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.

Identification of patterns of transmission of Salmonella within swine production systems using pulsed field gel electrophoresis (PFGE) and repetitive sequence polymerase chain reaction (REP-PCR): a quantitative analysis.

Pulsed field gel electrophoresis (PFGE) using 3 enzymes (Spe I, Xba I, Avr II) and repetitive sequence polymerase chain reaction (REP-PCR) with 3 primers (BOX, ERIC, REP) were compared with respect to their validity as a method for identifying transmission of Salmonella on swine farms. Sixty-eight isolates of Salmonella were obtained from feces of swine, cats, mice, and birds, insect body parts, water and floor samples, and boot scrapings collected on 9 swine farms in Illinois USA. Genetic distances between isolates were calculated using the Dice matching coefficient. Cluster analysis of distance matrices was conducted using the UPG-MA algorithm. There was no significant difference between PFGE and REP-PCR in the genetic diversity detected; however, REP-PCR differentiated between 14 pairs of isolates which PFGE identified as identical. There were no significant differences between PFGE and REP-PCR in identifying all or most close genetic links as isolates from the same farm, the same building, and from the same sampling visit, suggesting ecological validity for both methods. Thus, REP-PCR should be considered as an acceptable and perhaps preferable alternative to PFGE as a genotyping method for studies of Salmonella transmission.

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.

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.

Identification and characterization of a phase-variable nonfimbrial Salmonella typhimurium gene that alters O-antigen production.

Salmonella typhimurium 798, which was isolated from a pig, is known to phase vary from a nonadhesive to an adhesive phenotype. Cells of the adhesive phenotype adhere to porcine enterocytes, are more readily phagocytized by porcine neutrophils and macrophages, and once phagocytized can survive intracellularly, while cells of the nonadhesive phenotype die rapidly. The effect of phenotypic switching also can be visualized by changes in colony morphologies and the presence of between 10 and 15 proteins in the envelopes of cells in the adhesive phenotype. Mutants previously constructed with cells in the adhesive phenotype and the transposon TnphoA were screened to identify mutants lacking one or more of the unique proteins. One mutation was cloned and sequenced, and the mutation was shown to be in rfaL (O-antigen ligase). Expression of O antigen was shown to be phase variable. The adhesive strain expressed an O antigen that was at least eightfold longer than that for the nonadhesive strain and by virtue of O-antigen production was resistant to porcine complement. The mutant survived intracellularly in phagocytic cells as well as its wild-type parent.

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.

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.

In vivo expression of the beta-glucoside (bgl) operon of Escherichia coli occurs in mouse liver.

An Escherichia coli DNA fragment was identified that contained part of the beta-glucoside (bgl) operon. This fragment was identified because it contained a promoter that was responsible for the expression of a reporter gene, the chloramphenicol acetyltransferase gene, in a mouse liver during bacterial infection but not when a bacterial clone was grown in vitro. This fragment contained a promoter and a rho-independent transcription terminator which were flanked by the 3' end of bglG and the 5' end of bglF. Reverse transcription-PCR confirmed that cat-specific mRNA was produced in infected mouse liver but not in vitro. mRNA encoding the positive regulator of the bgl operon, bglG, also was detected in mouse liver infected with an E. coli strain. These results demonstrated that expression of the bgl operon occurs in infected mouse liver and suggests a unique role for this operon in vivo.

Effects of mastitis on the volume and composition of colostrum produced by Holstein cows.

The effects of mastitis during the late nonlactating period on colostral volume and concentrations and total yields of immunoglobulin (Ig) G1, fat, and protein in colostrum were investigated using matched pairs of mammary glands from multiparous Holstein cows. Samples of mammary secretions were collected at approximately 14 and 7 d prepartum and within 3 h after calving. At each sampling time, the glands and secretions were examined for gross abnormalities, and the California Mastitis Test was performed. Duplicate secretion samples from each gland were cultured, and somatic cell count, pH, and fat and protein concentrations were determined. The volume of colostrum obtained at the first milking of each gland was quantified using a quarter milking device, and its IgG1 concentration was measured. Colostral volume from persistently infected mammary glands was lower than that from matched uninfected glands, as was the total mass of IgG1. However, infection did not alter IgG1 concentration in colostrum. Fat and protein percentages were lower in prepartum secretions but not in colostrum from infected glands. Persistent infection was associated with increased somatic cell count and pH of secretions at all sampling times, and California Mastitis Test scores were higher for colostrum from infected glands. The appearance of secretions was extremely variable, but the presence of flakes or clots in colostrum was associated with infection. We concluded that mastitis during the late nonlactating period alters mammary gland function but is unlikely to be an important contributor to the high rate of failure of passive transfer of immunoglobulins in calves.

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.

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.

Expression of the gene cluster associated with the Escherichia coli pilus adhesin K99.

