Comparison of anti-Campylobacter activity of free thymol and thymol-ß-D-glucopyranoside in absence or presence of ß-glycoside-hydrolysing gut bacteria.

Thymol is a natural product that exhibits antimicrobial activity in vitro but in vivo results indicate that absorption within the proximal alimentary tract precludes its delivery to the distal gut. Presently, the anti-Campylobacter activity of thymol was compared against that of thymol-ß-D-glucopyranoside, the latter being resistant to absorption. When treated with 1 mM thymol, Campylobacter coli and jejuni were reduced during pure or co-culture with a ß-glycoside-hydrolysing Parabacteroides distasonis. Thymol-ß-D-glucopyranoside treatment (1 mM) did not reduce C. coli and jejuni during pure culture but did during co-culture with P. distasonis or during mixed culture with porcine or bovine faecal microbes possessing ß-glycoside-hydrolysing activity. Fermentation acid production was reduced by thymol-ß-D-glucopyranoside treatment, indicating that fermentation was inhibited, which may limit its application to just before harvest. Results suggest that thymol-ß-D-glucopyranoside or similar ß-glycosides may be able to escape absorption within the proximal gut and become activated by bacterial ß-glycosidases in the distal gut.

Altered egos: antibiotic effects on food animal microbiomes.

The human food chain begins with upwards of 1,000 species of bacteria that inhabit the intestinal tracts of poultry and livestock. These intestinal denizens are responsible for the health and safety of a major protein source for humans. The use of antibiotics to treat animal diseases was followed by the surprising discovery that antibiotics enhanced food animal growth, and both led to six decades of antibiotic use that has shaped food animal management practices. Perhaps the greatest impact of antibiotic feeding in food animals has been as a selective force in the evolution of their intestinal bacteria, particularly by increasing the prevalence and diversity of antibiotic resistance genes. Future antibiotic use will likely be limited to prudent applications in both human and veterinary medicine. Improved knowledge of antibiotic effects, particularly of growth-promoting antibiotics, will help overcome the challenges of managing animal health and food safety.

Biochemical and molecular characterization of Treponema phagedenis-like spirochetes isolated from a bovine digital dermatitis lesion.

BACKGROUND: Bovine papillomatous digital dermatitis (DD) is the leading cause of lameness in dairy cattle and represents a serious welfare and economic burden. Found primarily in high production dairy cattle worldwide, DD is characterized by the development of an often painful red, raw ulcerative or papillomatous lesion frequently located near the interdigital cleft and above the bulbs of the heel. While the exact etiology is unknown, several spirochete species have been isolated from lesion material. Four isolates of Treponema phagedenis-like spirochetes were isolated from dairy cows in Iowa. Given the distinct differences in host, environmental niche, and disease association, a closer analysis of phenotypic characteristics, growth characteristics, and genomic sequences of T. phagedenis, a human genitalia commensal, and the Iowa DD isolates was undertaken. RESULTS: Phenotypically, these isolates range from 8.0 to 9.7 µm in length with 6-8 flagella on each end. These isolates, like T. phagedenis, are strictly anaerobic, require serum and volatile fatty acids for growth, and are capable of fermenting fructose, mannitol, pectin, mannose, ribose, maltose, and glucose. Major glucose fermentation products produced are formate, acetate, and butyrate. Further study was conducted with a single isolate, 4A, showing an optimal growth pH of 7.0 (range of 6-8.5) and an optimal growth temperature of 40 °C (range of 29 °C-43 °C). Comparison of partial genomic contigs of isolate 4A and contigs of T. phagedenis F0421 revealed > 95% amino acid sequence identity with amino acid sequence of 4A. In silico DNA-DNA whole genome hybridization and BLAT analysis indicated a DDH estimate of >80% between isolate 4A and T. phagedenis F0421, and estimates of 52.5% or less when compared to the fully sequenced genomes of other treponeme species. CONCLUSION: Using both physiological, biochemical and genomic analysis, there is a lack of evidence for difference between T. phagedenis and isolate 4A. The description of Treponema phagedenis should be expanded from human genital skin commensal to include being an inhabitant within DD lesions in cattle.

High-impact animal health research conducted at the USDA's National Animal Disease Center.

