Clinical and research experiences with probiotics in cats (Sponsored by Nestlé Purina)

Probiotics are live microorganisms that when administered in adequate amounts confer a health effect on the host.1 There have been many studies of the effects of probiotics on the health of people, but very few in small animals. In a recent review of human studies involving probiotics, it was stated that "well-established probiotic effects include:2

1. Prevention and/or reduction of duration and complaints of rotavirus-induced or antibiotic-associated diarrhea as well as alleviation of complaints due to lactose intolerance;

2. Reduction of the concentration of cancer-promoting enzymes and/or putrefactive (bacterial) metabolites in the gut;

3. Prevention and alleviation of unspecific and irregular complaints of the gastrointestinal tracts in healthy people;

4. Beneficial effects on microbial aberrancies, inflammation, and other complaints in connection with inflammatory diseases of the gastrointestinal tract, Helicobacter pylori infection, or bacterial overgrowth;

5. Normalization of passing stool and stool consistency in subjects suffering from obstipation or an irritable colon;

6. Prevention or alleviation of allergies and atopic diseases in infants; and

7. Prevention of respiratory tract infections (common cold, influenza) and other infectious diseases as well as treatment of urogenital infections."

Infectious diseases are very common in small animals, so the potential beneficial effects of probiotics could impact veterinary practice significantly. All mechanisms of immune modulation have not been characterized and it is likely these effects vary by the probiotic. It is known that many probiotics in the lactic acid bacteria group help balance the endogenous microbiota and some can inhibit replication of pathogenic bacteria. The proposed mechanisms of action include competition for essential nutrients or receptor sites, binding with pathogenic bacteria, and production of inhibitory substances. It is also now known that some probiotics can beneficially influence innate and acquired immunity by a variety of proposed mechanisms including inducing cytokine production, natural killer cell activity, and both specific and nonspecific immunoglobulin production.2

Several recent review articles in human medicine suggest that the evidence to support the theory that probiotics are beneficial in a variety of human conditions, such as Clostridiumdifficile diarrhea and hospital-acquired pneumonia, is minimal and that larger, more rigorously controlled multicenter studies should be performed.3-5 These findings emphasize that biological effects of individual probiotics will vary and that each probiotic introduced should be rigorously evaluated in a controlled fashion to define the potential for clinical utility. In addition, the source of the probiotic should also be considered. For example, in a recent study in Canada, the majority of diets claiming to contain probiotics generally did not meet the label claim when evaluated.6

SF68 and immune stimulation in puppies

Enterococcus faecium strain SF68 (NCIMB 10415) was originally isolated from the feces of a healthy baby and was initially shown to inhibit the growth of a number of enteropathogens.7 This bacterium is now the probiotic in the Nestlé Purina PetCare Company product named FortiFlora™.

In one dog study, E. faecium strain SF68 was fed to a group of puppies vaccinated with canine distemper virus (CDV) and the effects compared over time with a control group that was similarly vaccinated, but not fed the probiotic.8 A number of findings suggested that the probiotic had an immune-modulating effect. The puppies supplemented with SF68 had increased serum and fecal total IgA concentrations, increased CDV-specific IgG and IgA serum concentrations, and an increased percentage of circulating B lymphocytes when compared with puppies in the control group. The effect on CDV-specific IgG and IgA antibodies in serum was only seen after the puppies had been supplemented for 31 and 44 weeks, respectively. It was believed that SF68 prevented the decline in antibody titers observed in the control group by maintaining high levels of antibodies.

SF68 and immune stimulation in cats

After publication of the puppy study, a similar collaborative study with Nestlé Purina PetCare was performed in healthy kittens.9 In that study, it was hypothesized that feeding E. faecium SF68 to kittens would enhance nonspecific immune responses; humoral immune responses to feline herpesvirus type 1 (FHV-1), feline calicivirus (FCV), and feline panleukopenia virus (FPV); and FHV-1-specific cell-mediated immune responses.

Twenty 6-week-old specific-pathogen-free kittens were divided into two groups. Starting at 7 weeks of age, one group was fed SF68 daily and the other group was fed a placebo. At 9 and 12 weeks of age, a commercial FVRCP modified-live vaccine was administered subcutaneously and the kittens were followed until 27 weeks of age. The attitudes and behavior of the kittens were monitored daily throughout the study, and body weight was measured weekly. Blood, saliva, and feces were collected from all cats throughout the study, and fecal extracts from samples taken at 9 and 27 weeks of age were analyzed for total IgA and total IgG.

