Mucosa-associated microbiota from different regions of the gastrointestinal (GI) tract of adult broilers was studied by analysis of 16S rRNA gene sequences. The microbiota mainly comprised Gram-positive bacteria along the GI tract. Fifty-one operational taxonomic units (OTUs) (from 98 clones) were detected in the ceca, as compared with 13 OTUs (from 49 clones) in the crops, 11 OTUs (from 51 clones) in the gizzard, 14 OTUs (from 52 clones) in the duodenum, 12 OTUs (from 50 clones) in the jejunum and nine OTUs (from 50 clones) in the ileum. Ceca were dominantly occupied by clostridia-related sequences (40%) with other abundant sequences being related to Faecalibacterium prausnitzii (14%), Escherichia coli (11%), lactobacilli (7%) and Ruminococcus (6%). Lactobacilli were predominant in the upper GI tract and had the highest diversity in the crop. Both Lactobacillus aviarius and Lactobacillus salivarius were the predominant species among lactobacilli. Candidatus division Arthromitus was also abundant in the jejunum and ileum.

Aims:  To assess the potential of essential oils and structurally related synthetic food additives in reducing bacterial pathogens in swine intestinal tract. Methods and Results:  The antimicrobial activity of essential oils/compounds was measured by determining the inhibition of bacterial growth. Among 66 essential oils/compounds that exhibited ≥80% inhibition towards Salmonellatyphimurium DT104 and Escherichia coli O157:H7, nine were further studied. Most of the oils/compounds demonstrated high efficacy against S. typhimurium DT104, E. coli O157:H7, and E. coli with K88 pili with little inhibition towards lactobacilli and bifidobacteria. They were also tolerant to the low pH. When mixed with pig cecal digesta, these oils/compounds retained their efficacy against E. coli O157:H7. In addition, they significantly inhibited E. coli and coliform bacteria in the digesta, but had little effect on the total number of lactobacilli and anaerobic bacteria. Conclusions:  Some essential oils/compounds demonstrated good potential, including efficacy, tolerance to low pH, and selectivity towards bacterial pathogens, in reducing human and animal bacterial pathogens in swine intestinal tract. Significance and Impact of the Study:  This study has identified candidates of essential oils/compounds for in vivo studies to develop antibiotic substitutes for the reduction of human and animal bacterial pathogens in swine intestinal tract.

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Aims: To determine the effect of pig diets in vitro on the antimicrobial activity of carvacrol, thymol and cinnamaldehyde, and to identify an emulsifier/stabilizer that can stabilize the essential oil (EO) components in aqueous solution and retain their antimicrobial activity in the presence of the diets. Methods and Results: Emulsification of essential oil components with hydrocolloid solution was achieved by blending with a Polytron. Antimicrobial activity was measured through in vitro assays to determine the inhibition of bacterial growth by measuring the optical density at 600 nm or plating on nutrition agar after incubation of the mixtures of an EO component with the culture of Salmonella serotype Typhimurium DT104 in the presence or absence of pig diets. The results generated through the in vitro assays indicated that pig diets were able to abolish the antimicrobial activity of EOs. Xanthan, fenugreek and yellow mustard gums were the best in forming stable emulsions of five different EO components among ten different plant polysaccharides and surfactants examined. Emulsification of all the EO components in the fenugreek gum solution did not alter their antimicrobial activity. However, the antimicrobial activity of geraniol was significantly reduced when emulsified with other polysaccharides and surfactants. Both fenugreek and xanthan gum solutions were unable to protect the antimicrobial activity of carvacrol and thymol when mixed with the diets. Although cinnamaldehyde required no emulsification, but a high concentration (equivalent to at least three times of minimum bactericidal concentration for cinnamon oil) to inhibit Salmonella growth significantly in the presence of the diets, emulsification in fenugreek gum appeared to be essential for cinnamaldehyde solution to retain its antimicrobial activity during storage. Conclusions: The diets for newly weaned pigs were a significant factor limiting the antimicrobial activity of EOs and their components. Cinnamaldehyde required a high concentration to retain its antimicrobial activity in the diets, in addition to its requirement for emulsification to stabilize its activity during the storage. Significance and Impact of the Study: The assay with the diets used in this study for measuring the antimicrobial activity can be used in vitro for rapid and effective screening of potential antimicrobials for swine production. This study has identified polysaccharides that are able to stabilize EO component solutions. It has also identified cinnamaldehyde for further in vivo studies that may have potential in future application in controlling Salmonella and possibly other enteric pathogens in swine production.

Aims:  To assess the potential of essential oils and structurally related synthetic food additives in inhibiting the growth of Clostridium perfringens for the control of necrotic enteritis in chickens. Methods and Results:  The antimicrobial activity of essential oils/compounds was measured by determining the inhibition of bacterial growth. Thirty‐three of 66 oils/compounds exhibited ≥80% inhibition. Seven with the highest potency were further studied. The oils/compounds had MIC95 values between 167 and 425 μg ml−1. Most of them were tolerant to low pH (2·0) and exhibited minor or no inhibition of Lactobacillus isolates from the chicken intestine. When mixed with chicken ileal digesta, the oils/compounds retained their efficacy against C. perfringens, but had little effect on the total number of lactobacilli and anaerobic bacteria in the digesta. Conclusions:  Some essential oils/compounds demonstrated good potential in controlling C. perfringens. Significance and Impact of the Study:  This study has identified candidates of essential oils/compounds for in vivo studies for the control of necrotic enteritis in chickens.

