Association of obesity with serum leptin, adiponectin, and serotonin and gut microflora in beagle dogs.

BACKGROUND: Serotonin (5-hydroxytryptamine, 5HT) is involved in hypothalamic regulation of energy consumption. Also, the gut microbiome can influence neuronal signaling to the brain through vagal afferent neurons. Therefore, serotonin concentrations in the central nervous system and the composition of the microbiota can be related to obesity. OBJECTIVE: To examine adipokine, and, serotonin concentrations, and the gut microbiota in lean dogs and dogs with experimentally induced obesity. ANIMALS: Fourteen healthy Beagle dogs were used in this study. METHODS: Seven Beagle dogs in the obese group were fed commercial food ad libitum, over a period of 6 months to increase their weight and seven Beagle dogs in lean group were fed a restricted amount of the same diet to maintain optimal body condition over a period of 6 months. Peripheral leptin, adiponectin, 5HT, and cerebrospinal fluid (CSF-5HT) levels were measured by ELISA. Fecal samples were collected in lean and obese groups 6 months after obesity was induced. Targeted pyrosequencing of the 16S rRNA gene was performed using a Genome Sequencer FLX plus system. RESULTS: Leptin concentrations were higher in the obese group (1.98 ± 1.00) compared to those of the lean group (1.12 ± 0.07, P = .025). Adiponectin and 5-hydroytryptamine of cerebrospinal fluid (CSF-5HT) concentrations were higher in the lean group (27.1 ± 7.28) than in the obese group (14.4 ± 5.40, P = .018). Analysis of the microbiome revealed that the diversity of the microbial community was lower in the obese group. Microbes from the phylum Firmicutes (85%) were predominant group in the gut microbiota of lean dogs. However, bacteria from the phylum Proteobacteria (76%) were the predominant group in the gut microbiota of dogs in the obese group. CONCLUSIONS AND CLINICAL IMPORTANCE: Decreased 5HT levels in obese group might increase the risk of obesity because of increased appetite. Microflora enriched with gram-negative might be related with chronic inflammation status in obese dogs.

Analysis of K99 plasmids from enterotoxigenic Escherichia coli.

Evaluation of 9 wild-type K99 positive strains of Escherichia coli showed that each had a plasmid of approximately 87.8 kb that hybridized with two DNA probes specific for K99 genes. The K99 reference plasmid from E. coli also is 87.8 kb. Each of these strains had a conserved 7.15-kb BamHI fragment that also hybridized to these probes. Several K99 negative mutants and three 3P- strains also contained K99 plasmids as well as the 7.15-kb BamHI fragment. These results suggest that there is a conservation in size of the K99 plasmids of diverse strains.

Pili of Pasteurella multocida of porcine origin.

Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pasteurella multocida isolated from pigs with atrophic rhinitis. Rigid pili were found on 60-80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocida and as a result frequently were difficult to see. After growth in vitro, piliated P. multocida cells produced few pili (approx. 3-5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.

Transmission of Salmonella typhimurium to swine.

These studies were designed to determine the rate of transmission and the colonization pattern of Salmonella typhimurium in swine. Two experiments were conducted. In experiment 1, swine challenged per os with either S. typhimurium strain 798T + or strain 798N + were exposed to heterologous feces. Following exposure to heterologous strains, heterologous Salmonella were recovered from the feces of infected swine within 3 days and from the tonsil and ileum at necropsy. Bacterial populations in swine initially challenged with Salmonella remained constant. In experiment 2, Salmonella-free swine were commingled with a population of pigs that were shedding 2.69 log10 CFU Salmonella/gram feces. Salmonella was recovered from pooled fecal samples from the commingled swine on day 2 post-exposure to the infected group. Low numbers of Salmonella were detected in the ileocolic lymph node, ileum, cecum or spleen of all commingled swine throughout the necropsy period. These data provide a means for evaluating transmission of Salmonella to a population of swine which may be used to study the mechanisms involved in transmission and maintenance of the disease.

Prevalence of pathogenic Yersinia enterocolitica in groups of swine at slaughter.

In order to estimate the prevalence of swine herds infected with pathogenic Yersinia enterocolitica, 103 lots of market swine were randomly selected at slaughter during six 1-month intervals. Pigs within each lot were sampled by swabbing the oral-pharyngeal surface, poststunning and postexsanguination but prescalding. Ninety-five lots (92.2%) contained at least one pig infected with Y. enterocolitica. Pathogenic strains were defined as those harboring the ail gene which has been identified in Y. enterocolitica that causes human clinical disease. Identification of those strains harboring the ail gene was accomplished using a polymerase chain reaction technique. Twenty-nine lots (28.2%) contained at least one pig from which ail-containing (pathogenic) Y. enterocolitica were isolated. Of the 107 pathogenic Y. enterocolitica isolates identified, 89.7% were serotype O:5 and 3.7% were serotype O:3. The results from this study will aid in the design of future epidemiological investigations concerning on-farm prevalence and associated risk factors for pathogenic Y. enterocolitica. Additionally, the results support the hypothesis that swine are a significant potential reservoir for human infections by Y. enterocolitica.