Other tests included randomly amplified polymorphic DNA (RAPD)-PCR on feces. RAPD-PCR was done to determine if viable E. faecium SF68 was in the stools of treated cats and to assess whether the probiotic was accidentally transmitted by laboratory staff from the treated kittens to the control kittens. Complete blood counts, serum biochemical panels, and urinalyses were performed to detect adverse events induced by the probiotic.

Antigen-specific humoral immune responses were estimated by measuring FHV-1-specific IgG, FHV-1-specific IgA, FCV-specific IgG, and FPV-specific IgG in sera, as well as FHV-1 specific IgG and IgA levels in saliva using adaptations of previously published ELISA methods. Total IgG and IgA concentrations in sera, fecal extracts, and saliva were estimated using a commercial ELISA or radial immunodiffusion assay. Cellular immune responses were assessed via flow cytometry and whole blood proliferation assays. Lymphocytes were stained for expression of CD4, CD8, CD44, MHC Class II, and B cells. In addition, lymphocyte proliferation in response to concanavalin A and FHV-1 antigens was assessed.

Body weight and fecal scores were not statistically different between the two groups. Feces from seven of nine treatment cats were positive for SF68 at some point during the study, whereas feces from all control cats were negative for SF68 throughout the study. SF68 DNA was not amplified from feces of any treated cat one week after stopping supplementation (Week 28). Complete blood counts and biochemical profiles were within normal limits for the age group for all cats at all time points.

At 21 and 27 weeks of age, the mean levels of FHV-1-specific IgA in serum and saliva were numerically greater in the treatment group when compared with the placebo group. Moreover, the mean FHV-1-specific serum IgG levels were numerically greater in the treatment group when compared with the placebo group at 15, 21, and 27 weeks of age. However, these differences in FHV-1 antibody levels did not reach statistical significance. No FHV-1-specific IgG was detected in saliva, and FCV-specific IgG levels in serum were similar between the groups. At 15 weeks of age, the treatment group's serum mean FPV-specific IgG levels were numerically greater than those of the placebo group, but the differences were not statistically significant. There were no significant differences in serum, fecal, or saliva IgG or IgA concentrations between the two groups. However, at 27 weeks of age, the treatment group had a significantly higher percentage of gated lymphocytes positive for CD4 than the placebo group.

In this study, it was concluded that SF68 was safe to administer to cats and the increase in CD4+ cell counts in the treatment group (without a concurrent increase in CD8+ counts) demonstrated a systemic immune-modulating effect by the probiotic. Because the results did not show a significant increase in lymphocyte stimulation by FHV-1 or an increase in the expression of the memory cell marker (CD44) on the CD4+ lymphocytes in the treatment group, the increase in CD4+ T lymphocytes may have been nonspecific as the cells appeared to be unprimed.

Two major subsets of CD4+ T cells are the Th1 and Th2 subsets. Th1 cells stimulate cell-mediated immune functions (cell-mediated immunity). Th2 cells produce interleukin-4, which stimulates IgE production by B cells (humoral immunity). The CD4+ T lymphocytes of kittens in this study were not additionally characterized via cytokine production profiles or additional cell surface marker characterization. Therefore, it could not be determined whether a Th1 or Th2 response predominated. Furthermore, either the sample size or the duration of this study may have precluded detection of statistical differences between the groups in regards to FPV, FCV, and FHV-1 antibody titers.

Figure 1. Kitten with conjunctivitis secondary to feline herpesvirus 1 i(FHV-1) nfection.

SF68 and management of feline herpesvirus type 1

The results of this study prompted a follow-up study on FHV-1.10 This virus is extremely common in cats and frequently results in recurrent ocular and respiratory clinical signs (see Figure 1). There is no known drug therapy that consistently eliminates the carrier state, and vaccination does not provide sterilizing immunity. It was hypothesized that feeding SF68 to cats with chronic FHV-1 infection would decrease clinical disease and reduce both episodes of FHV-1 shedding and the numbers of FHV-1 DNA copies shed over time. In this study, 12 cats with chronic FHV-1 infection were administered either SF68 or a placebo. The cats were then monitored for clinical signs of disease, analyzed for FHV-1 shedding, and evaluated for FHV-1-specific humoral and cell-mediated immune responses and fecal microbiome stability. After an equilibration period, mild stress was induced over time by changing the housing of the cats from cages to gang housing repeatedly over a five-month period. The SF68 was well tolerated by all cats. Fecal microbial diversity was maintained throughout the study in cats given SF68, but decreased in cats fed the placebo, indicating a more stable microbiome in cats fed SF68. Upper respiratory signs of disease were not exacerbated by the induced stress, but those fed SF68 had fewer episodes of conjunctivitis than the placebo group during the supplementation period, suggesting that administration of the probiotic lessened morbidity from chronic FHV-1 infection (see Figure 2).