Commissioned by President Dwight Eisenhower in 1958 and opened with a dedication ceremony in December 1961, the USDA, Agricultural Research Service (ARS), National Animal Disease Center (NADC) celebrated its 50-year anniversary in November 2011. Over these 50 years, the NADC established itself among the world's premier animal health research centers. Its historic mission has been to conduct basic and applied research on selected endemic diseases of economic importance to the U.S. livestock and poultry industries. Research from NADC has impacted control or management efforts on nearly every major animal disease in the United States since 1961. For example, diagnostic tests and vaccines developed by NADC scientists to detect and prevent hog cholera were integral in the ultimate eradication of this costly swine disease from the U.S. Most major veterinary vaccines for critical diseases such as brucellosis and leptospirosis in cattle, porcine respiratory and reproductive syndrome (PRRS), porcine parvovirus and influenza in swine had their research origins or were developed and tested at the NADC. Additional discoveries made by NADC scientists have also resulted in the development of a nutritional approach and feed additives to prevent milk fever in transition dairy cattle. More recently, NADC's archive of historic swine influenza viruses combined with an established critical mass of influenza research expertise enabled NADC researchers to lead an effective national research response to the pandemic associated with the novel 2009 H1N1 influenza virus. This review commemorates some of the key animal health contributions in NADC's first 50 years, recaps the newly completed modernization of the center into new facilities, and offers highlights of the ongoing research that will define NADC's mission going forward.

Butyrate-producing bacteria, including mucin degraders, from the swine intestinal tract.

To identify bacteria with potential for influencing gut health, 980 anaerobes were cultured from the swine intestinal tract and analyzed for butyrate production. Fifteen isolates in the order Clostridiales produced butyrate and had butyryl coenzyme A (CoA):acetate CoA transferase activity. Three of the isolates grew on mucin, suggesting an intimate association with host intestinal mucosa.

Mitsuokella jalaludinii inhibits growth of Salmonella enterica serovar Typhimurium.

Salmonella continues to be a significant human health threat, and the objective of this study was to identify microorganisms with the potential to improve porcine food-safety through their antagonism of Salmonella. Anaerobic culture supernatants of 973 bacterial isolates from the gastrointestinal tract and feces of swine were screened for their capacity to inhibit the growth of Salmonella enterica serovar Typhimurium. Growth inhibition of 1000-fold or greater was observed from 16 isolates, and 16S rRNA sequencing identified the isolates as members of the genera Mitsuokella, Escherichia/Shigella, Anaerovibrio, Selenomonas, and Streptococcus. Four isolates were identified as Mitsuokella jalaludinii, and the mechanism of Salmonella Typhimurium growth inhibition by M. jalaludinii was further investigated. M. jalaludinii stationary phase culture supernatants were observed to significantly inhibit growth, and featured the production of lactic, succinic, and acetic acids. Aerobic and anaerobic S. Typhimurium growth was restored when the pH of the culture supernatants (pH 4.6) was increased to pH 6.8. However, S. Typhimurium growth in fermentation acid-free media was the same at pH 4.6 and pH 6.8 - indicating a synergistic effect between fermentation acid production and low pH as the cause of S. Typhimurium growth inhibition. Furthermore, exposure of S. Typhimurium to M. jalaludinii culture supernatants inhibited Salmonella invasion of HEp-2 cells by 10-fold. The results identify M. jalaludinii as a possible inhibitor of Salmonella growth and invasion in swine, and thus a potential probiotic capable of improving food safety.

In-feed antibiotic effects on the swine intestinal microbiome.

Antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and growth promotion for over 50 y. The impact of such antibiotic use on the treatment of human diseases is hotly debated. We raised pigs in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics [chlortetracycline, sulfamethazine, and penicillin (known as ASP250)] and the other portion receiving the same diet but without the antibiotics. We used phylogenetic, metagenomic, and quantitative PCR-based approaches to address the impact of antibiotics on the swine gut microbiota. Bacterial phylotypes shifted after 14 d of antibiotic treatment, with the medicated pigs showing an increase in Proteobacteria (1-11%) compared with nonmedicated pigs at the same time point. This shift was driven by an increase in Escherichia coli populations. Analysis of the metagenomes showed that microbial functional genes relating to energy production and conversion were increased in the antibiotic-fed pigs. The results also indicate that antibiotic resistance genes increased in abundance and diversity in the medicated swine microbiome despite a high background of resistance genes in nonmedicated swine. Some enriched genes, such as aminoglycoside O-phosphotransferases, confer resistance to antibiotics that were not administered in this study, demonstrating the potential for indirect selection of resistance to classes of antibiotics not fed. The collateral effects of feeding subtherapeutic doses of antibiotics to agricultural animals are apparent and must be considered in cost-benefit analyses.

Chlortetracycline-resistant intestinal bacteria in organically raised and feral Swine.

Organically raised swine had high fecal populations of chlortetracycline (CTC)-resistant (growing at 64 µg CTC/ml) Escherichia coli, Megasphaera elsdenii, and anaerobic bacteria. By comparison, CTC-resistant bacteria in feral swine feces were over 1,000-fold fewer and exhibited lower taxonomic diversity.