Development of vaccines for bacterial diseases using recombinant DNA technology.

A vaccine was prepared using recombinant DNA techniques to prevent fatal enterotoxigenic Escherichia coli diarrhea in swine. The product, which is a subunit vaccine, was prepared by mechanical and chemical removal of pilus adhesins from the surface of genetically engineered strains of E. coli. The vaccine contains the pilus adhesins K88, K99, and 987P plus an adjuvant. The genes responsible for production of K88 and K99 were separately cloned into the multicopy vector pBR322. K88 was found to be encoded on a 7.6-kilobase HindIII-EcoRI fragment, and K99 was found to be encoded on a 7.15-kilobase BamHI fragment. Strains containing the recombinant plasmid for K99 produced up to ten times more K99 than strains containing the wild-type plasmid. Vaccination of pregnant pigs with the vaccine led to production of pilus-adhesin-specific antibodies that were transferred to the piglets in colostrum and milk. Pilus-adhesin-specific antibodies neutralized the adhesiveness of the pili on enterotoxigenic E. coli, thus preventing attachment, colonization, and disease. Mortality of pigs in litters from vaccinated pigs due to experimentally induced enterotoxigenic E. coli diarrhea was reduced 10-to-20-fold (depending upon the challenge strain), and the incidence, severity, and duration of diarrhea were also reduced.

Regulators of Escherichia coli K99 region 1 genes.

Expression of K99 is highly regulated being dependent on 8 K99-specific genes and several host-specific genes including cyclic AMP receptor protein (Crp) and leucine responsive protein (Lrp). The 8 K99-specific genes are organized into 3 separately regulated clusters (regions I-III) with region I and II genes being dependent on Crp. Using TnphoA tagged K99 genes, Lrp was shown to be required for expression of region I genes. Differential methylation of GATC boxes is a common method by which Lrp functions as a regulator. Two GATC boxes are present adjacent to the 5' end of fanA, the first gene. Using the restriction enzymes Dpn I and Mbo I which recognize methylated and non-methylated GATC boxes, respectively, it was shown that differential methylation was not a mechanism regulating K99 expression. Using a gel mobility shift assay and protein extracts from various strains, it was shown that a 625 bp DNA fragment adjacent to the 5' end of fanA bound protein prepared from Lrp+ strains but not from Lrp- strains. While region I genes also require CRP for expression, the same degree of gel shift was observed when the extract was prepared from a Crp- strain. The products of fanA and fanB are believed to be positive regulators. However, protein extracts from strains with or without fanA and fanB caused the same degree of gel shift. Thus, while there are a variety of regulators necessary for region I gene expression, only Lrp or Lrp regulated proteins bind to the promoter region 5' to fanA.

Phase variable switching of in vivo and environmental phenotypes of Salmonella typhimurium.

Previously it was shown that S. typhimurium strain 798, which is known to cause persistent asymptomatic infections in pigs, exists in two phenotypes. One phenotype, which is called adhesive, was shown to produce pili, is adhesive to porcine enterocytes, is readily phagocytized, and then survives intracellularly in phagocytes. The other phenotype, termed non-adhesive, does not produce pili, does not attach to enterocytes, is phagocytized less efficiently, and does not survive within the phagocyte. Cells in each phenotype can freely switch to the other phenotype at a fairly high frequency and thus the shift between each phenotype is phase variation. Further analysis of these phenotypes identified 4 additional characteristics that were co-regulated by phase variation. The first is the enterocyte-specific adhesin, which was shown to be type 1 fimbriae. Mutations in fimA, the major pilin molecule, led to a decreased ability to colonize the gut of pigs and mice. The second characteristic is O-antigen production. Adhesive cells produce a long O-antigen (up to 18 subunits) while non-adhesive cells do not (only 1-2 subunits). The long O-antigen produced by the adhesive cells leads to resistance to serum and appears to be the result of phase variable expression of rfaL. A third locus, ebu, has been identified based on differential color production of colonies growing on Evans blue-Uranine plates. The relationship of this trait to in vivo survival or virulence is not known but ebu is genetically related to a family of transcriptional activators. The fourth locus, prv is located on the virulence plasmid and a mutation in prv results in delayed time to death in mice. It is hypothesized that the adhesive phenotype is the in vivo, virulent form, while the non-adhesive phenotype is the environmental, avirulent form. By modulating the fraction of cells in each phase, persistent asymptomatic infections can be promoted.