Figure 2. Cumulative conjunctivitis scores in FHV-1-infected kittens.

SF68 and diarrhea in shelter animals

In previous research, mice administered SF68 and then infected with Giardia intestinalis shed fewer trophozoites and Giardia antigen than the placebo group.11 In addition, when compared with untreated mice, supplemented mice had increased CD4+ cells in Peyer's patches and the spleen, as well as increased anti-Giardia intestinal IgA and serum IgG. This work prompted a study on diarrhea in cats and dogs housed in an animal shelter.12

The hypothesis was that cats and dogs housed in an animal shelter and fed SF68 would have fewer episodes of diarrhea and improved fecal scores than untreated cats and dogs in the same environment. The study dogs and cats were divided into groups and housed in separate rooms (two groups of dogs and two groups of cats). The cats and dogs were all fed a standardized diet by species. Animals in one room were supplemented daily with FortiFlora and animals in the alternate room were supplemented daily with placebo. Otherwise, management of the rooms was identical.

Table 1. Description of the Purina Fecal Scoring System

Before the room was cleaned each morning, one of the investigators would score the feces in each animal's cage using the Purina Fecal Scoring System for Dogs and Cats (see Table 1). Feces from dogs and cats with a score of 4 or greater were collected and transported to Colorado State University (CSU) for infectious disease testing. The testing at CSU included microscopic examination for parasite eggs, cysts, and oocysts and immunofluorescent antibody testing for Cryptosporidium oocysts and Giardia cysts.

Diarrhea prevalence rates were low for all dogs in the study and so statistical differences were not detected. However, the percentage of cats with diarrhea of two or more days was 7.7% for the probiotic group and 20.7% for the placebo group. These results suggest that administering SF68 to cats housed in shelters may reduce the number of days with diarrhea. Since this was a short-term study, this effect was likely from the probiotic's influences on intestinal flora rather than its systemic immune-enhancing effects.

Summary

Controlled studies evaluating the use of probiotics in cats is limited and inconclusive. However, there is evidence that SF68 is well tolerated and may have various clinical applications. Further research is ongoing in this area.

References

1. Schrezenmeir J, de Vrese M. Probiotics, prebiotics, and synbiotics-approaching a definition. Am J Clin Nutr 2001;73:361S-364S.

2. De Vrese M, Scherezenmeir J. Probiotics, prebiotics, and synbiotics. Adv Biochem Eng Biotechnol 2008;111:1-66.

3. McNabb B, Isakow W. Probiotics for the prevention of nosocomial pneumonia: Current evidence and opinions. Curr Opin Pul Med 2008;14:168-175.

4. Dendukuri N, Costa V, McGregor M, et al. Probiotic therapy for the prevention and treatment of Clostridium difficile-associated diarrhea: A systematic review. Can Med Assoc J 2005;173:167-170.

5. Isakow W, Morrow LE, Kollef MH. Probiotics for preventing and treating nosocomial infections: Review of current evidence and recommendations. Chest 2007;132:286-294.

6. Weese JS, Arroyo L. Bacteriological evaluation of dog and cat diets that claim to contain probiotics. Can Vet J 2003;44:212-216.

7. Lewenstein A, Frigerio G, Moroni M. Biological properties of SF68, a new approach for the treatment of diarrhoeal disease. Curr Ther Res 1979;26:967-974.

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

9. Veir JV, Knorr R, Cavadini C, et al. Effect of supplementation with Enterococcus faecium (SF68) on immune functions in cats. Vet Therap 2007;8:229-238.

10. Lappin MR, Veir JK, Satyaraj E, et al. Pilot study to evaluate the effect of oral supplementation of Enterococcus faecium SF68 on cats with latent feline herpesvirus 1. J Feline Med Surg 2009;11:650-654.

11. Benyacoub J, Perez PF, Rochat F, et al. Enterococcus faecium SF68 enhances the immune response to Giardiaintestinalis in mice. J Nutr 2005;135:1171-1176.

12. Bybee SN, Scorza V, Lappin MR. Effect of Enterococcus faecium SF68 supplementation on diarrhea in cats housed in a northern Colorado animal shelter, in press, J Vet Int Med 2011; in press.