Persistence of antibiotic resistance: evaluation of a probiotic approach using antibiotic-sensitive Megasphaera elsdenii strains to prevent colonization of swine by antibiotic-resistant strains.

Megasphaera elsdenii is a lactate-fermenting, obligately anaerobic bacterium commonly present in the gastrointestinal tracts of mammals, including humans. Swine M. elsdenii strains were previously shown to have high levels of tetracycline resistance (MIC=64 to >256 µg/ml) and to carry mosaic (recombinant) tetracycline resistance genes. Baby pigs inherit intestinal microbiota from the mother sow. In these investigations we addressed two questions. When do M. elsdenii strains from the sow colonize baby pigs? Can five antibiotic-sensitive M. elsdenii strains administered intragastrically to newborn pigs affect natural colonization of the piglets by antibiotic-resistant (AR) M. elsdenii strains from the mother? M. elsdenii natural colonization of newborn pigs was undetectable (<10(4) CFU/g [wet weight] of feces) prior to weaning (20 days after birth). After weaning, all pigs became colonized (4 × 10(5) to 2 × 10(8) CFU/g feces). In a separate study, 61% (76/125) of M. elsdenii isolates from a gravid sow never exposed to antibiotics were resistant to chlortetracycline, ampicillin, or tylosin. The inoculation of the sow's offspring with mixtures of M. elsdenii antibiotic-sensitive strains prevented colonization of the offspring by maternal AR strains until at least 11 days postweaning. At 25 and 53 days postweaning, however, AR strains predominated. Antibiotic susceptibility phenotypes and single nucleotide polymorphism (SNP)-based identities of M. elsdenii isolated from sow and offspring were unexpectedly diverse. These results suggest that dosing newborn piglets with M. elsdenii antibiotic-sensitive strains delays but does not prevent colonization by maternal resistant strains. M. elsdenii subspecies diversity offers an explanation for the persistence of resistant strains in the absence of antibiotic selection.

Experimental inoculation of raccoons (Procyon lotor) with Spiroplasma mirum and transmissible mink encephalopathy (TME).

The primary objective of this study was to determine whether or not Spiroplasma mirum would be capable of producing lesions of transmissible spongiform encephalopathy (TSE) when inoculated in raccoons (Procyon lotor) and, if that was possible, to compare the clinicopathological findings with those of transmissible mink encephalopathy (TME) in the same experimental model. For this purpose, 5 groups (n = 5) of raccoon kits were inoculated intracerebrally with either S. mirum and/or TME. Two other groups (n = 5) of raccoon kits served as sham-inoculated controls. All animals inoculated with TME, either alone or in combination, showed clinical signs of neurologic disorder and were euthanized within 6 mo post-inoculation (MPI). None of the carcasses revealed gross lesions. Spongiform encephalopathy was observed by light microscopy and the presence of abnormal disease-causing prion protein (PrP(d)) was detected by immunohistochemistry (IHC) and Western blot (WB) techniques in only the raccoons administered TME. Raccoons inoculated with Spiroplasma, but not administered TME agent, were euthanized at 30 MPI. They did not show clinical neurologic signs, their brains did not have lesions of spongiform encephalopathy, and their tissues were negative for S. mirum by polymerase chain reaction (PCR) and for PrP(d) by IHC and WB techniques. The results of this study indicate that Spiroplasma mirum does not induce TSE-like disease in raccoons.

Induction and transcription of VSH-1, a prophage-like gene transfer agent of Brachyspira hyodysenteriae.

The anaerobic spirochete Brachyspira hyodysenteriae is host to a bacteriophage-like agent known as VSH-1. VSH-1 is a novel gene transfer mechanism which does not self-propagate and transfers random 7.5kb fragments of host DNA between B. hyodysenteriae cells. In these investigations early events during VSH-1 induction by mitomycin C were examined. Quantitative PCR analysis revealed that VSH-1 hvp38 and hvp53 genes did not detectably increase in copy numbers during induction. Based on Northern blot hybridization assays, transcription of VSH-1 genes hvp38, hvp53, hvp45, hvp101, and lys increased fivefold to tenfold between 2 and 4h after induction whereas mRNA levels for B. hyodysenteriae flaA1 declined over the same time period. Chloramphenicol prevented the mitomycin C-induced increases in VSH-1 gene transcription. Hydrogen peroxide (300muM) substituted for mitomycin C as an inducer of VSH-1 gene transcription and is a possible 'natural' inducer of VSH-1 production in vivo. Northern blot hybridization, RT PCR, and primer extension analyses showed that VSH-1 genes are co-transcribed at an initiation site upstream of the VSH-1 gene operon. Two direct heptanucleotide repeats (ACTTATA) were identified between the putative -35 and -10 positions of the VSH-1 gene operon and are likely to represent a binding site for transcription proteins. These findings indicate VSH-1 virion production does not require genome replication, consistent with the inability of VSH-1 to self-propagate. Early events in VSH-1 induction include de novo synthesis of protein(s) essential for transcription of VSH-1 genes as polycistronic mRNA initiating upstream of the hvp45 gene.