Effect of transportation and feed withdrawal on shedding of Salmonella typhimurium among experimentally infected pigs.

OBJECTIVE: To determine whether stress associated with transportation or feed withdrawal increased fecal shedding of Salmonella Typhimurium among pigs experimentally infected with the organism. ANIMALS: 86 healthy pigs. PROCEDURE: Pigs were challenge exposed with Salmonella Typhimurium at 4 weeks old and reared conventionally. When pigs reached market weight, they were assigned to groups and subjected to various combinations of transportation and feed withdrawal. Ileocecal contents were collected after slaughter and tested for Salmonella Typhimurium. RESULTS: Salmonella Typhimurium was not detected in feces collected from pigs just prior to slaughter. When feed was withheld for 24 hours prior to slaughter, the proportion of transported pigs with Salmonella Typhimurium in ileocecal contents at the time of slaughter was not significantly different from the proportion of nontransported pigs. However, when feed was not withheld prior to slaughter, the proportion of transported pigs with Salmonella Typhimurium in ileocecal contents at the time of slaughter was significantly higher than the proportion of nontransported pigs. CONCLUSIONS AND CLINICAL RELEVANCE: When carrier pigs remained on feed, transportation stress increased the proportion positive for Salmonella sp. On the basis of results reported here, it is suggested that producers withhold feed from pigs for 24 hours prior to transportation to a slaughter plant.

Pathogenic relationships of rotavirus, Escherichia coli, and other agents in mixed infections in calves.

Infection with agents interpreted as causing or contributing to diarrhea (rotavirus, coronavirus, enterotoxigenic Escherichia coli, and cryptosporidia) were demonstrated in 24 of 32 newborn calves that had naturally occurring diarrheal disease. The calves were from 12 herds in Iowa. Infections as well as enteric lesions and hypoglobulinemia occurred more frequently among diarrheal calves than among nondiarrheal calves from these same herds. In most calves, infections were mixed; ie, both viruses, one or both viruses plus cryptosporidia, or rotavirus plus enterotoxigenic E coli.

Enteric bacterial pathogens, villus atrophy and microbial growth.

The effects of three bacterial pathogens on the villus architecture of small intestines and the role that bacterial virulence factors play in pathogenesis are described. Bacterial pathogens cause a spectrum of effects ranging from severe tissue damage to a lack of perceptible damage. Enterotoxigenic Escherichia coli, which cause acute and severe diarrhea, does so by producing potent toxins, but these toxins act by altering the biological activity in epithelial cells. However, the cells are not damaged. Enteropathogenic E. coli and Salmonella, on the other hand cause various degrees of tissue damage. As part of their pathogenesis, they employ a type III protein secretion system to orchestrate internal changes in target cells. The expression of many virulence related genes is tightly regulated and appears to be turned on in response to cues found in the intestinal tract. The consequences of this level of regulation also is discussed.

Factors affecting expression of the Escherichia coli pilus K99.

An enzyme-linked antibody centrifuge assay for the detection of Escherichia coli pilus K99 was developed and shown to be a specific and quantitative assay for the detection of cell-bound K99. The data presented demonstrate the usefulness of the assay as a diagnostic tool. Using the assay, several factors that affect expression of K99 were investigated. Expression of K99 was dependent upon the degree of aeration provided: nonaerated bacteria produced little or no K99, whereas aerated bacteria produced large amounts of K99. K99 also appeared to be produced only by logarithmically growing cells, whereas there was a demonstrable decline in the amount of K99 per cell during stationary phase. Glucose was shown to repress K99 expression. At 0.5% glucose, K99 expression was highly repressed. Glucose-mediated repression could be overcome by the addition of cyclic adenosine 3',5'-monophosphate. Several other carbon sources also inhibited K99 expression, including pyruvate, arabinose, and lactose; glycerol was stimulatory.

Novel targets for antibiotics--an update.

The emergence of widespread antibiotic resistance as an impediment in the treatment of bacterial diseases is of growing concern. In some instances, clinicians are left with few or no antibiotics for treatment of infections and this problem will more than likely grow in magnitude. One approach to get around the problem of antibiotic resistance is to develop new drugs with novel targets and mechanisms of action. Due to the 'newness' of these novel targets as therapeutic targets, the likelihood that resistance will initially be widespread is low. Three approaches are discussed in this overview: discovery of new essential genes that are expressed exclusively in vivo development of compounds that act on global bacterial gene regulators; and interference with virulence determinants. By exploiting virulence related attributes or genes expressed exclusively in vivo, the risk of resistance is reduced since inhibiting these products will probably alter the ecology (habitats) of these organisms rather than causing direct cell death. This might also lead to a selective targeting of pathogens with the beneficial consequence of ignoring organisms growing in their normal habitat, such as in the gastrointestinal tract or skin.