Host-specific probiotics have been used to control enteric pathogens, including foodborne pathogens, in food animal production. However, evaluation of the efficacy of these probiotics requires costly in vivo assays in the target animal. The nematode Caenorhabditis elegans has been used for prescreening of antimicrobial agents and for studies of host-pathogen interactions. In the present study, 17 Lactobacillus isolates from chicken and pig intestines were tested with C. elegans, and the ability of these isolates to prevent death from Salmonella infection was variable. Two Lactobacillus isolates (S64, which gave full protection, and CL11, which gave no protection) were further studied. Both isolates exhibited a similar colonization profile in the C. elegans intestine. Although different culture fractions of CL11 were not protective, both live and heat-killed S64 cells provided full or partial protection of C. elegans from death caused by Salmonella infection. In contrast, different culture fractions from both isolates had similar effects on the colonization of the nematode intestine by Salmonella Typhimurium DT104. Our preliminary results from a pig performance trial revealed a correlation between the degree of protection in the C. elegans survival assay and the performance of 35-day-old weaned piglets that were treated with the same Lactobacillus isolates, suggesting that C. elegans can be used as a laboratory animal model for preselecting probiotics for control of Salmonella infections.

VP1, the putative virion-associated RNA-dependent RNA polymerase (RdRp) of infectious pancreatic necrosis virus (IPNV) can be guanylylated in vitro whereupon it becomes a primer for in vitro RNA synthesis [Virology 208 (1995) 19]. The role of a template or other virion polypeptides in the reaction is unknown. To shed light on this question, his-tagged recombinant VP(1) (rVP1) was expressed both in Escherichia coli and insect cells and used in the guanylylation reaction. Unlike other viral VPg polypeptides, the purified rVP1 alone could guanylylate itself in vitro in a template-independent manner. Chemical and enzymatic cleavage in combination with site-directed mutagenesis mapped the site of guanylylation to serine 163. The purified rVP1 functioned as a primer as well as an RdRp in vitro, producing labeled dsRNA in the presence of [alpha(32)P] NTP and synthetically produced viral ss + RNA as a template. Only a single cycle of replication was observed and labeled VPg could be recovered from the dsRNA by RNase V(1) digestion. Denaturation of the dsRNA yielded genome-length labeled ssRNA, indicating that RNA synthesis was not initiated by 3'-end snap-back self-priming. Mutating serine 163 to alanine of rVP1 abolished both its self-guanylylating and polymerizing activity.

Initial events in double-strand break repair by homologous recombination in vivo involve homology searching, 39 strand invasion, and new DNA synthesis. While studies in yeast have contributed much to our knowledge of these processes, in comparison, little is known of the early events in the integrated mammalian system. In this study, a sensitive PCR procedure was developed to detect the new DNA synthesis that accompanies mammalian homologous recombination. The test system exploits a well-characterized gene targeting assay in which the transfected vector bears a gap in the region of homology to the single-copy chromosomal immunoglobulin m heavy chain gene in mouse hybridoma cells. New DNA synthesis primed by invading 39 vector ends copies chromosomal m-gene template sequences excluded by the vector-borne double-stranded gap. Following electroporation, specific 39 extension products from each vector end are detected with rapid kinetics: they appear after 0.5 hr, peak at 3-6 hr, and then decline, likely as a result of the combined effects of susceptibility to degradation and cell division. New DNA synthesis from each vector 39 end extends at least $1000 nucleotides into the gapped region, but the efficiency declines markedly within the first $200 nucleotides. Over this short distance, an average frequency of 39 extension for the two invading vector ends is $0.007 events/vector backbone. DNA sequencing reveals precise copying of the cognate chromosomal m-gene template. In unsynchronized cells, 39 extension is sensitive to aphidicolin supporting involvement of a replicative polymerase. Analysis suggests that the vast majority of 39 extensions reside on linear plasmid molecules. Homologous recombination is an important pathway of repairing DSBs and its proper function is integral to cell survival (Wyman and Kanaar 2006). The repair of a DSB by homologous recombination involves 59-to-39 resection of the DNA ends, homology searching, strand invasion of the homologous template, and new DNA synthesis. Later steps in the recombination process can involve unwinding of the newly synthesized strand from the repair template to yield a noncrossover product or formation of a joint molecule bearing Holliday junctions that may be resolved to yield either crossover or noncrossover products (Pâques and Haber 1999;Li and Heyer 2008).Studies of homologous recombination in yeast, mammalian cells, and other organisms have benefitted from analyzing repair of DSBs generated at specific sites in vivo by the endonucleases HO and I-SceI (Haber 2000;Johnson and Jasin 2001;Wei and Rong 2007). Current insight into the early initiation events of homolSupporting information is available online at