Lactobacilli in the gastrointestinal tract of dog and wolf
isolation, identification and characterization of L. reuteri
Since the first wolf was taken as a companion animal a lot have happened with the biology of these animals. Is it possible that changes in the gut microbiota of the domestic dog are involved in the increasing numbers of animals with gastrointestinal disorders? Probiotics have been proven to promote the immune system of dogs by increasing concentrations of neutrophils and monocytes, decreasing the fragility of erythrocytes and increasing the serum IgG concentrations. Probiotics have also been shown to improve the fecal consistency, fecal dry matter and defecation frequency of dogs. The aim of this study was to find out if the bacterial counts in the gut of dogs differed from the gut of wolves, with the focus on lactobacilli. The presence of Lactobacillus reuteri and characterization of such strains in wolves and dogs was also analyzed to find a possible probiotic candidate. Fecal specimens from five dogs and intestine content from four wolves were collected and lactobacilli were analyzed and isolated by cultivation on the selective substrate Rogosa. Bacterial enumerations were noted and colonies with morphological differences were collected and isolated before the characterization was done using reuterin assay, rep-PCR and gel electrophoresis. A representative for each rep-PCR type was identified using 16S rRNA gene sequencing. Bacteria identified as L. reuteri was characterized further using methods for testing bile tolerance, detection of the genes ureC and pduC, mucus binding capacity and antibiotic susceptibility. A handful of isolates were also characterized by determining the gene sequence of a cell surface protein specific for L. reuteri. The content of lactobacilli in the fecal samples of dogs ranged from 106 to 108 cfu g-1 feces and in intestine of wolves from 103 to 105 cfu g-1 gut content. Nevertheless, not all of the bacteria grown on the Rogosa agar were lactobacilli, species of Streptococcus were also isolated. Even though the total counts of bacteria were higher in dog, the number of different species of isolated lactobacilli was higher in the wolf. In the samples from dog, two species of Lactobacillus were isolated; L. animalis and L. reuteri while in the wolf samples another five species were isolated. A total of 32 L. reuteri were isolated from the subjects and these could be divided into 10-12 different strains according to the characterization methods. Among the 13 isolated L. reuteri analyzed by the different characterization methods, four were reuterin positive and two were urease positive. Some of the strains bound well to intestinal mucus while others did not bind at all. The bile tolerance also differed between the strains but most of them, except for one strain, survived a concentration of 5% bile. The results from the antibiotic susceptibility test showed a close relation between the strains, with resistance against streptomycin and tetracycline. Some of these strains were isolated from both dog and wolf whereas other strains could only be found in dog or wolf. This was established using the different characterization methods as well as analyzing the gene sequence of a cell surface protein referred to as ?protein x?. A new method for isolating L. reuteri were also tested, called immunomagnetic isolation. The results from this method were both positive and negative, this since the beads bound to some strains of L. reuteri but not all. The Dynabeads also bound to lactobacilli in a dog sample but not to any bacteria in a wolf sample.