K99 surface antigen of Escherichia coli: purification and partial characterization.

K99, a presumed colonizing factor of enterotoxigenic Escherichia coli of calf origin, has been purified. K99 was removed from K99+ bacteria by salt extraction and subsequently purified by ammonium sulfate precipitation and column chromatography on diethylaminoethyl-Sephadex. The purified material was homogenous in size, having an s20,w of 13 to 15 S. It was composed of two subunits: a major component with a molecular weight of 22,500 and a minor component of 29,500. When observed in the electron microscope, K99 appeared to be rod-shaped, with a strong tendency for self-aggregation. At concentrations where aggregation was minimized, individual rods were observed with diameters of 8.4 nm and mean lengths of 130 nm. Based on the subunit structure, exterior location, and rod-like shape of K99, it is concluded that K99 is a pilus or pilus-like structure. Chemically, K99 is composed primarily of protein and has an isoelectric point of greater than 10. Purified K99 did not hemagglutinate guinea pig erythrocytes.

K99 surface antigen of Escherichia coli: antigenic characterization.

K99 prepared by acid precipitation hemagglutinated guinea pig erythrocytes, whereas K99 prepared by chromatography on diethylaminoethyl-Sephadex did not. K99 purified by either procedure hemagglutinated horse erythrocytes. K99 prepared by acid precipitation contained a second antigen not presnet in the K99 prepared by chromatography on diethylaminoethyl-Sephadex. This antigen could be detected by immunoprecipitation with some, but not all, sera prepared against K99-positive Escherichia coli strains. It was assumed that this second antigen is not K99 and is responsible for the guinea pig erythrocyte hemagglutination reaction. Furthermore, the second antigen has an isoelectric point of 4.2, which has been reported by Morris and co-workers to be the isoelectric point of K99.

Bacterial adherence to mucosal surfaces: an attribute of virulence.

Colonization of mucosal habitats is, with very few exceptions, a necessary prerequisite that must be satisfied for a bacterial organism to be virulent. The mechanism(s) whereby bacteria colonize such habitats is, for the most part, by association with the mucosa and proliferation at that site. However, the precise mechanism(s) of association is not known for most organisms. Direct adherence to the mucosal surface of the small intestine by some enterotoxigenic strains of Escherichia coli (ETEC) has been demonstrated both in vivo and in vitro. Specific surface appendages (pili) on the bacterial cell surface facilitate the direct attachment of bacteria to microvilli and as such have been termed adhesins. The adhesins of ETEC that cause diarrheal disease in pigs have been most extensively studied. Two adhesins, K88 and K99, are genetically encoded on plasmids while a third one, 987P, appears to be encoded on the chromosome. All three adhesins are composed of identical repeating protein subunits with molecular weights of 18,100-26,000 that undergo specific aggregation to form large polymers. These polymers are the active adhesins and appear as pili (synonym: fimbriae) when observed in the electron microscope. The function of these adhesins has been established by construction of mutants or plasmidless strains that do not produce the adhesin and by reintroduction of the adhesin genes back into the mutants. Only cells that produce the adhesins colonize and adhere to the mucosa of the pig intestine in vivo and thus produce diarrheal disease. The interaction of adhesin with the mucosal surface is mediated by specific receptors. Current data indicate that these receptors are glycoconjugates.

Regulation of expression of Escherichia coli pilus K99.

An immunoassay demonstrated that the assembled K99 pilus on the surface of Escherichia coli grown in minimal medium appeared during the logarithmic phase of growth, but the synthesis of K99 subunits, as measured by nonequilibrium two-dimensional gel electrophoresis, occurred throughout the life cycle of the cell. Contrary to other reports, the addition of glucose to the growth medium did not affect K99 pilus assembly or subunit synthesis, although in a K99+ adenyl cyclase (cya) mutant, subunit synthesis was reduced. There was no reduction in the amount of assembled K99 on the cell surface of the cya mutant compared with the wild-type parent. The addition of L-alanine to minimal medium repressed K99 synthesis. However, if L-threonine or L-isoleucine was also included in the growth medium, the effect of L-alanine was reduced. Chloramphenicol caused a complete inhibition of K99 subunit synthesis, but assembly proceeded normally. Growth at 18 degrees C inhibited both subunit synthesis and pilus assembly. Approximately 92% of all cellular K99 was associated with the outer membrane, and 4% was associated with the inner membrane. No K99 was detected in the cytoplasm.