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Title:   Author  item
                                            OAKLEY, BRIAN - Western University Of Health Sciencesitem
                                            Kogut, Michael

                                                         - *Mike*item
                                            Donovan, Daviditem
                                            COX, NELSON - US Department Of Agriculture (USDA)item
                                            Berrang, Markitem
                                            CHALGHOUMI, RAJA - University Of Carthage, Tunisiaitem
                                            Seal, Bruce
  Submitted to: Meeting Abstract
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 6/23/2015
                                    Publication  Date: N/A
                                    Citation: N/AInterpretive Summary:
                                Technical Abstract: The removal of antibiotics from animal feed as mandated by the European Union in 2006 and recently proposed by the U.S. Food and Drug Administration will require new strategies to maintain animal health, optimize nutrition, and control foodborne pathogens.  We have recently pursued three main approaches to managing the GI microbiome of poultry with potential application to other food animals.  First, we have documented the taxonomic composition and diversity of naturally occurring bacterial communities through the life span of a commercial broiler.  Second, we have begun to use these data to inform cultivation efforts with the goal of assembling probiotic consortia to mimic the natural community.  Finally, genome sequencing of novel bacteria isolated from the chicken GI tract has been used to identify endolysins of bacteriophage origin that may be used as targeted antimicrobials against specific animal or human pathogens.

Methods
Microbiome characterizations were conducted with next-gen (454 and Illumina MiSeq) sequencing of bar-coded 16S rRNA gene amplicons.  Data analyses have combined standard tools with our own in-house methods to document taxonomic composition, sequence richness and diversity, and correlations of specific taxa with feed treatments, maturation of the gut, bird performance parameters, and immune responses.  
Selective cultivation for members of the Clostridiales was performed by treating digesta with chloroform to eliminate vegetative bacterial cells and cultured under anaerobic conditions.  Briefly, samples were suspended in phosphate-buffered saline (PBS) and mixed with chloroform (final concentration 3%).  The chloroform was separated by centrifugation and the treated samples were drawn off with a pipette for culture.  
Genomic DNA was sequenced on an Illumina MiSeq instrument with PE250 or PE300 reads and data processed using standard genomics tools including trimmomatic, the fastx toolkit, flash, assembly with Velvet and Spades, and annotation by RAST and IMG.

Results
	In several independent studies, temporal changes in the GI microbiome have been highly significant and independent of various feed additives which have generally had non-significant effects on GI microbial communities.  Although distinct communities are present at 1, 3, and 6 wk post-hatch, all of these are dominated by various members of the Clostridiales whose relative abundance was generally negatively correlated with the expression of pro-inflammatory cytokines. 
Selective cultivation efforts have yielded a variety of novel Clostridial isolates, including strain MG20-2 which is most closely related to the H2/CO2 acetogen Blautia hydrogenotrophica (<93% 16S rRNA sequence similarity).  Genome sequencing of 44 genomes from a collection of Clostrdium perfringens isolates has yielded a variety of interesting endolysins that are the subject of ongoing investigations.

Discussion
Identifying taxa commonly present at different stages of gut maturation may help design probiotic consortia that reflect natural communities.  The use of naturally occurring gut bacteria as probiotics has several advantages including viability in the host, ability to compete against ecologically similar taxa from the same environment, and a natural rationale for regulatory approval.  Proper management of the gut microbiome of food animals will require rational design of probiotics and targeted antimicrobials.


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