF. Effects of probiotic Lactobacillus acidophilus strain DSM 13241 in healthy adult dogs. Am J Vet Res 65: 338-343, 2004.

Benyacoub J, Czarnecki-Maulden GI, Cavadini C, et al. Supplementation of food with Enterococcus faecium SF68 stimulates immune functions in young dogs. J Nutr 133: 1158-1162, 2003.

Rinkinen M, Jalava K, Westermarck E, et al. Interaction between probiotic lactic acid bacteria and canine enteric pathogens: a risk factor for intestinal Enterococcus faecium colonization? Vet Microbiol 92: 111-119, 2003.

Weese JS, Sharif S, Rodriguez-Palacios A. Probiotics in veterinary medicine. In: Therapeutic Microbiology: Probiotics and Related Strategies, Versalovic J and Wilson M eds. ASM Press, Washington DC, pp 341-356, 2008.

Marshall-Jones ZV, Baillon MI, Croft JM, et al. Effects of lactobacillus acidophilus dSM 13241 as a probiotic in healthy adult cats. Am J Vet Res 67: 1005-1012, 2006.

Veir JK, Knorr R, Cavadini D, et al. Effect of supplementation with Enterococcus faecium (SF68) on immune function in cats. Vet Therp 8: 229-238, 2007.

Koeppel KN, Bertschinger H, van Vuuren M, et al. The use of a probiotic in captive cheetahs (Acinonyx jubatus). JS Afr Vet Assoc 77: 127-130, 2006.