The biogenesis of the pilus adhesin K99 is dependent on the expression of eight contiguous genes, fanA to fanH. Transposon mutants were prepared by using TnlacZ and TnphoA, and selected transposon mutants were used to measure expression of each K99 gene. Expression of the K99 genes is likely controlled at the transcription level, since in general, there were no differences between the results obtained with the two transposons. fanC was the most highly expressed, and fanD was expressed at very low levels. The expression of TnlacZ fusions in fanA and fanB fusions was high. Deletion of fanA, fanB, and part of fanC abolished the expression of fanD but had no effect on the distal genes fanE to fanH. To locate the DNA regions required for expression of fanE to fanH, deletion mutations were prepared and the effects on expression of fanE to fanH were determined. The deletion of a segment between fanD and fanE abolished fanE and fanF expression but did not affect fanG and fanH. The deletion of a portion of fanF (approximately 1 kb proximal to fanG) abolished the expression of fanG and fanH. These results indicate the presence of regulatory elements proximal to fanE and to fanG. Putative promoters were identified in these regions by DNA homology and by primer extension. A stem-loop structure that may act as a transcriptional attenuator of fanF was also found at the beginning of fanF. These data confirm our previous model of K99 transcriptional organization.

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.

Analysis of a naturally occurring K99+ enterotoxigenic Escherichia coli strain that fails to produce K99.

A spontaneously occurring field isolate of enterotoxigenic Escherichia coli that was genotypically K99+ but phenotypically K99- was analyzed for the reason that it did not express K99. The defect, which was cis active, was located within an area 5' to the first gene required for K99 biogenesis and was the result of the deletion of a single base pair.

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.

Transcriptional organization of the Escherichia coli pilus adhesin K99.

The production of the Escherichia coli pilus adhesin K99 requires the expression of eight unique proteins: FanA-H. The transcriptional organization of the K99 operon was investigated by Northern blot analysis. Four RNAs of 0.54, 1.4, 2.5 and 3.5 kb were detected. When a fanC probe was used all four RNAs were detected while the use of fanD, fanF and fanG probes detected two RNAs each. Using several deletion and TnphoA insertion mutants it was concluded that there were seven unique K99-specific transcripts, several of which were of the same approximate sizes (1.4 and 2.5 kb). It also was concluded that K99 was comprised of at least three complementation groups, two of which were regulated by catabolite repression.

Adhesion of Salmonella typhimurium to porcine intestinal epithelial surfaces: identification and characterization of two phenotypes.

Salmonella typhimurium 798 is known to persistently colonize swine. A key step required to initiate colonization of intestines is adhesion of the organism to the intestinal epithelium. However, S. typhimurium 798 initially failed to attach to porcine enterocytes in vitro. An enrichment procedure was used to select adhesive S. typhimurium, and when cells of one colony type were grown in tryptone phosphate broth they were adhesive. Cells from a colony with a different morphology were not adhesive. Adhesion was time dependent, with maximal adhesion occurring at 1 h. As determined by electron microscopy, cells of the adhesive phenotype had pili while none of the cells with the nonadhesive phenotype produced pili. The pili on the adhesive cells were morphologically similar to type 1 pili. Mannose (0.5%) did not affect adhesion, suggesting that the adhesin on strain 798 did not recognize mannose as a receptor. An analysis of envelope proteins from cells of both phenotypes showed that the adhesive-phenotype cells expressed at least 10 unique proteins ranging in size from 20 to 60 kDa. Absorbed antiserum against cells of the adhesive phenotype agglutinated adhesive cells and was used to detect unique surface antigens on the cells of the adhesive phenotype by Western blots (immunoblots). These antigens were in the range of 30 kDa in size. An envelope extract competitively inhibited the binding of S. typhimurium to enterocytes, as did Fab fragments prepared from the absorbed serum. Cells of both phenotypes contained two plasmids, and each had identical restriction digestion patterns. Cells of the adhesive phenotype consistently were found to be more readily phagocytosed by pig leukocytes, and once in the phagocytes they survived better than cells of the nonadhesive phenotype.

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.

In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection.

Azithromycin, a novel azalide antibiotic, concentrated in human and mouse polymorphonuclear leukocytes (PMNs), murine peritoneal macrophages, and mouse and rat alveolar macrophages, attaining intracellular concentrations up to 226 times the external concentration in vitro. In murine peritoneal macrophages, azithromycin achieved concentration gradients (internal to external) up to 26 times higher than erythromycin. The cellular uptake of azithromycin was dependent on temperature, viability, and pH and was decreased by 2,4-dinitrophenol. Azithromycin did not decrease phagocyte-mediated bactericidal activity or affect PMN or macrophage oxidative burst activity (H2O2 release or Nitro Blue Tetrazolium reduction, respectively). Azithromycin remained in cells for several hours, even after extracellular drug was removed. However, its release was significantly enhanced by phagocytosis of Staphylococcus aureus (82 versus 23% by 1.5 h). In vivo, 0.05 micrograms of azithromycin was found in peritoneal fluids of mice 20 h after oral treatment with a dose of 50 mg/kg. Following caseinate-induced PMN infiltration, there was a sixfold increase in peritoneal cavity azithromycin to 0.32 micrograms, most of which was intracellular. Therefore, the uptake, transport, and later release of azithromycin by these cells demonstrate that phagocytes may deliver active drug to sites of infection.

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.