Prophage-like gene transfer agents-novel mechanisms of gene exchange for Methanococcus, Desulfovibrio, Brachyspira, and Rhodobacter species.

Gene transfer agents (GTAs) are novel mechanisms for bacterial gene transfer. They resemble small, tailed bacteriophages in ultrastructure and act like generalized transducing prophages. In contrast to functional prophages, GTAs package random fragments of bacterial genomes and incomplete copies of their own genomes. The packaged DNA content is characteristic of the GTA and ranges in size from 4.4 to 13.6kb. GTAs have been reported in species of Brachyspira, Methanococcus, Desulfovibrio, and Rhodobacter. The best studied GTAs are VSH-1 of the anaerobic, pathogenic spirochete Brachyspira hyodysenteriae and RcGTA of the nonsulfur, purple, photosynthetic bacterium Rhodobacter capsulatus. VSH-1 and RcGTA have likely contributed to the ecology and evolution of these bacteria. The existence of GTAs in phylogenetically diverse bacteria suggests GTAs may be more common in nature than is now appreciated.

Identification of genes of VSH-1, a prophage-like gene transfer agent of Brachyspira hyodysenteriae.

VSH-1 is a mitomycin C-inducible prophage of the anaerobic spirochete Brachyspira hyodysenteriae. Purified VSH-1 virions are noninfectious, contain random 7.5-kb fragments of the bacterial genome, and mediate generalized transduction of B. hyodysenteriae cells. In order to identify and sequence genes of this novel gene transfer agent (GTA), proteins associated either with VSH-1 capsids or with tails were purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequences of 11 proteins were determined. Degenerate PCR primers were designed from the amino acid sequences and used to amplify several VSH-1 genes from B. hyodysenteriae strain B204 DNA. A lambda clone library of B. hyodysenteriae B204 DNA was subsequently screened by Southern hybridization methods and used to identify and sequence overlapping DNA inserts containing additional VSH-1 genes. VSH-1 genes spanned 16.3 kb of the B. hyodysenteriae chromosome and were flanked by bacterial genes. VSH-1 identified genes and unidentified, intervening open reading frames were consecutively organized in head (seven genes), tail (seven genes), and lysis (four genes) clusters in the same transcriptional direction. Putative lysis genes encoding endolysin (Lys) and holin proteins were identified from sequence and structural similarities of their translated protein products with GenBank bacteriophage proteins. Recombinant Lys protein hydrolyzed peptidoglycan purified from B. hyodysenteriae cells. The identified VSH-1 genes exceed the DNA capacity of VSH-1 virions and do not encode traditional bacteriophage early functions involved in DNA replication. These genome properties explain the noninfectious nature of VSH-1 virions and further confirm its resemblance to known prophage-like, GTAs of other bacterial species, such as the GTA from Rhodobacter capsulatus. The identification of VSH-1 genes will enable analysis of the regulation of this GTA and should facilitate investigations of VSH-1-like prophages from other Brachyspira species.

Detection of bacteriophage VSH-1 svp38 gene in Brachyspira spirochetes.

VSH-1 is a mitomycin C-inducible, non-lytic, phage-like agent that packages random 7.5-kb fragments of the Brachyspira hyodysenteriae genome. VSH-1 is the first recognized mechanism for gene transfer between B. hyodysenteriae cells. To analyze the distribution of VSH-1 among spirochetes, a 344-bp probe for gene svp38, encoding the VSH-1 major head protein, was amplified by polymerase chain reaction and used in Southern blot hybridizations with genomic DNA from various spirochete genera. The svp38 probe hybridized to a 40-kb SalI-SmaI fragment of the B. hyodysenteriae B78(T) chromosome, indicating VSH-1 DNA insertion into the chromosome at a unique site. Restriction endonuclease digested DNAs of 27 spirochete strains representing six Brachyspira species (B. hyodysenteriae, B. innocens, B. pilosicoli, B. murdochii, B. intermedia, B. alvinipulli) contained a single fragment hybridizing with the svp38 probe. DNAs from spirochete species of the genera Treponema, Spirochaeta, Borrelia, and Leptospira did not hybridize with the probe. VSH-1-like agents appear to be widely distributed among Brachyspira species and, as has been demonstrated for B. hyodysenteriae, may serve as useful gene transfer agents for those other species.